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Introduction: Fibrillar collagens accumulate in the breast cancer stroma and appear as poorly defined spiculated masses in mammography imaging. The prognostic value of tissue type I collagen remains elusive in treatment-naïve and chemotherapy-treated breast cancer patients. Here, type I collagen mRNA and protein expression were analysed in 2 large independent breast cancer cohorts. Levels were related to clinicopathological parameters, prognostic biomarkers, and outcome. Method: COL1A1 mRNA expression was analysed in 2509 patients with breast cancer obtained from the cBioPortal database. Type I collagen protein expression was studied by immunohistochemistry in 1395 women diagnosed with early invasive breast cancer. Results: Low COL1A1 mRNA and protein levels correlated with poor prognosis features, such as hormone receptor negativity, high histological grade, triple-negative subtype, node positivity, and tumour size. In unadjusted analysis, high stromal type I collagen protein expression was associated with improved overall survival (OS) (HR = 0.78, 95% CI = 0.61-0.99, p = .043) and trended towards improved breast cancer–specific survival (BCSS) (HR = 0.65, 95% CI = 0.42-1.01, P = 0.053), although these findings were lost after adjustment for other clinical variables. In unadjusted analysis, high expression of type I collagen was associated with better OS (HR = 0.70, 95% CI = 0.55-0.90, P = .006) and BCSS (HR = 0.55, 95% CI = 0.34-0.88, P = .014) among patients not receiving chemotherapy. Strikingly, the opposite was observed among patients receiving chemotherapy. There, high expression of type I collagen was instead associated with worse OS (HR = 1.83, 95% CI = 0.65-5.14, P = .25) and BCSS (HR = 1.72, 95% CI = 0.54-5.50, P = .357). Conclusion: Low stromal type I collagen mRNA and protein expression are associated with unfavourable tumour characteristics in breast cancer. Stromal type I collagen might predict chemotherapy response.

Introduction: Fibrillar collagens accumulate in the breast cancer stroma and appear as poorly defined spiculated masses in mammography imaging. The prognostic value of tissue type I collagen remains elusive in treatment-naïve and chemotherapy-treated breast cancer patients. Here, type I collagen mRNA and protein expression were analysed in 2 large independent breast cancer cohorts. Levels were related to clinicopathological parameters, prognostic biomarkers, and outcome. Method: COL1A1 mRNA expression was analysed in 2509 patients with breast cancer obtained from the cBioPortal database. Type I collagen protein expression was studied by immunohistochemistry in 1395 women diagnosed with early invasive breast cancer. Results: Low COL1A1 mRNA and protein levels correlated with poor prognosis features, such as hormone receptor negativity, high histological grade, triple-negative subtype, node positivity, and tumour size. In unadjusted analysis, high stromal type I collagen protein expression was associated with improved overall survival (OS) (HR = 0.78, 95% CI = 0.61-0.99, p = .043) and trended towards improved breast cancer–specific survival (BCSS) (HR = 0.65, 95% CI = 0.42-1.01, P = 0.053), although these findings were lost after adjustment for other clinical variables. In unadjusted analysis, high expression of type I collagen was associated with better OS (HR = 0.70, 95% CI = 0.55-0.90, P = .006) and BCSS (HR = 0.55, 95% CI = 0.34-0.88, P = .014) among patients not receiving chemotherapy. Strikingly, the opposite was observed among patients receiving chemotherapy. There, high expression of type I collagen was instead associated with worse OS (HR = 1.83, 95% CI = 0.65-5.14, P = .25) and BCSS (HR = 1.72, 95% CI = 0.54-5.50, P = .357). Conclusion: Low stromal type I collagen mRNA and protein expression are associated with unfavourable tumour characteristics in breast cancer. Stromal type I collagen might predict chemotherapy response.

Despite the innate regenerative capabilities of bone tissue, self-repair is impaired when an injury exceeds the critical size threshold because of trauma, congenital, or pathological conditions. Autologous transplantation is the gold standard to reconstruct large bone defects. However, this method has drawbacks such as limited amount of graftable material, limited accessibility, and donor site morbidity. For these reasons, alternative regenerative medicine and tissue engineering approaches are being developed, including implantable scaffolds.The use of in vitro-made scaffolds containing biomaterials that mimic the functional characteristics and composition of extracellular bone matrix has been favored in 3D vs 2D in vitro culture systems. Adult stem cells such as mesenchymal stem cells (MSCs), that give rise to bone building cells, have been used in combination with various biomaterials. The development of an implantable scaffold with or without cells requires extensive in vitro validation and optimization prior to its testing in vivo. Thus, the primary aim of this thesis was to develop a 3D model for the optimization of MSC differentiation. Further aims were to utilize this 3D model to evaluate the MSC response to a novel osteogenic biomaterial. To achieve these objectives human bone marrow MSCs (BMSCs) were utilized in various hydrogels in combination with chemical differentiation factors or biomaterials. Moreover, the osteogenic capability of the tested biomaterials and their induced inflammatory/angiogenic responses were investigated, and the culture conditions were optimized for clinical application. In this thesis, a comparison between 2D and 3D (hydrogels) in vitro culture models was developed with the purpose of studying osteogenic differentiation in BSMCs. Testing various hydrogels revealedt he superiority of type I collagen hydrogels for the osteogenic 3D in vitro culture system. Further, cell culture conditions were improved for the expansion and differen

Despite the innate regenerative capabilities of bone tissue, self-repair is impaired when an injury exceeds the critical size threshold because of trauma, congenital, or pathological conditions. Autologous transplantation is the gold standard to reconstruct large bone defects. However, this method has drawbacks such as limited amount of graftable material, limited accessibility, and donor site morbidity. For these reasons, alternative regenerative medicine and tissue engineering approaches are being developed, including implantable scaffolds.The use of in vitro-made scaffolds containing biomaterials that mimic the functional characteristics and composition of extracellular bone matrix has been favored in 3D vs 2D in vitro culture systems. Adult stem cells such as mesenchymal stem cells (MSCs), that give rise to bone building cells, have been used in combination with various biomaterials. The development of an implantable scaffold with or without cells requires extensive in vitro validation and optimization prior to its testing in vivo. Thus, the primary aim of this thesis was to develop a 3D model for the optimization of MSC differentiation. Further aims were to utilize this 3D model to evaluate the MSC response to a novel osteogenic biomaterial. To achieve these objectives human bone marrow MSCs (BMSCs) were utilized in various hydrogels in combination with chemical differentiation factors or biomaterials. Moreover, the osteogenic capability of the tested biomaterials and their induced inflammatory/angiogenic responses were investigated, and the culture conditions were optimized for clinical application. In this thesis, a comparison between 2D and 3D (hydrogels) in vitro culture models was developed with the purpose of studying osteogenic differentiation in BSMCs. Testing various hydrogels revealedt he superiority of type I collagen hydrogels for the osteogenic 3D in vitro culture system. Further, cell culture conditions were improved for the expansion and differen

