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Effect of Molecular Weight and Functionality on Acrylated Poly(caprolactone) for Stereolithography and Biomedical Applications.

Sun, 2019-09-08 21:00
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Effect of Molecular Weight and Functionality on Acrylated Poly(caprolactone) for Stereolithography and Biomedical Applications.

Biomacromolecules. 2018 09 10;19(9):3682-3692

Authors: Green BJ, Worthington KS, Thompson JR, Bunn SJ, Rethwisch M, Kaalberg EE, Jiao C, Wiley LA, Mullins RF, Stone EM, Sohn EH, Tucker BA, Guymon CA

Abstract
Degradable polymers are integral components in many biomedical polymer applications. The ability of these materials to decompose in situ has become a critical component for tissue engineering, allowing scaffolds to guide cell and tissue growth while facilitating gradual regeneration of native tissue. The objective of this work is to understand the role of prepolymer molecular weight and functionality of photocurable poly(caprolactone) (PCL) in determining reaction kinetics, mechanical properties, polymer degradation, biocompatibility, and suitability for stereolithography. PCL, a degradable polymer used in a number of biomedical applications, was functionalized with acrylate groups to enable photopolymerization and three-dimensional printing via stereolithography. PCL prepolymers with different molecular weights and functionalities were studied to understand the role of molecular structure in reaction kinetics, mechanical properties, and degradation rates. The mechanical properties of photocured PCL were dependent on cross-link density and directly related to the molecular weight and functionality of the prepolymers. High-molecular weight, low-functionality PCLDA prepolymers exhibited a lower modulus and a higher strain at break, while low-molecular weight, high-functionality PCLTA prepolymers exhibited a lower strain at break and a higher modulus. Additionally, degradation profiles of cross-linked PCL followed a similar trend, with low cross-link density leading to degradation times up to 2.5 times shorter than those of more highly cross-linked polymers. Furthermore, photopolymerized PCL showed biocompatibility both in vitro and in vivo, causing no observed detrimental effects on seeded murine-induced pluripotent stem cells or when implanted into pig retinas. Finally, the ability to create three-dimensional PCL structures is shown by fabrication of simple structures using digital light projection stereolithography. Low-molecular weight, high-functionality PCLTA prepolymers printed objects with feature sizes near the hardware resolution limit of 50 μm. This work lays the foundation for future work in fabricating microscale PCL structures for a wide range of tissue regeneration applications.

PMID: 30044915 [PubMed - indexed for MEDLINE]

Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

Sun, 2019-09-01 19:26
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Helper-Dependent Adenovirus Transduces the Human and Rat Retina but Elicits an Inflammatory Reaction When Delivered Subretinally in Rats.

Hum Gene Ther. 2019 Aug 28;:

Authors: Han IC, Burnight E, Ulferts MJ, Worthington KS, Russell SR, Sohn EH, Mullins RF, Stone E, Tucker BA, Wiley LA

Abstract
The identification of over 100 genes causing inherited retinal degeneration and the promising results of recent gene augmentation trials have led to an increase in the number of studies investigating the preclinical efficacy of viral-mediated gene transfer. Despite success using adeno-associated viruses (AAV), many disease-causing genes, like ABCA4 or USH2A, are too large to be packaged into these vectors. One option for large gene delivery is the family of integration deficient helper-dependent adenoviruses (HDAds), which efficiently transduce post-mitotic neurons. However, HDAds have been shown in other organ systems to elicit an immune response, and the immunogenicity of HDAds in the retina has not been characterized. In this study, HDAd5 was found to successfully transduce rod and cone photoreceptors in ex vivo human retinal organ cultures. The ocular inflammatory response to subretinal injection of the HDAd serotype 5 (HDAd5) was evaluated using a rat model. Subretinal injection of HDAd5 carrying cytomegalovirus promoter-driven enhanced Green Fluorescent Protein (HDAd5-CMVp-eGFP) elicited a robust inflammatory response by 3-days post-injection. This reaction included vitreous infiltration of Iba1-positive monocytes and increased expression of the pro-inflammatory protein, ICAM-1. By 7-days post-injection, most Iba1-positive infiltrates migrated into the neural retina, including the outer nuclear layer, and ICAM-1 expression was significantly increased compared to buffer-injected contralateral control eyes. At 14-days post-injection, Iba1-positive cells persisted in the retinas of HDAd5-injected eyes, and there was thinning of the outer nuclear layer indicative of photoreceptor cell loss. Subretinal injection of an empty HDAd5 virus was used to confirm that the inflammatory response was in response to the HDAd5 vector and not due to eGFP-induced overexpression cytotoxicity. Despite their larger carrying capacity, further work is needed to elucidate the inflammatory pathways involved and to identify an immunomodulation paradigm sufficient for safe and effective transfer of large genes to the retina using the HDAd5 system.

PMID: 31456426 [PubMed - as supplied by publisher]

Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Sun, 2019-06-30 05:28
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Connective Tissue Growth Factor Promotes Efficient Generation of Human Induced Pluripotent Stem Cell-Derived Choroidal Endothelium.

Stem Cells Transl Med. 2017 06;6(6):1533-1546

Authors: Songstad AE, Worthington KS, Chirco KR, Giacalone JC, Whitmore SS, Anfinson KR, Ochoa D, Cranston CM, Riker MJ, Neiman M, Stone EM, Mullins RF, Tucker BA

Abstract
Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of retinal pigment epithelial (RPE) and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be lost in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific, stem cell-derived CECs will be required. In this study, we report for the first time a stepwise differentiation protocol suitable for generating human iPSC-derived CEC-like cells. RNA-seq analysis of the monkey CEC line, RF/6A, combined with two statistical screens allowed us to develop media comprised of various protein combinations. In both screens, connective tissue growth factor (CTGF) was identified as the key component required for driving CEC development. A second factor tumor necrosis factor (TNF)-related weak inducer of apoptosis receptor was also found to promote iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CEC-like cells formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. In combination with RPE and photoreceptor cells, patient-specific iPSC derived CEC-like cells will enable scientists to accurately evaluate AMD pathophysiology and develop effective cell replacement therapies. Stem Cells Translational Medicine 2017;6:1533-1546.

PMID: 28474838 [PubMed - indexed for MEDLINE]