Higher Amounts Of Collagen Research Articles

Differential effects of macrophage subtype-specific cytokines on fibroblast proliferation and endothelial cell function in co-culture system.

Macrophages are involved in several critical activities associated with tissue repair and regeneration. Current approaches in regenerative medicine are focusing on leveraging the innate immune response to accelerate tissue regeneration and improve long-term healing outcomes. Of particular interest in this regard are the currently known, four main M2 macrophage subtypes: M2interleukin (IL)-4,IL-13, M2IC, M2IL-10, M2non-selective adenosine receptor agonists (NECA) (M2IL-4,IL-13 → M2NECA). In this study, rat bone marrow-derived macrophages (M0) were polarized to each of the four subtypes M2IL-4,IL-13 → M2NECA and cultured for 72 h in vitro. Luminex assay results highlighted increased production of tissue inhibitor of metalloproteinases-1 (TIMP-1) for M2IL-4,IL-13, higher amounts of transforming growth factor-beta 1 (TGF-β1) for M2IL-10, and elevated vascular endothelial growth factor A (VEGF-A) from M2NECA. Co-culture experiments performed with M2IL-10 macrophages and L929 fibroblasts highlighted the increased production of soluble collagen within the media as well as higher amounts of collagen in the extracellular matrix. Human umbilical vein endothelial cells (HUVECs) were co-cultured with M2NECA macrophages, which demonstrated an increase in intercellular adhesion molecule (ICAM) and platelet endothelial cell adhesion molecule (PECAM), as well as increased formation of endothelial tubes. The findings of this study emphasize a critical demand for further characterization and analyses of distinct M2 subtypes and careful selection of specific macrophage populations for regeneration of specific tissue types. The current, broad classification of "M2" may be sufficient in many general tissue engineering applications, but, as conditions are constantly in flux within the microenvironment in vivo, a higher degree of specificity and control over the initial M2 subtype could result in more consistent long-term outcomes where macrophages are utilized as part of an overall regenerative strategy.

Unravelling the impact of active and passive contributors to arterial stiffness in male mice and their role in vascular aging

Arterial stiffness, a key indicator of vascular health, encompassing active (vascular tone) and passive (extracellular matrix) components. This study aims to address how these different components affect arterial stiffness along the aorta and the influence of aging. Aortic segments of 12 week and 24 month old (both n = 6) male C57BL/6J mice were mounted in a Rodent Oscillatory Set-up to study Arterial Compliance, in order to measure arterial stiffness and vascular reactivity. Regional variations in arterial stiffness were evident, with abdominal infrarenal aorta (AIA) exhibiting highest stiffness and smallest diameters. AIA displayed both the highest amount of collagen and collagen:elastin ratio. Regional ex vivo vascular reactivity revealed heightened AIA contractions and lowered NO availability. Aging is a significant factor contributing towards vessel remodelling and arterial stiffness. Aging increased arterial stiffness, aortic diameters, collagen content, and reduced VSMC contraction. The results of this study could identify specific regions or mechanisms to target in the development of innovative therapeutic interventions aimed at enhancing overall vascular health.

Effect of packaging thickness and muscle type on ultrasound-assisted beef quality

High-intensity ultrasound (HIU) can modify muscle structure, leading to improvements in tenderness. However, factors such as packing type and muscle complexity may attenuate the acoustic cavitation. In this research, the effect of packing thickness (40.6–70 μm) on the quality of bovine Gluteus medius and Biceps femoris treated with HIU (37 kHz, 90 W/cm2, 40 min) was evaluated. The hardness of G. medius decreased significantly as the thickness of the packing bag decreased. The wide interfibrillar and intermyofibrillar spaces corroborated the tenderizing effect. These effects are related to damage of cell structure and changes in the collagen content (3.37 ± 0.1 µg/mL). In addition, the HIU decrease the variability in the water holding capacity of the muscle produced by the use of low thickness bags during storage. The trained sensory panel described the sonicated samples in 50.8 μm bags as less hard and juicier. Contrarily, in B. femoris no significant effects were reported in the variables evaluated. B. femoris is a white muscle, with a high amount of collagen (3.59 ± 0.1 µg/mL) and little intramuscular fat. Consequently, the effect of the HIU on muscle quality is associated with the composition of the muscle fibers and the thickness of the packing bag. HIU application is recommended to improve the quality of leg muscles whenever low-thickness bags (50.8 μm or less) are used.