Despite the innate regenerative capabilities of bone tissue, self-repair is impaired when an injury exceeds the critical size threshold because of trauma, congenital, or pathological conditions. Autologous transplantation is the gold standard to reconstruct large bone defects. However, this method has drawbacks such as limited amount of graftable material, limited accessibility, and donor site morbidity. For these reasons, alternative regenerative medicine and tissue engineering approaches are being developed, including implantable scaffolds.The use of in vitro-made scaffolds containing biomaterials that mimic the functional characteristics and composition of extracellular bone matrix has been favored in 3D vs 2D in vitro culture systems. Adult stem cells such as mesenchymal stem cells (MSCs), that give rise to bone building cells, have been used in combination with various biomaterials. The development of an implantable scaffold with or without cells requires extensive in vitro validation and optimization prior to its testing in vivo. Thus, the primary aim of this thesis was to develop a 3D model for the optimization of MSC differentiation. Further aims were to utilize this 3D model to evaluate the MSC response to a novel osteogenic biomaterial. To achieve these objectives human bone marrow MSCs (BMSCs) were utilized in various hydrogels in combination with chemical differentiation factors or biomaterials. Moreover, the osteogenic capability of the tested biomaterials and their induced inflammatory/angiogenic responses were investigated, and the culture conditions were optimized for clinical application. In this thesis, a comparison between 2D and 3D (hydrogels) in vitro culture models was developed with the purpose of studying osteogenic differentiation in BSMCs. Testing various hydrogels revealedt he superiority of type I collagen hydrogels for the osteogenic 3D in vitro culture system. Further, cell culture conditions were improved for the expansion and differen

Collagen is a cornerstone protein for tissue engineering and 3D bioprinting due to its outstanding biocompatibility, low immunogenicity, and natural abundance in human tissues. Nonetheless, it still poses some important challenges, such as complicated and limited extraction processes, usually accompanied by batchto-batch reproducibility and influence of factors, such as temperature, pH, and ionic strength. In this work, we evaluated the suitability and performance of new, fibrillar type I collagen as standardized and reproducible collagen source for 3D printing and bioprinting. The acidic, native fibrous collagen formulation (5% w/w) performed remarkably during 3D printing, which was possible to print constructs of up to 27 layers without collapsing. On the other hand, the fibrous collagen mass has been modified to provide a fast, reliable, and easily neutralizable process. The neutralization with TRIS-HCl enabled the inclusion of cells without hindering printability. The cell-laden constructs were printed under mild conditions (50–80 kPa, pneumatic 3D printing), providing remarkable cellular viability (>90%) as well as a stable platform for cell growth and proliferation in vitro. Therefore, the native, type I collagen masses characterized in this work offer a reproducible and reliable source of collagen for 3D printing and bioprinting purposes.

Type I collagen is a high-value polymer found naturally in animal species and with many applications in the biomedical field. Collagen is frequently obtained from bovine tendons, but this source presents the risk of disease transmission, thus marine collagen is becoming an alternative source of this valuable material. In the present work, we report the successful extraction and characterization of the natural collagen found in the scales of Biajaiba, a highly consumed fish native to the Gulf of Mexico. We obtained acid- and pepsin-soluble type I collagens with high denaturation temperatures, high hydroxyproline contents, and yields in the ranges reported for other fish scales. Our work proposes a useful alternative for transforming the huge quantities of discarded fish scales in our country to extract a high-value product for biomedical applications. © 2023 the author(s), published by De Gruyter.

The morphological method—based on the topology and singularity theory and originally developed for the analysis of the scattering experiments—was extended to be applicable for the analysis of biological data. The usefulness of the topological viewpoint was demonstrated by quantification of the changes in collagen fiber straightness in the human colon mucosa (healthy mucosa, colorectal cancer, and uninvolved mucosa far from cancer). This has been done by modeling the distribution of collagen segment angles by the polymorphic beta-distribution. Its shapes were classified according to the number and type of critical points. We found that biologically relevant shapes could be classified as shapes without any preferable orientation (i.e. shapes without local extrema), transitional forms (i.e. forms with one broad local maximum), and highly oriented forms (i.e. forms with two minima at both ends and one very narrow maximum between them). Thus, changes in the fiber organization were linked to the metamorphoses of the beta-distribution forms. The obtained classification was used to define a new, shape-aware/based, measure of the collagen straightness, which revealed a slight and moderate increase of the straightness in mucosa samples taken 20 and 10 cm away from the tumor. The largest increase of collagen straightness was found in samples of cancer tissue. Samples of healthy individuals have a uniform distribution of beta-distribution forms. We found that this distribution has the maximal information entropy. At 20 cm and 10 cm away from cancer, the transition forms redistribute into unoriented and highly oriented forms. Closer to cancer the number of unoriented forms decreases rapidly leaving only highly oriented forms present in the samples of the cancer tissue, whose distribution has minimal information entropy. The polarization of the distribution was followed by a significant increase in the number of quasi-symmetrical forms in samples 20 cm away from cancer which decr

Background: In 2020, more than 2,260,000 women were diagnosed with breast cancer. Most patients are cured with surgery and adjuvant treatment, but despite that, approximately 700,000 women die of the disease every year. The historical focus on breast cancer progression has been on the malignant epithelial cell. However, cancer cells do not grow in isolation. In recent years, the importance of the tumour microenvironment in cancer progression has been highlighted. Perlecan and type IV collagen are basement membrane (BM) proteins in the normal mammary gland, and type I collagen is the main fibrillar collagen in the interstitial extracellular matrix (ECM). In cancer development, perlecan and type IV collagen have multifunctional roles and when degraded from the BM, bioactive substances and other fragments are released in the circulation. Significant ECM changes also occur that lead to an accumulation of fibrillar collagens. Given their abundance in the ECM; perlecan, type IV and type I collagen are of interest for breast cancer progression and may be of importance as new biomarkers to monitor disease, predict patient outcome and the treatment effect. Aim: In this thesis, the protein and mRNA expression of perlecan, type IV and I collagen in breast cancer tissue is studied. The aim is to characterize the expression pattern of these proteins in breast cancer tissue and to see whether there is a correlation to known prognostic biomarkers and to the patient prognosis. Moreover, to evaluate circulating perlecan and type IV collagen as diagnostic and prognostic biomarkers in breast cancer patients. Methods: In this thesis project, eight different patient cohorts were used. In freshly frozen normal breast and breast cancer tissue, perlecan protein expression was visualized using immunofluorescence. Type IV and I collagen protein expression were studied with immunohistochemistry in formalin-fixed, paraffin embedded primary breast cancer tissue, and type IV collagen in metastat

