Techniques For Type I Collagen Organization

Tissue Engineering is a process in which cells, engineering, and material methods are used in amalgamation to improve biological functions. The purpose of tissue engineering is to develop alternative solutions to treat or cure tissues and organs that have been severely altered or damaged by diseases, congenital defects, trauma, or cancer. One of the most common and most promising biological materials for tissue engineering to develop scaffolds is Type I collagen. A major challenge in biomedical research is aligning Type I collagen to mimic biological structures, such as ligaments, tendons, bones, and other hierarchal aligned structures within the human body. The intent of this research is to examine possible techniques for organizing Type I collagen and to assess which of the techniques is effective for potential biological applications. The techniques used in this research to organize collagen are soft lithography with solution-assisted sonication embossing, directional freezing, and direct poling. The final concentration used for both soft lithography with solution-assisted sonication embossing and direct poling was 1 mg/ml, whereas for directional freezing the final concentration varied between 4mg/ml, 2mg/ml, and 1 mg/ml. These techniques were characterized using the Atomic Force Microscope (AFM) and Helium Ion Microscope (HIM). In this study, we have found that out of the three techniques, the soft lithography and directional freezing techniques have been successful in organizing collagen in a particular pattern, but not alignment. We concluded alignment may be dependent on the pH of collagen and the amount of acetic acid used in collagen solution. However, experiments are still being conducted to optimize all three techniques to align collagen in a unidirectional arrangement

Techniques For Type I Collagen Organization

Tissue Engineering is a process in which cells, engineering, and material methods are used in amalgamation to improve biological functions. The purpose of tissue engineering is to develop alternative solutions to treat or cure tissues and organs that have been severely altered or damaged by diseases, congenital defects, trauma, or cancer. One of the most common and most promising biological materials for tissue engineering to develop scaffolds is Type I collagen. A major challenge in biomedical research is aligning Type I collagen to mimic biological structures, such as ligaments, tendons, bones, and other hierarchal aligned structures within the human body. The intent of this research is to examine possible techniques for organizing Type I collagen and to assess which of the techniques is effective for potential biological applications. The techniques used in this research to organize collagen are soft lithography with solution-assisted sonication embossing, directional freezing, and direct poling. The final concentration used for both soft lithography with solution-assisted sonication embossing and direct poling was 1 mg/ml, whereas for directional freezing the final concentration varied between 4mg/ml, 2mg/ml, and 1 mg/ml. These techniques were characterized using the Atomic Force Microscope (AFM) and Helium Ion Microscope (HIM). In this study, we have found that out of the three techniques, the soft lithography and directional freezing techniques have been successful in organizing collagen in a particular pattern, but not alignment. We concluded alignment may be dependent on the pH of collagen and the amount of acetic acid used in collagen solution. However, experiments are still being conducted to optimize all three techniques to align collagen in a unidirectional arrangement

Techniques For Type I Collagen Organization

Tissue Engineering is a process in which cells, engineering, and material methods are used in amalgamation to improve biological functions. The purpose of tissue engineering is to develop alternative solutions to treat or cure tissues and organs that have been severely altered or damaged by diseases, congenital defects, trauma, or cancer. One of the most common and most promising biological materials for tissue engineering to develop scaffolds is Type I collagen. A major challenge in biomedical research is aligning Type I collagen to mimic biological structures, such as ligaments, tendons, bones, and other hierarchal aligned structures within the human body. The intent of this research is to examine possible techniques for organizing Type I collagen and to assess which of the techniques is effective for potential biological applications. The techniques used in this research to organize collagen are soft lithography with solution-assisted sonication embossing, directional freezing, and direct poling. The final concentration used for both soft lithography with solution-assisted sonication embossing and direct poling was 1 mg/ml, whereas for directional freezing the final concentration varied between 4mg/ml, 2mg/ml, and 1 mg/ml. These techniques were characterized using the Atomic Force Microscope (AFM) and Helium Ion Microscope (HIM). In this study, we have found that out of the three techniques, the soft lithography and directional freezing techniques have been successful in organizing collagen in a particular pattern, but not alignment. We concluded alignment may be dependent on the pH of collagen and the amount of acetic acid used in collagen solution. However, experiments are still being conducted to optimize all three techniques to align collagen in a unidirectional arrangement

Second-harmonic generation microscopy analysis reveals proteoglycan decorin is necessary for proper collagen organization in prostate.