A dual-crosslinked injectable hydrogel derived from muscular decellularized matrix promoting myoblasts proliferation and myogenic differentiation

To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. Firstly, hyaluronic acid was oxidized with NaIO 4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young's modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). FTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young's modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point ( P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group ( P<0.05). The MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.

Clitoria ternatea Flower Extract-Based Gel Elevates TGF-β1 Gene Expression and Collagen Density in UVB-Induced Collagen Loss Rat Skin

Ultraviolet B (UVB) irradiation on the skin induces collagen loss by the ROS pathways and pro-collagen factors, mainly the TGF-β family. Many studies of antioxidant compounds in Clitoria ternatea have shown to induce decreasing ROS production, in the collagen loss process. However, the molecular mechanism of action is still not clearly studied, inducing the mechanism of TGF-β1 activation and regulation of collagen loss need to be explored. This study aimed to investigate the effect of Clitoria ternatea flower extract-based gel on TGF-β1 gene expression and collagen density in UVB-induced rat skin. A randomized control group post-test-only design was conducted on 20 male Wistar rats aged 8-10 weeks (150-250 grams). Rats were divided into four groups: Untreated group (Healthy Control), UVB+Based gel group (Negative Control), 5% Clitoria ternatea flower extract-based gel group (T-5%), and 10% Clitoria ternatea flower extract-based gel group (T-10%). Rats exposed to UVB for 5 consecutive days and treated with gel every day for two weeks. At week 3, rat skins were isolated for TGF-β1 gene expression using qRT-PCR, and Masson Trichrome-collagen specific staining. Comparative analysis of qRT-PCR showed that the lowest mean TGF-β1 expression was seen in the Negative Control group (0.01±0.00), followed by T-5% (0.11±0.22), Healthy Control group (1.01±0.01) and T-10% (2.32±2.46). The highest amount of collagen is in the Healthy Control group (43.83±5.3), followed by T-10% (38.39±3.1), T-5% (29.04±3.2), and the Negative Control group. (13.87±2.7). Clitoria ternatea flower extract-based gel may increase TGF-β1 gene expression and collagen deposition in the UVB-induced collagen loss rat skin.

Chelation of the Collagen Peptide of Seabass (Lates calcarifer) Scales with Calcium and Its Product Development

Seabass (Lates calcarifer) is one of the top farmed and raised fish in Taiwan, and fish scales are the main by-product after processing. Fish scales contain high amounts of collagen, which can chelate with minerals and enhance mineral absorption in the human body. Hence, fish scales from seabass were enzymatically hydrolyzed to obtain seabass scale collagen peptide (SBSCP). Calcium, the most consumed mineral supplement, was chelated with SBSCP to form SBSCP-Ca. The optimal conditions for chelation were a peptide/calcium ratio: 1:1 (w/w), pH 5.0, and 50 °C for 20 min. The conjugated sites were carboxyl and amino groups based on Fourier transform infrared (FTIR) spectroscopy. Scanning electronic microscope/energy-dispersive X-ray spectroscopy (SEM/EDS) evidently showed the alternation of SBSCP’s molecular structure after chelation and increased concentrations of metal ions. SBSCP-Ca was stable up to 90 °C and from pH 2.0 to 5.0. The retention rate was 70%, as determined after in vitro digestion. The extracts of blackcurrant or berry-grape seeds were added to neutralize the fishy odor and provide antioxidant ability for commercialization. This is the first complete study of the characteristics of SBSCP-Ca as well as their commercialization.