Background: In 2020, more than 2,260,000 women were diagnosed with breast cancer. Most patients are cured with surgery and adjuvant treatment, but despite that, approximately 700,000 women die of the disease every year. The historical focus on breast cancer progression has been on the malignant epithelial cell. However, cancer cells do not grow in isolation. In recent years, the importance of the tumour microenvironment in cancer progression has been highlighted. Perlecan and type IV collagen are basement membrane (BM) proteins in the normal mammary gland, and type I collagen is the main fibrillar collagen in the interstitial extracellular matrix (ECM). In cancer development, perlecan and type IV collagen have multifunctional roles and when degraded from the BM, bioactive substances and other fragments are released in the circulation. Significant ECM changes also occur that lead to an accumulation of fibrillar collagens. Given their abundance in the ECM; perlecan, type IV and type I collagen are of interest for breast cancer progression and may be of importance as new biomarkers to monitor disease, predict patient outcome and the treatment effect. Aim: In this thesis, the protein and mRNA expression of perlecan, type IV and I collagen in breast cancer tissue is studied. The aim is to characterize the expression pattern of these proteins in breast cancer tissue and to see whether there is a correlation to known prognostic biomarkers and to the patient prognosis. Moreover, to evaluate circulating perlecan and type IV collagen as diagnostic and prognostic biomarkers in breast cancer patients. Methods: In this thesis project, eight different patient cohorts were used. In freshly frozen normal breast and breast cancer tissue, perlecan protein expression was visualized using immunofluorescence. Type IV and I collagen protein expression were studied with immunohistochemistry in formalin-fixed, paraffin embedded primary breast cancer tissue, and type IV collagen in metastat

The host immunologic response to a specific material is a critical aspect when considering it for clinical implementation. Collagen and gelatin extracted from marine sources have been proposed as biomaterials for tissue engineering applications, but there is a lack of information in the literature about their immunogenicity. In this work, we evaluated the immune response to collagen and/or gelatin from blue shark and codfish, previously extracted and characterized. After endotoxin evaluation, bone marrow-derived macrophages were exposed to the materials and a panel of pro- and anti-inflammatory cytokines were evaluated both for protein quantification and gene expression. Then, the impact of those materials in the host was evaluated through peritoneal injection in C57BL/6 mice. The results suggested shark collagen as the less immunogenic material, inducing low expression of pro-inflammatory cytokines as well as inducible nitric oxide synthase (encoded by Nos2) and high expression of Arginase 1 (encoded by Arg1). Although shark gelatin appeared to be the material with higher pro-inflammatory expression, it also presents a high expression of IL-10 (anti-inflammatory cytokine) and Arginase (both markers for M2-like macrophages). When injected in the peritoneal cavity of mice, our materials demonstrated a transient recruitment of neutrophil, being almost non-existent after 24 hours of injection. Based on these findings, the studied collagenous materials can be considered interesting biomaterial candidates for regenerative medicine as they may induce an activation of the M2-like macrophage population, which is involved in suppressing the inflammatory processes promoting tissue remodeling

Background: In 2020, more than 2,260,000 women were diagnosed with breast cancer. Most patients are cured with surgery and adjuvant treatment, but despite that, approximately 700,000 women die of the disease every year. The historical focus on breast cancer progression has been on the malignant epithelial cell. However, cancer cells do not grow in isolation. In recent years, the importance of the tumour microenvironment in cancer progression has been highlighted. Perlecan and type IV collagen are basement membrane (BM) proteins in the normal mammary gland, and type I collagen is the main fibrillar collagen in the interstitial extracellular matrix (ECM). In cancer development, perlecan and type IV collagen have multifunctional roles and when degraded from the BM, bioactive substances and other fragments are released in the circulation. Significant ECM changes also occur that lead to an accumulation of fibrillar collagens. Given their abundance in the ECM; perlecan, type IV and type I collagen are of interest for breast cancer progression and may be of importance as new biomarkers to monitor disease, predict patient outcome and the treatment effect. Aim: In this thesis, the protein and mRNA expression of perlecan, type IV and I collagen in breast cancer tissue is studied. The aim is to characterize the expression pattern of these proteins in breast cancer tissue and to see whether there is a correlation to known prognostic biomarkers and to the patient prognosis. Moreover, to evaluate circulating perlecan and type IV collagen as diagnostic and prognostic biomarkers in breast cancer patients. Methods: In this thesis project, eight different patient cohorts were used. In freshly frozen normal breast and breast cancer tissue, perlecan protein expression was visualized using immunofluorescence. Type IV and I collagen protein expression were studied with immunohistochemistry in formalin-fixed, paraffin embedded primary breast cancer tissue, and type IV collagen in metastat

The morphological method—based on the topology and singularity theory and originally developed for the analysis of the scattering experiments—was extended to be applicable for the analysis of biological data. The usefulness of the topological viewpoint was demonstrated by quantification of the changes in collagen fiber straightness in the human colon mucosa (healthy mucosa, colorectal cancer, and uninvolved mucosa far from cancer). This has been done by modeling the distribution of collagen segment angles by the polymorphic beta-distribution. Its shapes were classified according to the number and type of critical points. We found that biologically relevant shapes could be classified as shapes without any preferable orientation (i.e. shapes without local extrema), transitional forms (i.e. forms with one broad local maximum), and highly oriented forms (i.e. forms with two minima at both ends and one very narrow maximum between them). Thus, changes in the fiber organization were linked to the metamorphoses of the beta-distribution forms. The obtained classification was used to define a new, shape-aware/based, measure of the collagen straightness, which revealed a slight and moderate increase of the straightness in mucosa samples taken 20 and 10 cm away from the tumor. The largest increase of collagen straightness was found in samples of cancer tissue. Samples of healthy individuals have a uniform distribution of beta-distribution forms. We found that this distribution has the maximal information entropy. At 20 cm and 10 cm away from cancer, the transition forms redistribute into unoriented and highly oriented forms. Closer to cancer the number of unoriented forms decreases rapidly leaving only highly oriented forms present in the samples of the cancer tissue, whose distribution has minimal information entropy. The polarization of the distribution was followed by a significant increase in the number of quasi-symmetrical forms in samples 20 cm away from cancer which decr

