Silk apatite composites from electrospun fibers

Li, Chunmei,Jin, Hyoung-Joon,Botsaris, Gregory D.,Kaplan, David L. Cambridge University Press (Materials Research Soc 2005 Journal of materials research Vol.20 No.12

<P>Human bone is a three-dimensional composite structure consisting of inorganic apatite crystals and organic collagen fibers. An attractive strategy for fabricating mimics of these types of composite biomaterials is to selectively grow apatite on polymers with control of structure, mechanical properties, and function. In the present study, silk/apatite composites were prepared by growing apatite on functionalized nanodiameter silk fibroin fibers prepared by electrospinning. The functionalized fibers were spun from an aqueous solution of silk/polyethylene oxide (PEO) (78/22 wt/wt) containing poly(L-aspartate) (poly-Asp), which was introduced as an analogue of noncollageous proteins normally found in bone. Silk fibroin associated with the acidic poly-Asp and acted as template for mineralization. Apatite mineral growth occurred preferentially along the longitudinal direction of the fibers, a feature that was not present in the absence of the combination of components at appropriate concentrations. Energy dispersive spectroscopy and x-ray diffraction confirmed that the mineral deposits were apatite. The results suggest that this approach can be used to form structures with potential utility for bone-related biomaterials based on the ability to control the interface wherein nucleation and crystal growth occur on the silk fibroin. With this level of inorganic-organic control, coupled with the unique mechanical properties, slow rates of biodegradation, and polymorphic features of this type of proteins, new opportunities emerge for utility of biomaterials.</P>

Mass Production of Pullulan with Optimized Concentrations of Carbon and Nitrogen Sources by Aureobasidium pullulans HP-2001 in a 100-L Bioreactor with the Inner Pressure

( Seo Hyung Pil ),( Chung Han Chung ),( Sung Koo Kim ),( Richard A Gross ),( David L. Kaplan ),( Jin Woo Lee ) 한국미생물생명공학회 2004 Journal of microbiology and biotechnology Vol.14 No.2

Exopolymers from curdlan production: Incorporation of glucose-related sugars by Agrobacterium sp. strain ATCC 31749

Lee, Jin W.,Allen, Alfred L.,Deng, Flank,Yeomans, Walter G.,Kaplan, David L.,Gross, Richard A. 東亞大學校附設遺傳工學硏究所 1998 遺傳工學硏究 Vol.- No.5

Three different exopolymers were purified from cultures of Agrobacterium sp. strain ATCC 31749 grown in a mineral salts medium containing 2% glucose at 30℃ for 5 days under aerobic culture conditions. These exopolymers were curdlan (extracellular, homo-B-(1-3)-gulcan.water insoluble at neutral pH), a water-soluble noncurdlan-type exopolymer A (WSNCE-A), and a water-soluble noncurdlan-type exopolymer B (WSNCE-B). Curdlan. WSNCE-A. and WSNCE-B composed by weight 61,27,and 12%,respectively, of the exopolymer produced from goucose. Compositions of all polymers were confirmed by gas chromatography (GC) and gas chromatography -mass spectrometry(GC-MS). The WSNCE-A is composed of glucose and galactose with lower contents of rhamnose. The WSNCE-B consists of glucose and mannose. To biosynthesize modified biopolymers, glucose-related sugars including 3-0-methyl-D-glucose, 2-amino-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-glucose(N-acetylgucosamine)were fed separately as the sole carbon source. Using 3-O-methyl-D-glucose, 8-12 mol% of the curdlan repeats were 3-O-methyl-D-glucose based on GC and H-nuclear magnetic resonance spectrometry. N-Acetylgucosamine was incorporated into WSNCE-A at 10 mol% based on the GC-MS but was not found in curdlan or WSNCE-B. Trois exopolymeres differents ont ete purifies a partir de cultures d'Agrobacterium sp. souche ATCC 31749, dont la croissance s'est effectuee a 30℃ durant 5 jours sur un milieu contenant des sels mineraux et 2% de glucose dans des conditions aerobies. Ces trois exopolymeres se sontreveles etre:un type coagule(curdlan) extracellulaire. insoluble dans 1'eau a pH neutre et constitue d'homo-B-(1-3)-glucane, et deux types solubles, non coagules, soit 1'exopolymere A (WSNCE-A) et 1'exopolyere B(WSNCE-B). Les poids respectifs du type coagule esdes deux types noncoagules A et B ont represente 61,27 et 12% de 1'exopolymere produit a partir du glucose. La composition de chacun des polymeres a ete identifiee par chromatographie en phase gazeuse(GC) et par chromatographie en phage gazeuse-spectrometrie de masse (GC-MS). Le WSNCE-A s'est revele constitue de glucose et de galactose avec faible teneur en rhamnose, et lw WSNCE-B, de glucose et de mannose. Dans le but de biosynthetiser des biopolymeres modifies, des sucres apparentes au glucose ont ete incorpores separement aux milieux de culture comme seule source de carbone:le 3-O-methyl-D-glucose, le 2-amino-2-desoxy-D-glucose, et le 2-acetamido-2-desoxy-D-glucose(N-acetylgulcosamine). Utilisant le 3-O-methyl-D-glucose, 8-12 mol% des repetitions du type coagule ont ete du 3-O-methyl-D-glucose, d'apres la GC et la spectrometrie de resonnance magnetique nucleaire-'H. La N-acetylglucosamine a ete incorporee dans le WSNCE-A a 10 mol% d'apres les GC-MS, mails il n'a pas ete trouve chez les exopolymeres coagules ou le WSNCE-B.