Quantitative comparison between carotid plaque hardness and histopathological findings: an observational study

BackgroundPlaque hardness in carotid artery stenosis correlates with cerebral infarction. This study aimed to quantitatively compare plaque hardness with histopathological findings and identify the pathological factors involved in plaque hardness.MethodsThis study included 84 patients (89 lesions) undergoing carotid endarterectomy (CEA) at our institution. Plaque hardness was quantitatively measured immediately after excision using a hardness meter. Collagen and calcification were evaluated as the pathological factors. Collagen was stained with Elastica van Gieson stain, converted to a gray-scale image, and displayed in a 256-step histogram. The median gray-scale median (GSM) was used as the collagen content. The degree of calcification was defined by the hematoxylin–eosin stain as follows: "0:" no calcification, "1:" scattered microcalcification, or "2:" calcification greater than 1 mm or more than 2% of the total calcification. Carotid echocardiographic findings, specifically echoluminance or the brightness of the narrowest lesion of the plaque, classified as hypo-, iso-, or hyper-echoic by comparison with the intima-media complex surrounding the plaque, and clinical data were reviewed.ResultsPlaque hardness was significantly negatively correlated with GSM [Spearman's correlation coefficient: -0.7137 (p < 0.0001)]: the harder the plaque, the higher the collagen content. There were significant differences between plaque hardness and degree of calcification between "0" and "2" (p = 0.0206). For plaque hardness and echoluminance (hypo-iso-hyper), significant differences were found between hypo-iso (p = 0.0220), hypo-hyper (p = 0.0006), and iso-hyper (p = 0.0015): the harder the plaque, the higher the luminance. In single regression analysis, GSM, sex, and diabetes mellitus were significant variables, and in multiple regression analysis, only GSM was extracted as a significant variable.ConclusionsPlaque hardness was associated with a higher amount of collagen, which is the main component of the fibrous cap. Greater plaque hardness was associated with increased plaque stability. The degree of calcification may also be associated with plaque hardness.

Effects of morphometric and biochemical parameters on collagen and pepsin-solubilized collagen yields of Holothuria tubulosa (Gmelin, 1790) and Holothuria (Roweothuria) poli (Delle Chiaje, 1823)

Due to their unique biochemical composition, sea cucumbers are highly prized marine echinoderm species. One of their most important properties is that they contain a high amount of collagen in their body wall. In this study, the relationship between collagen and pepsin-solubilized collagen yields from Holothuria tubulosa and Holothuria poli and morphometric and biochemical parameters were investigated.&#x0D; Collagen yields were in the range of 10.63–16.04% for H. tubulosa and 7.12–13.10% for H. poli. It was determined that they may be related to length, body wall weight, and biochemical composition at different length frequencies. Moreover, maturity may have a direct effect on the yield, as mature specimens were found to have lower content of collagen, whereas immature small specimens contained a higher percentage of collagen. It was found that with increasing pepsin concentration, the PSC yield increased to 1.83–1.89% in H. tubulosa and H. poli, respectively. It was determined that collagen from smaller individuals, which contained more moisture and ash, was likely more susceptible to pepsin hydrolyzation.&#x0D; This is the first published study demonstrating that collagen yield of sea cucumbers can vary with length, weight, maturity, and biochemical composition, in addition to species-specific differences.

Abstract 12112: Near Infrared Spectroscopic Characterization of Cardiac and Renal Fibrosis

Introduction: Fibrosis is significantly associated with nearly all forms of heart and kidney disease. Clinical diagnosis of fibrosis is currently reliant on conventional methods that do not have the sensitivity and specificity required for effective diagnosis. Hypothesis: An handheld portable near-infrared (NIR) spectrometer, usable intraoperatively coupled to machine learning algorithms can discriminate between fibrotic and healthy cardiac and renal tissue. We sought to validate this in an animal model. Methods: 10 Male Sprague Dawley rats (SDR) with either induced cardiac (SDR-H) and renal (SDR-K) fibrosis (n=5) compare to normal controls (n=5). Hearts from all rats were used as tissue screening in model validation as they contain a high amount of collagen. Multiple tissue samples were harvested from SDR-K ( n fibrosis = 12, n control = 4) and SDR-H ( n fibrosis = 12, n control = 4) groups. NIR spectra (1350 - 2500 nm) were acquired from all tissue sections. Results: Stained sections showed insignificant differences between the fibrotic SDR-H and their corresponding controls as collagen fibrils dominated both groups. SDR-K showed distinguishable features between examined groups. NIR absorption at 1509, 1725, 2055, and 2306 nm were found to be highly indicative of fibrosis (Figure1). PCA (57% explained variance) and PLS-DA (sensitivity: 96%) showed excellent discrimination for SDR-K groups while the heart shows no meaningful discrimination for SDR-H groups. SVM and LR analysis corroborated these results by achieving a 98% classification accuracy for SDR-K and no discrimination for SDR-H. All machine learning models were cross-validated with outcomes of histological staining to establish a robust interpretation and underpin their pathological meanings. Conclusions: NIR accurately diagnoses cardiac and renal fibrosis in rats model. There is potential for this technology to be translated into an intraoperative instrument for tissue diagnosis

