The Role of \u3cem\u3eCol11a1\u3c/em\u3e Expression During Cartilage Development

It is currently a major scientific and medical goal to identify and characterize genetic defects and their impact in health and disease. For example, mutations in genes that encode collagen alpha chains can cause skeletal dysplasia and lead to premature degenerative joint disease. Collagen is the main structural protein in the ECM of connective tissues such as the cartilage, joints, ligaments and tendons. Therefore, the goal of this research is to define the impact of the alpha one chain of collagen type XI chain, encoded by the COL11A1 gene in humans, on chondrocyte behavior during development of the cartilage. We hypothesize that altered expression of COL11A1 dysregulates chondrocyte behavior during cartilage development by altering β-catenin dependent signaling pathways. To test this hypothesis, we inhibited the expression of the COL11A1 homolog col11a1a in transgenic zebrafish expressing green fluorescence protein in neural crest derived cells and osteoblast. Then, the col11a1a deficient zebrafish were imaged by confocal microscopy to analyze the organization of cells contributing to craniofacial development. Inhibiting col11a1a expression reduced the size and shape of the developing Meckel’s cartilage in zebrafish. These changes occurred because the cells failed to form the hallmark columns that normally promote longitudinal growth. In addition, premature and ectopic mineralization was found to occur in the cartilage tissue of the Meckel’s cartilage. Additional investigation using the well-established embryonic mouse cell line ATDC5 led to the identification of changes in post translational modification of proteins which regulate cell behavior. Specifically, the AKT/GSK3β/β-catenin proteins were modified to increase the activity of the transcription factor TCF/LEF. This activation was coupled to 1) changes in gene expression, 2) decreased cartilage growth, and 3) increased mineralization. In conclusion, the results confirm that loss of COL11A1 homolog expression in zebrafish (col11a1a) and mouse (Col11a1) cells leads to severe chondrodysplasia that affects cells behavior and tissue shape. Treatment enhancing COL11A1 protein production in humans may be useful in inhibiting excessive mineralization. On the other hand, inhibition of COL11A1 protein production could be useful for tissue engineers and researchers developing treatments to accelerate the mineralization of cartilage during fracture healing in patients

Extracellular Matrix in Development and Disease

The evolution of multicellular metazoan organisms was marked by the inclusion of an extracellular matrix (ECM), a multicomponent, proteinaceous network between cells that contributes to the spatial arrangement of cells and the resulting tissue organization. The development of an ECM that provides support in larger organisms may have represented an advantage in the face of selection pressure for the evolution of the ECM

Advances in Cartilage Tissue Engineering Using Bioinks with Decellularized Cartilage and Three-Dimensional Printing

Osteoarthritis, a chronic, debilitating, and painful disease, is one of the leading causes of disability and socioeconomic burden, with an estimated 250 million people affected worldwide. Currently, there is no cure for osteoarthritis and treatments for joint disease require improvements. To address the challenge of improving cartilage repair and regeneration, three-dimensional (3D) printing for tissue engineering purposes has been developed. In this review, emerging technologies are presented with an overview of bioprinting, cartilage structure, current treatment options, decellularization, bioinks, and recent progress in the field of decellularized extracellular matrix (dECM)–bioink composites is discussed. The optimization of tissue engineering approaches using 3D-bioprinted biological scaffolds with dECM incorporated to create novel bioinks is an innovative strategy to promote cartilage repair and regeneration. Challenges and future directions that may lead to innovative improvements to currently available treatments for cartilage regeneration are presented

Authentication of a Novel Antibody to Zebrafish Collagen Type XI Alpha 1 Chain (Col11a1a)

Objective: Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). Results: The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research

The Shape of the Jaw—Zebrafish Col11a1a Regulates Meckel’s Cartilage Morphogenesis and Mineralization