Background: In 2020, more than 2,260,000 women were diagnosed with breast cancer. Most patients are cured with surgery and adjuvant treatment, but despite that, approximately 700,000 women die of the disease every year. The historical focus on breast cancer progression has been on the malignant epithelial cell. However, cancer cells do not grow in isolation. In recent years, the importance of the tumour microenvironment in cancer progression has been highlighted. Perlecan and type IV collagen are basement membrane (BM) proteins in the normal mammary gland, and type I collagen is the main fibrillar collagen in the interstitial extracellular matrix (ECM). In cancer development, perlecan and type IV collagen have multifunctional roles and when degraded from the BM, bioactive substances and other fragments are released in the circulation. Significant ECM changes also occur that lead to an accumulation of fibrillar collagens. Given their abundance in the ECM; perlecan, type IV and type I collagen are of interest for breast cancer progression and may be of importance as new biomarkers to monitor disease, predict patient outcome and the treatment effect. Aim: In this thesis, the protein and mRNA expression of perlecan, type IV and I collagen in breast cancer tissue is studied. The aim is to characterize the expression pattern of these proteins in breast cancer tissue and to see whether there is a correlation to known prognostic biomarkers and to the patient prognosis. Moreover, to evaluate circulating perlecan and type IV collagen as diagnostic and prognostic biomarkers in breast cancer patients. Methods: In this thesis project, eight different patient cohorts were used. In freshly frozen normal breast and breast cancer tissue, perlecan protein expression was visualized using immunofluorescence. Type IV and I collagen protein expression were studied with immunohistochemistry in formalin-fixed, paraffin embedded primary breast cancer tissue, and type IV collagen in metastat

Background: In 2020, more than 2,260,000 women were diagnosed with breast cancer. Most patients are cured with surgery and adjuvant treatment, but despite that, approximately 700,000 women die of the disease every year. The historical focus on breast cancer progression has been on the malignant epithelial cell. However, cancer cells do not grow in isolation. In recent years, the importance of the tumour microenvironment in cancer progression has been highlighted. Perlecan and type IV collagen are basement membrane (BM) proteins in the normal mammary gland, and type I collagen is the main fibrillar collagen in the interstitial extracellular matrix (ECM). In cancer development, perlecan and type IV collagen have multifunctional roles and when degraded from the BM, bioactive substances and other fragments are released in the circulation. Significant ECM changes also occur that lead to an accumulation of fibrillar collagens. Given their abundance in the ECM; perlecan, type IV and type I collagen are of interest for breast cancer progression and may be of importance as new biomarkers to monitor disease, predict patient outcome and the treatment effect. Aim: In this thesis, the protein and mRNA expression of perlecan, type IV and I collagen in breast cancer tissue is studied. The aim is to characterize the expression pattern of these proteins in breast cancer tissue and to see whether there is a correlation to known prognostic biomarkers and to the patient prognosis. Moreover, to evaluate circulating perlecan and type IV collagen as diagnostic and prognostic biomarkers in breast cancer patients. Methods: In this thesis project, eight different patient cohorts were used. In freshly frozen normal breast and breast cancer tissue, perlecan protein expression was visualized using immunofluorescence. Type IV and I collagen protein expression were studied with immunohistochemistry in formalin-fixed, paraffin embedded primary breast cancer tissue, and type IV collagen in metastat

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.

Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.

The use of embedded cells within alginate matrices is a developing technique with great clinical applications in cell-based therapies. However, one feature that needs additional investigation is the improvement of alginate-cells viability, which could be achieved by integrating other materials with alginate to improve its surface properties. In recent years, the field of nanotechnology has shown the many properties of a huge number of materials. Graphene oxide (GO), for instance, seems to be a good choice for improving alginate cell viability and functionality. We previously observed that GO, coated with fetal bovine serum (FBS) within alginate hydrogels, improves the viability of embedded myoblasts. In the current research, we aim to study several proteins, specifically bovine serum albumin (BSA), type I collagen and elastin, to discern their impact on the previously observed improvement on embedded myoblasts within alginate hydrogels containing GO coated with FBS. Thus, we describe the mechanisms of the formation of BSA, collagen and elastin protein layers on the GO surface, showing a high adsorption by BSA and elastin, and a decreasing GO impedance and capacitance. Moreover, we described a better cell viability and protein release from embedded cells within hydrogels containing protein-coated GO. We conclude that these hybrid hydrogels could provide a step forward in regenerative medicine.

The use of embedded cells within alginate matrices is a developing technique with great clinical applications in cell-based therapies. However, one feature that needs additional investigation is the improvement of alginate-cells viability, which could be achieved by integrating other materials with alginate to improve its surface properties. In recent years, the field of nanotechnology has shown the many properties of a huge number of materials. Graphene oxide (GO), for instance, seems to be a good choice for improving alginate cell viability and functionality. We previously observed that GO, coated with fetal bovine serum (FBS) within alginate hydrogels, improves the viability of embedded myoblasts. In the current research, we aim to study several proteins, specifically bovine serum albumin (BSA), type I collagen and elastin, to discern their impact on the previously observed improvement on embedded myoblasts within alginate hydrogels containing GO coated with FBS. Thus, we describe the mechanisms of the formation of BSA, collagen and elastin protein layers on the GO surface, showing a high adsorption by BSA and elastin, and a decreasing GO impedance and capacitance. Moreover, we described a better cell viability and protein release from embedded cells within hydrogels containing protein-coated GO. We conclude that these hybrid hydrogels could provide a step forward in regenerative medicine.

The use of embedded cells within alginate matrices is a developing technique with great clinical applications in cell-based therapies. However, one feature that needs additional investigation is the improvement of alginate-cells viability, which could be achieved by integrating other materials with alginate to improve its surface properties. In recent years, the field of nanotechnology has shown the many properties of a huge number of materials. Graphene oxide (GO), for instance, seems to be a good choice for improving alginate cell viability and functionality. We previously observed that GO, coated with fetal bovine serum (FBS) within alginate hydrogels, improves the viability of embedded myoblasts. In the current research, we aim to study several proteins, specifically bovine serum albumin (BSA), type I collagen and elastin, to discern their impact on the previously observed improvement on embedded myoblasts within alginate hydrogels containing GO coated with FBS. Thus, we describe the mechanisms of the formation of BSA, collagen and elastin protein layers on the GO surface, showing a high adsorption by BSA and elastin, and a decreasing GO impedance and capacitance. Moreover, we described a better cell viability and protein release from embedded cells within hydrogels containing protein-coated GO. We conclude that these hybrid hydrogels could provide a step forward in regenerative medicine.