ACTIVIDAD ANTIOXIDANTE Y ANTIMICROBIANA DEL HIDROLIZADO DE GELATINA DE PIELES DE Sarda chiliensis chiliensis “bonito”

Los residuos de la actividad pesquera contienen altas cantidades de colágeno y gelatina, que sometidos a procesos de hidrólisis enzimáticas podrían manifestar alguna actividad biológica. El presente trabajo tuvo como objetivo determinar la actividad antioxidante y antimicrobiana del hidrolizado de gelatina obtenido del colágeno de las pieles de Sarda chiliensis chiliensis “bonito”. Para ello, la gelatina obtenida de las pieles de bonito se hidrolizó con la enzima alcalasa a diferentes condiciones de pH, temperatura, tiempo y relación enzima/sustrato. La actividad antioxidante in vitro de quince hidrolizados se evaluó mediante radical 2,2-difenil- 1-picrilhidracilo (DPPH) y radical catiónico 2,2-azino-bis-3-etilbenzotiazolin-6-sulfónico (ABTS). La actividad antimicrobiana se evaluó mediante difusión en disco y microdilución en placa para la concentración mínima inhibitoria (CMI). Los hidrolizados de gelatina de bonito presentaron grado de hidrólisis superiores a 10 %, con un máximo de 33,67 % y evidenciaron la IC50 para DPPH entre 0,459 y 1,830 mg/mL y para ABTS entre 0,075 y 0,200 mg/mL. El valor más bajo y el más alto de TEAC fue 61,76 y 165,38 μmol de trolox/g para los hidrolizados H7011 y H7012, respectivamente. Ocho hidrolizados (1000 μg/mL) presentaron efecto inhibitorio frente a Staphylococcus aureus y Bacillus subtilis con CMI igual o mayor a 500 μg/mL. Se puede concluir que los hidrolizados obtenidos mostraron actividad antioxidante y antimicrobiana para el modelo experimental aplicado.

Wound healing activity of neferine in experimental diabetic rats through the inhibition of inflammatory cytokines and nrf-2 pathway

The diabetic foot ulcer (DFU) may be associated with late healing and septic manifestation, subsequently lead to amputation which is an overpriced incident. Neferine is an alkaloid found lotus. Neferine possesses many physiological functions such as anti-inflammatory, antioxidant, antimicrobial activity and anticancer effect. The aim of the present study was to evaluate the effect of topical application based on neferine, in streptozotocin-induced diabetic incision wound models rats. The data demonstrated wound healing activities via macroscopic, biochemical, histological, immuno-histochemical, immunofluorescent and molecular methods. There was significant acceleration in wound closure rate, decrease in the period of re-epitalization, higher amount of collagen and protein content in neferine treated group when compared with diabetic wound control. Histological data evidence collagen formation in skin and marked granulation with more connective tissue markers. The augmentation of serum insulin and HDL was dissimilar with blood glucose reduction and decreased lipid level (TC, TG and LDL). The healing effect was additionally validated by decreased lipid peroxidation and enhanced antioxidants. Concurrently, the mRNA level of Nrf-2, collagen-1, TGF-β and α-SMA were decreased with Kaep-1 increased significantly. This enhancement was achieved through downregulation of inflammatory mediators such as nuclear factor kappa-light-chain-enhancer of activated B cells, tumour necrosis factor-α, interleukin-1β, interleukin-8, inducible nitric oxide synthase, and cyclooxygenase-2, and upregulation of growth factor such as in groups treated with neferine. The western blot results reveal the macrophage (CD 68 and CD 163) involved in wound healing markedly elevated. Hence, the results indicate that neferine significantly promotes a fast and efficient wound healing in diabetic rats.