Collagen remodeling occurs in many prostate pathologies; however, the underlying structural architecture in both normal and diseased prostatic tissues is largely unexplored. Here, we use second-harmonic generation (SHG) microscopy to specifically probe the role of the proteoglycan decorin (Dcn) on collagen assembly in a wild type (wt) and Dcn null mouse (Dcn  -    /    -  ). Dcn is required for proper organization of collagen fibrils as it regulates size by forming an arch-like structure at the end of the fibril. We have utilized SHG metrics based on emission directionality (forward-backward ratio) and relative conversion efficiency, which are both related to the SHG coherence length, and found more disordered fibril organization in the Dcn  -    /    -  . We have also used image analysis readouts based on entropy, multifractal dimension, and wavelet transforms to compare the collagen fibril/fiber architecture in the two models, where all these showed that the Dcn  -    /    -   prostate comprised smaller and more disorganized collagen structures. All these SHG metrics are consistent with decreased SHG phase matching in the Dcn  -    /    -   and are further consistent with ultrastructural analysis of collagen in this model in other tissues, which show a more random distribution of fibril sizes and their packing into fibers. As Dcn is a known tumor suppressor, this work forms the basis for future studies of collagen remodeling in both malignant and benign prostate disease

DDR2 controls breast tumor stiffness and metastasis by regulating integrin mediated mechanotransduction in CAFs

Biomechanical changes in the tumor microenvironment influence tumor progression and metastases. Collagen content and fiber organization within the tumor stroma are major contributors to biomechanical changes (e., tumor stiffness) and correlated with tumor aggressiveness and outcome. What signals and in what cells control collagen organization within the tumors, and how, is not fully understood. We show in mouse breast tumors that the action of the collagen receptor DDR2 in CAFs controls tumor stiffness by reorganizing collagen fibers specifically at the tumor-stromal boundary. These changes were associated with lung metastases. The action of DDR2 in mouse and human CAFs, and tumors in vivo, was found to influence mechanotransduction by controlling full collagen-binding integrin activation via Rap1-mediated Talin1 and Kindlin2 recruitment. The action of DDR2 in tumor CAFs is thus critical for remodeling collagen fibers at the tumor-stromal boundary to generate a physically permissive tumor microenvironment for tumor cell invasion and metastases

Quantification of Collagen Organization Using Fractal Dimensions and Fourier Transforms

Collagen fibers and fibrils that comprise tendons and ligaments are disrupted or damaged during injury. Fibrillogenesis during healing produces a matrix that is initially quite disorganized, but remodels over time to resemble, but not replicate, the original roughly parallel microstructure. Quantification of these changes is traditionally a laborious and subjective task. In this work we applied two automated techniques, fast Fourier transformation (FFT) and fractal dimension analysis (FA) to quantify the organization of collagen fibers or fibrils. Using multi-photon images of collagen fibers obtained from rat ligament we showed that for healing ligaments, FA differentiates more clearly between the different time-points during healing. Using scanning electron microscopy images of overstretched porcine flexor tendon, we showed that combining FFT and FA measures distinguishes the damaged and undamaged groups more clearly than either method separately

Effect of ultrasound and dexpanthenol on collagen organization in tegumentary lesions

OBJECTIVE: To analyze the effect of ultrasound (US), dexpanthenol (d-P) and a combination of these treatments (US+d-P) on collagen fiber organization in tegumentary lesions in rats by birefringence analysis. METHODS: Wistar rats (50) were anesthetized (Thionembutal - Sodic = 50mg/Kg), 1cm² of dorsal region skin was removed, and the animals were divided into five groups: control (C), gel (G), US (3 MHz, 0.1 W/cm2, 1 minute, continuous), d-P (10%) and US+d-P. After daily treatment for 7 and 14 days, 6µm thick sections of lesioned areas were stained in picrosirius and measurements of the collagen birefringent area (µm²) were obtained using polarized light microscopy (Zeiss Axiolab-ZEISS- Germany) with histological image analysis software (KS 400 2.0 - Kontrol Eletronics, Munique, Germany). The means were compared by ANOVA followed by the Tukey test (p<0.05). RESULTS: The US+d-P group showed a significantly greater (p<0.001) birefringent area (1586.43±162.14) than the other experimental groups: C (139.36±35.35), US (317.55±129.9) and d-P (192.41±3657) by the 7th day of treatment, indicating acceleration of the wound healing process. By the 14th day of treatment, the US+d-P, US and d-P groups presented greater birefringence than the control group, but did not differ from each other. CONCLUSION: The combination of treatments (US+d-P) accelerated collagen fiber synthesis and organization in the early stages of cutaneous repair.OBJETIVO: Analisar o efeito do ultrassom (US), do dexapantenol (d-P) e da associação dos tratamentos (US+d-P) na organização de fibras colágenas na lesão tegumentar em ratos por meio da análise da birrefringência. MÉTODOS: Foram utilizados 50 ratos Wistar, anestesiados com Thionembutal Sódico (50mg/Kg), dos quais foi retirado 1cm² de pele da região dorsal, divididos em cinco grupos: controle (C), gel (G), US (3 MHz, 0,1 W/cm², 1 minuto, modo contínuo), d-P (10%) e US+d-P. Após sete e 14 dias de tratamento diário, foram removidos segmentos dessas áreas e obtidos cortes de 6µm de espessura que, posteriormente, foram corados em Picrosirius. Os cortes foram observados em microscopia de polarização utilizando um software responsável pela medida de birrefringência das fibras colágenas (KS400 2.0 - Kontrol Eletronics). As médias das áreas birrefringentes (µm²) de cada grupo foram submetidas à análise de variância pela ANOVA, seguida do teste de Tukey (p<0,05). RESULTADOS: A média de área birrefringente do grupo US+d-P (1586,43±162,14) foi maior (p<0,001) que a dos grupos experimentais (C: 139,36±35,35, US: 317,55±129,9 e d-P: 192,41±36,57) no 7º dia de tratamento, indicando uma aceleração na síntese e organização das fibras colágenas na região lesionada. No 14º dia de tratamento, os grupos US+d-P (2858,47±510,17), US (1779,94±482,78) e d-P (2546,88±304,45) apresentaram área birrefringente maior que a do grupo C, porém não diferiram entre si. CONCLUSÃO: A associação dos tratamentos (US+d-P) acelerou a síntese e a organização das fibras colágenas apenas no estágio inicial de reparo tegumentar.Universidade Metodista de Piracicaba Faculdade de Ciências da SaúdeUniversidade Estadual de Campinas Faculdade de Odontologia de PiracicabaUniversidade Federal de São Paulo (UNIFESP) Farmácia e BioquímicaUNIARARASUNIFESP, Farmácia e BioquímicaSciEL