Non-mammalian collagens have attracted increasing attention for industrial and biomedical use. We have therefore evaluated extraction conditions and the biochemical properties of collagens from aquacultured sturgeon. Pepsin-soluble type I and type II collagen were respectively extracted from the skin and notochord of bester sturgeon by-products, with yields of 63.9 ± 0.19% and 35.5 ± 0.68%. Collagen extraction efficiency was improved by an alkaline pretreatment of the skin and notochord (fewer extraction cycles were required), but the final yields decreased to 56.2 ± 0.84% for type I and 31.8 ± 1.13% for type II. Alkaline pretreatment did not affect the thermal stability or triple-helical structure of both types of collagen. Types I and II collagen formed re-assembled fibril structures in vitro, under different conditions. Alkaline pretreatment slowed down the formation of type I collagen fibrils and specifically inhibited the formation of thick fibril-bundle structures. In contrast, alkaline pretreatment did not change type II collagen fibril formation. In conclusion, alkaline pretreatment of sturgeon skin and notochord is an effective method to accelerate collagen extraction process of types I and II collagen without changing their biochemical properties. However, it decreases the yield of both collagens and specifically changes the fibril-forming ability of type I collagen.

Keratins are a family of structural proteins that can be extracted from a variety of sources including wool, nails, skin, hooves, and hair. Keratin can be processed into different constructs such as coatings, scaffolds, and hydrogels, and has shown favorable results when placed in in vitro and in vivo settings for different tissue regeneration applications. Over three decades, keratin extraction technology has been continuously modified, and these differences in extraction processes have distinct effects on the characteristics of the end product. In this work, we examine the effect of keratin aggregation during a widely-used purification step, dialysis ultra-filtration, on material characteristics of the final keratin product when fabricated into a hydrogel. Two distinct dialysis procedures were applied during the extraction of oxidized keratin (keratose): one promoting protein aggregation and the other mitigating it. Analyses of material properties such as mechanical and enzymatic stability were conducted in addition to observing the differences in solution behavior between products. Data revealed that protein aggregation during the extraction process has a profound effect on keratose hydrogel material properties. After determination of the effect of protein aggregation during extraction on keratose hydrogels, investigation of how a blended material comprised of said keratose and type I collagen was undertaken. It was hypothesized that a blend would result in mixing at the molecular level, resulting in improved properties compared to either pure material alone. A protocol was created to make stable keratose/type I collagen blends and material characterization techniques were applied to determine the inherent properties of samples with differing ratios. Crosslinking density, mechanical properties, enzymatic degradation properties, water uptake capacity, structural architecture, and thermal properties were all assessed. In addition, the ability of this material to ma

Vocal fold scarring is the fibrotic manifestation of a variety of voice disorders, and is difficult to treat. Tissue engineering therapies provide a potential strategy to regenerate the native tissue microenvironment in order to restore vocal fold functionality. However, major challenges remain in capturing the complexity of the native tissue and sustaining regeneration. We hypothesized that hydrogels with tunable viscoelastic properties that present relevant biological cues to cells might be better suited as therapeutics. Herein, we characterized the response of human vocal fold fibroblasts to four different biomimetic hydrogels: thiolated hyaluronan (HA) crosslinked with poly(ethylene glycol) diacrylate (PEGDA), HA-PEGDA with type I collagen (HA-Col I), HA-PEGDA with type III collagen (HA-Col III) and HA-PEGDA with type I and III collagen (HA-Col I-Col III). Collagen incorporation allowed for interpenetrating fibrils of collagen within the non-fibrillar HA network, which increased the mechanical properties of the hydrogels. The addition of collagen fibrils also reduced hyaluronidase degradation of HA and hydrogel swelling ratio. Fibroblasts encapsulated in the HA-Col gels adopted a spindle shaped fibroblastic morphology by day 7 and exhibited extensive cytoskeletal networks by day 21, suggesting that the incorporation of collagen was essential for cell adhesion and spreading. Cells remained viable and synthesized new DNA throughout 21 days of culture. Gene expression levels significantly differed between the cells encapsulated in the different hydrogels. Relative fold changes in gene expression of MMP1, COL1A1, fibronectin and decorin suggest higher degrees of remodeling in HA-Col I-Col III gels in comparison to HA-Col I or HA-Col III hydrogels, suggesting that the former may better serve as a natural biomimetic hydrogel for tissue engineering applications. STATEMENT OF SIGNIFICANCE: Voice disorders affect about 1/3rd of the US population and significantly reduce

Non-mammalian collagens have attracted increasing attention for industrial and biomedical use. We have therefore evaluated extraction conditions and the biochemical properties of collagens from aquacultured sturgeon. Pepsin-soluble type I and type II collagen were respectively extracted from the skin and notochord of bester sturgeon by-products, with yields of 63.9 ± 0.19% and 35.5 ± 0.68%. Collagen extraction efficiency was improved by an alkaline pretreatment of the skin and notochord (fewer extraction cycles were required), but the final yields decreased to 56.2 ± 0.84% for type I and 31.8 ± 1.13% for type II. Alkaline pretreatment did not affect the thermal stability or triple-helical structure of both types of collagen. Types I and II collagen formed re-assembled fibril structures in vitro, under different conditions. Alkaline pretreatment slowed down the formation of type I collagen fibrils and specifically inhibited the formation of thick fibril-bundle structures. In contrast, alkaline pretreatment did not change type II collagen fibril formation. In conclusion, alkaline pretreatment of sturgeon skin and notochord is an effective method to accelerate collagen extraction process of types I and II collagen without changing their biochemical properties. However, it decreases the yield of both collagens and specifically changes the fibril-forming ability of type I collagen.