Human umbilical cord-derived scaffolds for cartilage tissue engineering.

Since articular cartilage is an avascular tissue, it has limited self-regeneration capacity after damage. Current methods for human bone marrow-derived mesenchymal stem cell (hBM-MSC) differentiation into cartilage result in tissues with a lower quality as compared to native articular cartilage. Decellularized biological scaffolds have the potential to provide appropriate signals, in order to support cellular retention, migration, proliferation, and differentiation. Given the high amount of collagen, hyaluronic acid (HA), and glycosaminoglycan (GAG) in umbilical cord, this tissue can be considered as an abundant natural biomaterial for tissue engineering applications. Human umbilical cord-derived scaffolds were prepared, and the chondrogenic induction of hBM-MSCs loaded onto the scaffolds was investigated. Gelatin-based scaffolds as a commercial material was used as a control. The results show that hBM-MSCs in tissue-derived scaffolds have an increased expression of chondrogenic markers compared with gelatin, whereas there are no significant differences between the expression of hypertrophic and osteogenic markers between tissue and gelatin scaffolds. In conclusion, it is confirmed that umbilical cord-derived scaffolds are able to provide a native environment for the cells and can promote cartilage differentiation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1793-1802, 2019.

Low-level laser and adipose-derived stem cells altered remodelling genes expression and improved collagen reorganization during tendon repair.

ObjectivesThe cellular therapy using adipose‐derived mesenchymal stem cells (ASCs) aims to improve tendon healing, considering that repaired tendons often result in a less resistant tissue. Our objective was to evaluate the effects of the ASCs combination with a low‐level laser (LLL), an effective photobiostimulation for the healing processes.Materials and methodsRats calcaneal tendons were divided into five groups: normal (NT), transected (T), transected and ASCs (SC) or LLL (L), or with ASCs and LLL (SCL).ResultsAll treated groups presented higher expression of Dcn and greater organization of collagen fibres. In comparison with T, LLL also up‐regulated Gdf5 gene expression, ASCs up‐regulated the expression of Tnmd, and the association of LLL and ASCs down‐regulated the expression of Scx. No differences were observed for the expression of Il1b, Timp2, Tgfb1, Lox, Mmp2, Mmp8 and Mmp9, neither in the quantification of hydroxyproline, TNF‐α, PCNA and in the protein level of Tnmd. A higher amount of IL‐10 was detected in SC, L and SCL compared to T, and higher amount of collagen I and III was observed in SC compared to SCL.ConclusionsTransplanted ASCs migrated to the transected region, and all treatments altered the remodelling genes expression. The LLL was the most effective in the collagen reorganization, followed by its combination with ASCs. Further investigations are needed to elucidate the molecular mechanisms involved in the LLL and ASCs combination during initial phases of tendon repair.

Transected Tendon Treated with a New Fibrin Sealant Alone or Associated with Adipose-Derived Stem Cells.