The expression of the col11a1a gene is essential for normal skeletal development, affecting both cartilage and bone. Loss of function mutations have been shown to cause abnormalities in the growth plate of long bones, as well as in craniofacial development. However, the specific effects on Meckel’s cartilage have not been well studied. To further understand the effect of col11a1a gene function, we analyzed the developing jaw in zebrafish using gene knockdown by the injection of an antisense morpholino oligonucleotide using transgenic Tg(sp7:EGFP) and Tg(Fli1a:EGFP) EGFP reporter fish, as well as wildtype AB zebrafish. Our results demonstrate that zebrafish col11a1a knockdown impairs the cellular organization of Meckel’s cartilage in the developing jaw and alters the bone formation that occurs adjacent to the Meckel’s cartilage. These results suggest roles for Col11a1a protein in cartilage intermediates of bone development, the subsequent mineralization of the bony collar of long bones, and that which occurs adjacent to Meckel’s cartilage in the developing jaw

REVIEW: The Effects of Parental Phthalate Exposure on Newborn Defects

Phthalates, or phthalate esters, are employed in the making of numerous products around the world, such as food containers, children’s toys, detergents, surfactants, household items, and even pharmaceutical tablets. With the flexibility, elasticity, and inexpensive price, companies started using phthalates without doubting their acute and chronic toxicity. Recent research suggests that phthalate esters are possible causes of asthma, obesity, type II diabetes, neurodevelopmental issues, heart diseases, cancer, and especially reproductive system issues. However, studies about the effects of phthalates on fetal development are rare. To summarize updated data on this field, this review poster covers the most prominent in vivo studies, both animal models and humans, regarding phthalates exposure during pregnancy to newborn defects. Additionally, novel hypotheses on molecular mechanisms and important suggestions are provided to minimize possible negative outcomes of phthalate applications

Compensation for Col11a1 Deficiency by Other Minor Collagens

Collagens are triple helical proteins found in the extracellular matrix and are of broad biomedical importance. Understanding collagens are essential in elucidating the mechanisms that take place during embryogenesis, in the development of diseases such as arthritis, cardiovascular disease, during tumor invasion, and in the applications of tissue repair and regenerative medicine. The most abundant collagens are type I and type II. In contrast, collagen XI and collagen V are minor constituents of the extracellular matrix and are essential in regulation of fibril assembly and diameter of collagen fibrils. Collagen II-containing fibrils cannot form in the absence of collagen XI. Likewise, collagen I fibrils do not form correctly in the absence of collagen V. Collagen XI shares structural homology with collagen V and may share functions. We investigated mRNA expression in a mouse model with a deficiency in Col11a1 to detect evidence for compensation by Col5a1 and Col5a3. Our studies allowed us to answer the questions, “do minor fibrillar collagens share functions during development and cell differentiation or, alternatively, do they perform unique functions?” To answer this research question, we extracted RNA from mouse tissues and generated cDNA by reverse transcription. The cDNA was then amplified by PCR and the products were detected using agarose gel electrophoresis. We show that the mouse model, UAB Col11a1deltaVR, differentially expressed Col11a1 mRNA when compared to the wild type. To address the possibility of compensation for the deficiency in Col11a1 by other minor collagens, we will carry out RT-PCR using primers specific for mRNA encoding the other minor collagen chains. Identifying differential expression of mRNA for other minor collagens may provide diagnostic medical tools and could improve early detection of diseases and disorders associated with minor collagens such as Ehlers-Danlos and Stickler syndromes

The Effects of Parental Phthalate Exposure on Newborn Defects

Phthalates, or phthalate esters, are employed in the making of numerous products around the world, such as food containers, children’s toys, detergents, surfactants, household items, and even pharmaceutical tablets. With the flexibility, elasticity, and inexpensive price, companies started using phthalates without doubting their acute and chronic toxicity. Recent research suggests that phthalate esters are possible causes of asthma, obesity, type II diabetes, neurodevelopmental issues, heart diseases, cancer, and especially reproductive system issues. However, studies about the effects of phthalates on fetal development are rare. To summarize updated data on this field, this review poster covers the most prominent in vivo studies, both animal models and humans, regarding phthalates exposure during pregnancy to newborn defects. Additionally, novel hypotheses on molecular mechanisms and important suggestions are provided to minimize possible negative outcomes of phthalate applications