Актуальність. Клітини проток становлять до 30 % від маси підшлункової залози (ПЗ) людини, а збільшення кількості бранчів проток відноситься до одних із перших сонологічних ознак хронічного панкреатиту (ХП). Мета дослідження — визначення морфологічних й імуногістохімічних особливостей протокової системи ПЗ хворих на ХП, яким були виконані резекційні та дренуючі операції з приводу ускладнень хронічного панкреатиту. Матеріали та методи. Проведено імуногістохімічне дослідження операційного матеріалу 40 хворих з ускладненим хронічним панкреатитом з використанням реакцій на a-ізоформу гладком’язового актину (a-SMA), десмін, фібронектин й колаген I типу. Результати. Характерна морфологічна картина розвитку ХП є поєднанням фіброзування з деформацією протокової системи за рахунок перидуктального фіброзу з розширенням міждолькових фіброзних септ, потовщенням інтралобулярних сполучно­тканинних тяжів, а також значною звивістістю і розширенням проток з ретенційними кістами. Значна структурна перебудова протокової системи ПЗ у хворих на ХП спостерігалася в 87,5 % випадків, в 17,5 % випадків картина доповнювалася явищами ацинарно-протокової метаплазії з трансформацією ацинарних клітин у протокові і формуванням дрібних дуктулоподібних структур, так званих тубулярних комплексів. У 7,5 % випадків в головній панкреатичній протоці визначалися вогнища атрофії протокового епітелію, які чергувалися з вогнищами папілярної інтраепітеліальної гіперплазії епітелію (PanIN). Розвитку панкреатичного фіброзу при ХП сприяє накопичення a-SMA+-активованих панкреатичних зірчастих клітин, що синтезують надлишок компонентів екстрацелюлярного матриксу, зокрема колагену I типу. Середня площа колагену І типу та інтенсивність його експресії були вірогідно нижче при зниженій експресії a-SMA (p < 0,05) і вірогідно вище у випадках вираженої експресії фібронектину (p < 0,05). Висновки. Вивчення структурних змін протокової системи ПЗ з використанням методів імуногістохімії є одним із способів покращення р, Актуальность. Клетки протоков составляют до 30 % от массы поджелудочной железы (ПЖ) человека, а увеличение количества бранчей протоков относится к одним из первых сонологических признаков хронического панкреатита (ХП). Цель исследования: определение морфологических и иммуногистохимических особенностей протоковой системы ПЖ больных ХП, которым были выполнены резекционные и дренирующие операции по поводу осложнений хронического панкреатита. Материалы и методы. Проведено иммуногистохимическое исследование операционного материала 40 больных с осложненным хроническим панкреатитом с использованием реакций на a-изоформу гладкомышечного актина (a-SMA), десмин, фибронектин и коллаген I типа. Результаты. Характерная морфологическая картина развития ХП представляет собой сочетание фиброзирования с деформацией протоковой системы за счет перидуктального фиброза с расширением междольковых фиброзных септ, утолщением интралобулярных соединительнотканных тяжей, а также со значительной извитостью и расширением протоков с ретенционными кистами. Значительная структурная перестройка протоковой системы ПЖ у больных ХП наблюдалась в 87,5 % случаев, в 17,5 % случаев картина дополнялась явлениями ацинарно-протоковой метаплазии с трансформацией ацинарных клеток в протоковые и формированием мелких дуктулоподобных структур, так называемых тубулярных комплексов. В 7,5 % случаев в главном панкреатическом протоке определялись очаги атрофии протокового эпителия, которые чередовались с очагами папиллярной интраэпителиальной гиперплазии эпителия (PanIN). Развитию панкреатического фиброза при ХП способствует накопление a-SMA+ активированных панкреатических звездчатых клеток, синтезирующих избыток компонентов экстрацеллюлярного матрикса, в частности коллагена I типа. Средняя площадь коллагена І типа и интенсивность его экспрессии были достоверно ниже при сниженной экспрессии a-SMA (p < 0,05) и достоверно выше в случаях выраженной экспрессии фибронектина (p < 0,05). Выводы. Изучение структурных и, Background. Chronic pancreatitis (CP) is characterized by a number of specific changes in the structure and function of the pancreas, including atrophy of functional tissue, fibrosis and ductal degeneration, a decrease in the number of islets. Despite a large number of possible etiological factors, the main pathophysiological mechanisms of CP development have much in common. Thus, with the preservation of secretory activity under obstruction of the ducts and their compensatory expansion, pancreatic secret infiltrates the surrounding tissue with the formation of edema, followed by intrapancreatic activation of digestive enzymes, in particular trypsinogen, which initiates proteolytic necrobiosis of the pancreocytes. The duct cells make up to 30 % of the human pancreas, compared to 1–2 % of endocrine cells. One of the first signs of CP is the abnormal increase in the number of ductal branches. It has also been shown that in the ducts of CP patients, the number of cells containing insulin increases, as well as cells that contain other endocrine and exocrine markers. Modeling chronic, acute and complicated pancreatitis in rats also confirmed that epithelial cells are involved in the regulation of extracellular matrix of the pancreas. Materials and methods. Comprehensive histopathological and immunohistochemical (IHC) study was performed in the laboratory of histological and immunohistochemical diagnosis of the university hospital of Zaporizhia State Medical University on postoperative material of 40 patients with CP aged 28 to 74 years, who were opera­ted at the clinic of surgery and minimally invasive technologies of Zaporizhia State Medical University. Immunohistochemical exa­mination was carried out according to standard procedures using a mouse monoclonal antibody Alpha Smooth Muscle Actin (Clone 1A4) against the a-isoform of smooth muscle actin (a-SMA) and Desmin (Clone D33) against desmin (DAKO, USA), monoclonal rabbit antibody against collagen I (Clone RAH C11) an

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were isolated from rohu skin with the yield of 64.2 and 6.8% (dry weight basis), respectively. Both collagens had glycine as the major amino acid with imino acid content of 196-202 residues/1,000 residues and were characterized as type I collagen with molecular composition of (1)(2)2-heterotrimer. Fourier transform infrared spectra of both collagens were similar, with no shift in wavenumber of all amide bands. The T-max value of ASC and PSC was 36.40 and 35.48 degrees C, respectively. The zero surface net charge of ASC and PSC was found at pH 5.9 and 5.3, respectively.

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were isolated from rohu skin with the yield of 64.2 and 6.8% (dry weight basis), respectively. Both collagens had glycine as the major amino acid with imino acid content of 196-202 residues/1,000 residues and were characterized as type I collagen with molecular composition of (1)(2)2-heterotrimer. Fourier transform infrared spectra of both collagens were similar, with no shift in wavenumber of all amide bands. The T-max value of ASC and PSC was 36.40 and 35.48 degrees C, respectively. The zero surface net charge of ASC and PSC was found at pH 5.9 and 5.3, respectively.

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) were isolated from rohu skin with the yield of 64.2 and 6.8% (dry weight basis), respectively. Both collagens had glycine as the major amino acid with imino acid content of 196-202 residues/1,000 residues and were characterized as type I collagen with molecular composition of (1)(2)2-heterotrimer. Fourier transform infrared spectra of both collagens were similar, with no shift in wavenumber of all amide bands. The T-max value of ASC and PSC was 36.40 and 35.48 degrees C, respectively. The zero surface net charge of ASC and PSC was found at pH 5.9 and 5.3, respectively.