Evaluation of a Surgical Intervention to Experimentally Compare CO2 Laser to Scalpel Incisions, Added Growth Factor, and Suture Material to Reduce Cutaneous Scarring

Introduction The goal of this study was to determine if the repair of full thickness skin incisions in an animal model could be improved by using a CO2 laser vs. scalpel, commercial vs. swine intestinal submucosa (SIS) sutures, and addition of exogenous nerve growth factor (NGF). Materials and Methods A rat model was used to evaluate the following tissue components: prevalence of mast cell granules, thickness of epidermis, organization of collagen, infiltration of tissue into SIS, neutrophil presence around suture holes, and granulation tissue production around suture holes. Results Added NGF led to a significant decrease in the number of granules in mast cells following laser incisions. A significant number of neutrophils were detected in skin following laser incision without added NGF. Added NGF significantly increased the band iv of granulation tissue for both types of incision methods however, the laser resulted in a significantly wider band of granulation tissue with or without added NGF. A thicker epithelium was apparent following use of laser as was the level of collagen organization. Added NGF significantly increased incorporation of skin elements into SIS sutures. The use of the laser without NGF resulted in greatest collagen organization, number of mast cell granules, and neutrophils, and significantly greater vascularization. Discussion The greater extent and duration of granulation tissue proliferation following laser incision may be attributable to an inappropriately high laser dosage. Collagen organization improves with laser use. Incorporation of tissue into SIS sutures was promoted by adding NGF, but unaffected by surgical technique. Increased vascularity following laser incision suggested blood vessels re-opened or angiogenesis occurred post-surgery. With added NGF, epidermal width following laser incision was even greater in contrast to the scalpel incised group. Depending upon the intent of the surgeon, the use of surgical modality, suture material, or additional exogenous NGFs has to be tailored to the specific patient and desired outcom

Osteocyte dysfunction promotes osteoarthritis through MMP13-dependent suppression of subchondral bone homeostasis.

Osteoarthritis (OA), long considered a primary disorder of articular cartilage, is commonly associated with subchondral bone sclerosis. However, the cellular mechanisms responsible for changes to subchondral bone in OA, and the extent to which these changes are drivers of or a secondary reaction to cartilage degeneration, remain unclear. In knee joints from human patients with end-stage OA, we found evidence of profound defects in osteocyte function. Suppression of osteocyte perilacunar/canalicular remodeling (PLR) was most severe in the medial compartment of OA subchondral bone, with lower protease expression, diminished canalicular networks, and disorganized and hypermineralized extracellular matrix. As a step toward evaluating the causality of PLR suppression in OA, we ablated the PLR enzyme MMP13 in osteocytes while leaving chondrocytic MMP13 intact, using Cre recombinase driven by the 9.6-kb DMP1 promoter. Not only did osteocytic MMP13 deficiency suppress PLR in cortical and subchondral bone, but it also compromised cartilage. Even in the absence of injury, osteocytic MMP13 deficiency was sufficient to reduce cartilage proteoglycan content, change chondrocyte production of collagen II, aggrecan, and MMP13, and increase the incidence of cartilage lesions, consistent with early OA. Thus, in humans and mice, defects in PLR coincide with cartilage defects. Osteocyte-derived MMP13 emerges as a critical regulator of cartilage homeostasis, likely via its effects on PLR. Together, these findings implicate osteocytes in bone-cartilage crosstalk in the joint and suggest a causal role for suppressed perilacunar/canalicular remodeling in osteoarthritis