Tissue engineering and cell-based therapy combine techniques that create biocompatible materials for cell survival, which can improve tendon repair. This study seeks to use a new fibrin sealant (FS) derived from the venom of Crotalus durissus terrificus, a biodegradable three-dimensional scaffolding produced from animal components only, associated with adipose-derived stem cells (ASC) for application in tendons injuries, considered a common and serious orthopedic problem. Lewis rats had tendons distributed in five groups: normal (N), transected (T), transected and FS (FS) or ASC (ASC) or with FS and ASC (FS + ASC). The in vivo imaging showed higher quantification of transplanted PKH26-labeled ASC in tendons of FS + ASC compared to ASC on the 14th day after transection. A small number of Iba1 labeled macrophages carrying PKH26 signal, probably due to phagocytosis of dead ASC, were observed in tendons of transected groups. ASC up-regulated the Tenomodulin gene expression in the transection region when compared to N, T and FS groups and the expression of TIMP-2 and Scleraxis genes in relation to the N group. FS group presented a greater organization of collagen fibers, followed by FS + ASC and ASC in comparison to N. Tendons from ASC group presented higher hydroxyproline concentration in relation to N and the transected tendons of T, FS and FS + ASC had a higher amount of collagen I and tenomodulin in comparison to N group. Although no marked differences were observed in the other biomechanical parameters, T group had higher value of maximum load compared to the groups ASC and FS + ASC. In conclusion, the FS kept constant the number of transplanted ASC in the transected region until the 14th day after injury. Our data suggest this FS to be a good scaffold for treatment during tendon repair because it was the most effective one regarding tendon organization recovering, followed by the FS treatment associated with ASC and finally by the transplanted ASC on the 21st day. Further investigations in long-term time points of the tendon repair are needed to analyze if the higher tissue organization found with the FS scaffold will improve the biomechanics of the tendons.

Quality parameters of frankfurter-type sausages with partial replacement of fat by hydrolyzed collagen

Frankfurters were manufactured to determine the effects of pork backfat replacement by hydrolyzed collagen on the quality parameters. Four formulations were developed: control (SS) and three levels of collagen replacements: 25% (CS25), 50% (SC50) and 75% (SC75). Appearance, aroma, texture, taste and overall acceptance were analyzed. Parameters like moisture, ash, lipid and protein, water activity (Aw), pH, water holding capacity (WHC), emulsion stability (ES), color (L*, a*, b*) and shear force (SF) was analyzed, as well as the texture properties. SC25, SC50 and SC75 had better texture and taste compared to SS, but the appearance, aroma and overall acceptability did not differ among treatments (p < 0.05). In SC75, a 43% increase in protein content compared to SS was observed, and increased ash content. There were no changes in pH and Aw in sausages. L* value decreased with fat replacement, and a* did not change, but SC75 showed higher b* value. From 50% of collagen addition, higher WHC and SF were obtained. In parallel, only hardness, and chewiness had effects significant, increasing in higher amounts of collagen, probably by aggregating water in the protein matrix. It was possible to obtain 50% reduction with hydrolyzed collagen without reducing the quality of sausages.

Optimization of Liver Decellularization Maintains Extracellular Matrix Micro-Architecture and Composition Predisposing to Effective Cell Seeding.

Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative to conventional transplantation. However, scaffolds are usually obtained using decellularization protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We aim to develop a decellularization technique that reliably maintains hepatic microarchitecture and ECM components. Isolated rat livers were decellularized by detergent-enzymatic technique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteomics confirmed decellularization with further DNA reduction with the addition of EDTA. Quantification, histology, immunostaining, and proteomics demonstrated preservation of extracellular matrix components in both scaffolds with a higher amount of collagen and glycosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete repopulation in 14 days, with cells proliferating at that time. Decellularization using DET preserves microarchitecture and extracellular matrix components whilst allowing for cell growth for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation of the scaffolds and scaling up of the methodology are the next steps for successful hepatic tissue engineering.

Odontogenic Myxoma of Mandible: Report of A Rare Case.

Odontogenic Myxoma of Mandible: Report of A Rare Case.

Optimization of optical and mechanical properties of real architecture for 3-dimensional tissue equivalents: Towards treatment of limbal epithelial stem cell deficiency