Tendon ruptures and retractions with an extensive tissue loss represent a major clinical problem and a great challenge in surgical reconstruction. Traditional approaches consist in autologous or allogeneic grafts, which still have some drawbacks. Hence, tissue engineering strategies aimed at developing functionalized tendon grafts. In this context, the use of xenogeneic tissues represents a promising perspective to obtain decellularized tendon grafts. This study is focused on the identification of suitable culture conditions for the generation of reseeded and functional decellularized constructs to be used as tendon grafts. Equine superficial digital flexor tendons were decellularized, reseeded with mesenchymal stem cells (MSCs) from bone marrow and statically cultured in two different culture media to maintain undifferentiated cells (U-MSCs) or to induce a terminal tenogenic differentiation (T-MSCs) for 24 hours, 7 and 14 days. Cell viability, proliferation, morphology as well as matrix deposition and type I and III collagen production were assessed by means of histological, immunohistochemical and semi-quantitative analyses. Results showed that cell viability was not affected by any culture conditions and active proliferation was maintained 14 days after reseeding. However, seeded MSCs were not able to penetrate within the dense matrix of the decellularized tendons. Nevertheless, U-MSCs synthesized a greater amount of extracellular matrix rich in type I collagen compared to T-MSCs. In spite of the inability to deeply colonize the decellularized matrix in vitro, reseeding tendon matrices with U-MSCs could represent a suitable method for the functionalization of biological constructs, considering also any potential chemoattractant capability of the newly deposed extracellular matrix to recruit resident cells. This bioengineering approach can be exploited to produce functionalized tendon constructs for the substitution of large tendon defects.

PurposeCurrent culture schema for human intestinal stem cells (hISCs) frequently rely on a 3D culture system using Matrigel™, a laminin-rich matrix derived from murine sarcoma that is not suitable for clinical use. We have developed a novel 2D culture system for the in vitro expansion of hISCs as an intestinal epithelial monolayer without the use of Matrigel.MethodsCadaveric duodenal samples were processed to isolate intestinal crypts from the mucosa. Crypts were cultured on a thin coat of type I collagen or laminin. Intestinal epithelial monolayers were supported with growth factors to promote self-renewal or differentiation of the hISCs. Proliferating monolayers were sub-cultured every 4-5days.ResultsIntestinal epithelial monolayers were capable of long-term cell renewal. Less differentiated monolayers expressed high levels of gene marker LGR5, while more differentiated monolayers had higher expressions of CDX2, MUC2, LYZ, DEF5, and CHGA. Furthermore, monolayers were capable of passaging into a 3D culture system to generate spheroids and enteroids.ConclusionThis 2D system is an important step to expand hISCs for further experimental studies and for clinical cell transplantation.Level of evidence1 Experimental.

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of clown featherback (Chitala ornata) were isolated and characterised. Yields of ASC and PSC were 27.64 and 44.63% (dry weight basis) with total collagen recovery of 82.08%. Both collagens contained glycine as the major amino acid with relatively high content of proline, hydroxyproline and glutamic acid/glutamine. Nevertheless, they had the low content of cysteine, histidine and tryrosine. The collagen was characterised as type I, comprising (1)(2)2-heterotrimer. Pepsin-aided process did not affect triple-helical structure of PSC as determined by FTIR spectra. Thermal transition temperature of ASC (36.28 degrees C) was slightly higher than that of PSC (35.23 degrees C). However, no differences in isoelectric point (5.54-5.68) between ASC and PSC were observed. Therefore, collagen from the skin of clown featherback could be successfully extracted for further applications.

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of clown featherback (Chitala ornata) were isolated and characterised. Yields of ASC and PSC were 27.64 and 44.63% (dry weight basis) with total collagen recovery of 82.08%. Both collagens contained glycine as the major amino acid with relatively high content of proline, hydroxyproline and glutamic acid/glutamine. Nevertheless, they had the low content of cysteine, histidine and tryrosine. The collagen was characterised as type I, comprising (1)(2)2-heterotrimer. Pepsin-aided process did not affect triple-helical structure of PSC as determined by FTIR spectra. Thermal transition temperature of ASC (36.28 degrees C) was slightly higher than that of PSC (35.23 degrees C). However, no differences in isoelectric point (5.54-5.68) between ASC and PSC were observed. Therefore, collagen from the skin of clown featherback could be successfully extracted for further applications.

Acid soluble collagen (ASC) and pepsin soluble collagen (PSC) from the skin of clown featherback (Chitala ornata) were isolated and characterised. Yields of ASC and PSC were 27.64 and 44.63% (dry weight basis) with total collagen recovery of 82.08%. Both collagens contained glycine as the major amino acid with relatively high content of proline, hydroxyproline and glutamic acid/glutamine. Nevertheless, they had the low content of cysteine, histidine and tryrosine. The collagen was characterised as type I, comprising (1)(2)2-heterotrimer. Pepsin-aided process did not affect triple-helical structure of PSC as determined by FTIR spectra. Thermal transition temperature of ASC (36.28 degrees C) was slightly higher than that of PSC (35.23 degrees C). However, no differences in isoelectric point (5.54-5.68) between ASC and PSC were observed. Therefore, collagen from the skin of clown featherback could be successfully extracted for further applications.

Background Matrix metalloproteinase-2 (MMP-2) is an endopeptidase that facilitates extracellular matrix remodeling and molecular regulation, and is implicated in tumor metastasis. Type I collagen (Col I) regulates the activation of MMP-2 through both transcriptional and post-transcriptional means; however gaps remain in our understanding of the involvement of collagen-binding ?1 integrins in collagen-stimulated MMP-2 activation. Methods Three ?1 integrin siRNAs were used to elucidate the involvement of ?1 integrins in the Col I-induced MMP-2 activation mechanism. ?1 integrin knockdown was analyzed by quantitative RT-PCR, Western Blot and FACS analysis. Adhesion assay and collagen gel contraction were used to test the biological effects of ?1 integrin abrogation. MMP-2 activation levels were monitored by gelatin zymography. Results All three ?1 integrin siRNAs were efficient at ?1 integrin knockdown and FACS analysis revealed commensurate reductions of integrins ?2 and ?3, which are heterodimeric partners of ?1, but not ?V, which is not. All three ?1 integrin siRNAs inhibited adhesion and collagen gel contraction, however only the siRNA showing the greatest magnitude of ?1 knockdown inhibited Col I-induced MMP-2 activation and reduced the accompanying upregulation of MT1-MMP, suggesting a dose response threshold effect. Re-transfection with codon-swapped ?1 integrin overcame the reduction in MMP-2 activation induced by Col-1, confirming the ?1 integrin target specificity. MMP-2 activation induced by TPA or Concanavalin A (Con A) was not inhibited by ?1 integrin siRNA knockdown. Conclusion Together, the data reveals that strong abrogation of ?1 integrin is required to block MMP-2 activation induced by Col I, which may have implications for the therapeutic targeting of ?1 integrin.

Three-dimensional culture was performed using gamma-irradiated type Ⅰ collagen gel prepared from rat tail tendon. The morphological outgrowing appearance with in type Ⅰ collagen gel provided a marked difference in accordance with histopathological diagnosis, subsequently suggesting the difference in adhesion ability to type Ⅰ collagen. On the other hand, type Ⅰ collagen may be involved in differentiation-related proteins expressed during in vitro morphogenesis, such as carcinoembryonic antigen for tubulogenesis of the salivary gland. The present experiment system would be useful for studying further the relation between type Ⅰ collagen matrix and metastatic potentiality and/or cellular differentiation.

Three-dimensional culture was performed using gamma-irradiated type Ⅰ collagen gel prepared from rat tail tendon. The morphological outgrowing appearance with in type Ⅰ collagen gel provided a marked difference in accordance with histopathological diagnosis, subsequently suggesting the difference in adhesion ability to type Ⅰ collagen. On the other hand, type Ⅰ collagen may be involved in differentiation-related proteins expressed during in vitro morphogenesis, such as carcinoembryonic antigen for tubulogenesis of the salivary gland. The present experiment system would be useful for studying further the relation between type Ⅰ collagen matrix and metastatic potentiality and/or cellular differentiation.

Fibronectin (FN) is a ubiquitous extracellular matrix protein (ECM) protein that is organized into fibrillar networks by cells through an integrin-mediated process that involves contractile forces. This assembly allows for the unfolding of the FN molecule, exposing cryptic domains that are not available in the native globular FN structure and activating intracellular signalling complexes. However, organization of FN into a physiological fibrillar network upon adsorption on a material surface has not been observed. Here we demonstrate cell-free, material-induced FN fibrillogenesis into a biological matrix with enhanced cellular activities. We found that simple FN adsorption onto poly(ethyl acrylate) surfaces, but not control polymers, triggered FN organization into a fibrillar network via interactions in the amino-terminal 70 kDa fragment, which is involved in the formation of cell-mediated FN fibrils. Moreover, the material-driven FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules and even type I collagen. Our results demonstrate that molecular assembly of FN can take place at the material interface, giving rise to a physiological protein network similar to fibrillar matrices assembled by cells. This research identifies material surfaces that trigger the organization of extracellular matrix proteins into biological active fibrils and establishes a new paradigm to engineer ECM-mimetic biomaterials.

Fibronectin (FN) is a ubiquitous extracellular matrix protein (ECM) protein that is organized into fibrillar networks by cells through an integrin-mediated process that involves contractile forces. This assembly allows for the unfolding of the FN molecule, exposing cryptic domains that are not available in the native globular FN structure and activating intracellular signalling complexes. However, organization of FN into a physiological fibrillar network upon adsorption on a material surface has not been observed. Here we demonstrate cell-free, material-induced FN fibrillogenesis into a biological matrix with enhanced cellular activities. We found that simple FN adsorption onto poly(ethyl acrylate) surfaces, but not control polymers, triggered FN organization into a fibrillar network via interactions in the amino-terminal 70 kDa fragment, which is involved in the formation of cell-mediated FN fibrils. Moreover, the material-driven FN fibrils exhibited enhanced biological activities in terms of myogenic differentiation compared to individual FN molecules and even type I collagen. Our results demonstrate that molecular assembly of FN can take place at the material interface, giving rise to a physiological protein network similar to fibrillar matrices assembled by cells. This research identifies material surfaces that trigger the organization of extracellular matrix proteins into biological active fibrils and establishes a new paradigm to engineer ECM-mimetic biomaterials.

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.

The authors report the use of synthetic beta-tricalcium phosphate with type I collagen immediately after tooth extraction for simple socket preservation indicated in the pre-implant management of alveolar bone. The bone material was used without a barrier membrane and forming a mucoperiosteal flap. Clinical examination revealed solid new bone formation with no changes in vertical and horizontal dimensions 9 months after the socket preservation. Immunohistochemical analysis demonstrated presence of active osteonectine-positive cells. The new bone formed after the use of beta-tricalcium phosphate and type I collagen in the socket preservation method can allow dental implant placement and implant loading.

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.

Formation and remodeling of type I collagen fibril networks are paradigms of biopolymer self-assembly, yet many of their aspects remain poorly understood. Type I collagen is the most abundant vertebrate protein which self assembles into fibrils and hierarchical fibril network structures, forming scaffolds of bone, skin, tendons and other tissues. The normal isoform of type I collagen is a heterotrimer of two &alpha1(I) and one &alpha2(I) chains, but homotrimers of three &alpha1(I) chains have been reported, e.g., in cancer and fibrosis. Despite their importance in various disorders, very little is known about potential effects of the type I collagen homotrimers on self-assembly, physical properties, and remodeling of collagen fibrils and fibril networks. Thus, we selected characterization of these effects and understanding the underlying physical mechanisms as the topic of the present thesis. Some of our most important findings were: (i) different nucleation mechanism and morphology in homotrimer fibrils compared to the normal heterotrimers fibrils; (ii) segregation of the homo- and heterotrimers within fibrils; (iii) increased bending rigidity of homotrimer fibrils; and (iv) homotrimer resistance to cleavage by enzymes responsible for fibril degradation and remodeling due to increased triple helix stability at the cleavage site. The corresponding in vitro experiments and theoretical analysis of the results suggested drastically different physics of the fibril networks composed of the homo/heterotrimer mixtures and pointed to a potential role of these physics in various disorders, e.g., in cancer and fibrosis pathology.

Little is known regarding the mechanisms that govern the structural organization of cancellous bone. In this study, we compare the nature of the collagen in vertebral cancellous bone with the structural organization of its trabecular network. Cylindrical specimens of cancellous bone from vertebrae were obtained from nine autopsy subjects (ages 46-88). In each subject, eight pairs of corresponding samples were obtained from three levels in the spine and three areas within the vertebral body, leading to a total of 68 pairs of samples. The cylinders from one side were used for morphometry and the classical morphometrical parameters were obtained (BV/TV, bone volume fraction; Tb.Th, trabecular thickness; Tb.N, number; Tb.Sp, trabecular spacing) and strut analysis (TSL, total strut length; Nd, number of nodes; Fe, number of free-ends). The amount of osteoid bone was also quantified. The cylinders from the other side were powdered and used for collagen assessment, including the amount of collagen (% w/w), and its content in immature cross-links; such as hydroxylysinonorleucine (mol/mol of collagen) and dihydroxylysinornorleucine, as well as stable mature cross-links, such as hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), and the pyrrole cross-links. A random regression model was used to explore the correlations. None of the biochemical parameters correlated with the BV/TV except the ratio between immature and mature cross-links (eta(2) = 0.34, p < 0.05). There was no relationship between the amount of osteoid bone and the cross-link profile. However, the concentration of pyrrole and HP cross-links in the bone samples correlated with the structural organization of its trabeculae, but in an opposite direction. Hence, the pyrrole/HP ratio was a good predictor of Tb.Th, Tb.N, Tb.Sp, and TSL (eta(2) > 0.65 and p < 0.01) as well as Fe and star marrow space (eta(2) > 0.45 and p < 0.05). The cylinders from subjects with high pyrrole or low HP in their bone collagen had a