Limbal epithelial stem cell (LESC) deficiency can cause blindness. Transplantation of cultured human limbal epithelial cells (hLE) on human amniotic membrane (HAM) can restore vision but clinical graft manufacture can be unreliable. We have developed a reliable and robust tissue equivalent (TE) alternative to HAM, Real Architecture for 3D Tissue (RAFT). Here, we aimed to optimize the optical and mechanical properties of RAFT TE for treatment of LESC deficiency in clinical application. The RAFT TE protocol is tunable; varying collagen concentration and volume produces differing RAFT TEs. These were compared with HAM samples taken from locations proximal and distal to the placental disc. Outcomes assessed were transparency, thickness, light transmission, tensile strength, ease of handling, degradation rates and suitability as substrate for hLE culture. Proximal HAM samples were thicker and stronger with poorer optical properties than distal HAM samples. RAFT TEs produced using higher amounts of collagen were thicker and stronger with poorer optical properties than those produced using lower amounts of collagen. The ‘optimal’ RAFT TE was thin, transparent but still handleable and was produced using 0.6ml of 3mg/ml collagen. Degradation rates of the ‘optimal’ RAFT TE and HAM were similar. hLE achieved confluency on ‘optimal’ RAFT TEs at comparable rates to HAM and cells expressed high levels of putative stem cell marker p63α. These findings support the use of RAFT TE for hLE transplantation towards treatment of LESC deficiency.

A novel bioreactor for mechanobiological studies of engineered heart valve tissue formation under pulmonary arterial physiological flow conditions.

The ability to replicate physiological hemodynamic conditions during in vitro tissue development has been recognized as an important aspect in the development and in vitro assessment of engineered heart valve tissues. Moreover, we have demonstrated that studies aiming to understand mechanical conditioning require separation of the major heart valve deformation loading modes: flow, stretch, and flexure (FSF) (Sacks et al., 2009, "Bioengineering Challenges for Heart Valve Tissue Engineering," Annu. Rev. Biomed. Eng., 11(1), pp. 289-313). To achieve these goals in a novel bioreactor design, we utilized a cylindrical conduit configuration for the conditioning chamber to allow for higher fluid velocities, translating to higher shear stresses on the in situ tissue specimens while retaining laminar flow conditions. Moving boundary computational fluid dynamic (CFD) simulations were performed to predict the flow field under combined cyclic flexure and steady flow (cyclic-flex-flow) states using various combinations of flow rate, and media viscosity. The device was successfully constructed and tested for incubator housing, gas exchange, and sterility. In addition, we performed a pilot experiment using biodegradable polymer scaffolds seeded with bone marrow derived stem cells (BMSCs) at a seeding density of 5 × 10(6) cells/cm(2). The constructs were subjected to combined cyclic flexure (1 Hz frequency) and steady flow (Re = 1376; flow rate of 1.06 l/min (LPM); shear stress in the range of 0-9 dynes/cm(2) for 2 weeks to permit physiological shear stress conditions. Assays revealed significantly (P < 0.05) higher amounts of collagen (2051 ± 256 μg/g) at the end of 2 weeks in comparison to similar experiments previously conducted in our laboratory but performed at subphysiological levels of shear stress (<2 dynes/cm(2); Engelmayr et al., 2006, "Cyclic Flexure and Laminar Flow Synergistically Accelerate Mesenchymal Stem Cell-Mediated Engineered Tissue Formation: Implications for Engineered Heart Valve Tissues," Biomaterials, 27(36), pp. 6083-6095). The implications of this novel design are that fully coupled or decoupled physiological flow, flexure, and stretch modes of engineered tissue conditioning investigations can be readily accomplished with the inclusion of this device in experimental protocols on engineered heart valve tissue formation.

Extraction and characterization of collagen from rabbit skin: partial characterization

Extraction and characterization of collagen were carried out in rabbit skins as a new alternative for collagen type I. Acetic acid and pepsin were for the extraction of soluble and insoluble collagen, respectively. The enzymatic treatment yielded higher amount of collagen (71%). The average pH value was 6.3, no matter what the method of extraction was. Denaturation temperature of collagen was found at 36°C approximately in two different techniques: Rheometer and Differential Scanning Calorimetry (DSC). Despite the amount of collagen in solution was low, its viscosity was high because of the hydrodynamic behaviour of collagen molecules. Sodium dodecyl sulphate polyacrylamide gel electrophoresis results showed three different bands that reflected two alpha-chains and one beta-chain with molecular weights of 102, 118 and 220 kDa, respectively. Determination of hydroxyproline gave evidence that the extracted material was collagen. It was concluded that rabbit skin could be an alternative source for the extraction of collagen.