Compositional consistancy of a heteropolysaccharide-7 produced by Beijerinckia indica

Lee, Jin W.,Yeomans, Walter G.,Allen, Alfred L.,Gross, Richard A.,Kaplan, David L. 東亞大學校附設遺傳工學硏究所 1998 遺傳工學硏究 Vol.- No.5

The component sugars of heteropolysaccharide-7(PS-7) produced by Beijerinckia indica were rhamnose and glucose (1.0:4.8,mol:mol) by gas chromatographic analysis. Galacturonic acid, previously reported as a repeat unit of PS-7, was not found in purified PS-7. The yield of PS-7 varied with physiological conditions, such as concentration of carbon source and initial pH of medium, but the molar ratio of rhamnose to glucose stayed within 1.0 to 4.6-5.1. B indica utilized glucose and some glucose analogs as carbon sources and produced exopolymers, although there was no direct incorporation of these sugars into PS-7. The molar ratio of rhamnose to glucose in each polymer synthesized from glucose-related sugars showed no significant variation(1.0 to 4.5-4.7).

Microbial production of Water-Soluble Non Curdlan Type Exopolymer-B with Controlled Composition by Agrobacterium sp.

Lee, Jin W.,Yeomans, Walter G.,Allen, Alfred L.,Gross, Richard A.,Kaplan, David L. 東亞大學校附設遺傳工學硏究所 1998 遺傳工學硏究 Vol.- No.5

The cell growth, production of exopolymers, and the molar ratio of glucose to mannose in the water-soluble non curdlan type exopolymer-B (WSNCE-B), which is one of three exopolymers purified from the culture of Agrobacterium sp. varied with carbon source, culture medium, and initial medium pH. The molar percentage of rhamnose, a ninor component in WSNCE-B, varied up to 13%, dependent on physiological condition. No rhamnose was found in the WSNCE-B purified from the culture with initial medium pH>6.8. The relative amount of mannose in WSNCE-B increased regularly with the amount of yeast extract added to the mineral salts medium. The relative amounts of glucose, mannose, and rhamnose in the WSNCE-B can be controlled by varying culture conditions.

Production of Chitosan- and Chitin-like Exopolymers by Acetobacter xylinum ATCC 10245

Lee,Jin W.,Deng,Fang,Yeomans,Walter G.,Allen,Alfred L.,Gross,Richard A.,Kaplan,David L. 한국생명과학회 1998 한국생명과학회 학술발표회 Vol.20 No.-

To biosynthesize modified cellulose, glucose analogs including 3-O-methyl-D-glucose, glucosamine, N-acetylglucosamine, and 2-deoxy-D-glucose were used separately as the carbon source. Incorporation of glucosamine and N-acetylglucosamine repeat units into cellulose by Acetobacter xylinum ATCC 10245 was confirmed by GC, GC/MS, FTIR and ¹H-NMR. Based on data, the average molar percentages of glucosamine and N-acetylglucosamine repeat units into exopolymers were 19% and 18%, respectively. The yields of exopolymers made with glucosamine and N-acetylglucosamine as carbon source after 7 day culture were 0.37 ㎎/㎖ and 0.67 ㎎/㎖ , respectively, whereas that of glucose was 3.9 ㎎/㎖. The yield of exopolymer made with the mixture of glucose (0.5%, v/v) and glucosamine (1.5%, v/v) as carbon sources was 1.75 ㎎/㎖, and the average molar percentage of glucosamine repeat unit into the exopolymer was 17%. Exopolymers made with glucosamine and N-acetylglucosamine were fractionated by 10% acetic acid and DW, respectively, The molar ratio of glucose to glucosamine in the acetic acid (10%, v/v) soluble fractionation of glucosamine incorporated exopolymer was 0.6: 1.0. The molar ratio of glucose to N-acetylglucosanune in the water soluble fraction of N-acetylglucosamine incorporated exopolymer was 0.8 : 1.0.

Biosynthesis of Novel Exopolymers by Aureobasidium pullulans

Lee,Jin W.,Deng,Fang,Yeomans,Walter G.,Allen,Alfred L.,Gross,Richard A.,Kaplan,David L. 한국생명과학회 1998 한국생명과학회 학술발표회 Vol.20 No.-

Exopolymers produced by A. pullulans ATCC 42023 under aerobic conditions with glucose, mannose, and glucose analogs including 3-O- methyl-D-glucose, glucosamine, N-acetylglucosamine, and 2-deoxy-D- glucose as carbon sources contained glucose and mannose. The molar ratio of glucose to mannose in exopolymers and the molecular weight of exopolymers varied with the carbon source and culture time. Exopolymers synthesized with glucose and mannose as carbon sources showed glucose contents of 87±3 and 89±2%, respectively, with a decreased molecular weight from 3.50 - 2.12 x 10^6 to 0.85 - 0.77 x 10^6 with culture time. The molar ratio of glucose to mannose in the exopolymer synthesized with glucosamine changed from 55±3 : 45±3 to 29±2 : 71±2 and its molecular weight increased from 2.73 x 10^6 to 4.86 x10^6 with culture time. The molar ratio of glucose to mannose in exopolymers ranged from 87±3 : 13±3 to 28±2 : 72±2 and can be controlled by carbon source. On the basis on the results from enzyme hydrolysis of the exopolymers and comparison of ¹H- and ^(13)C-NMR chromatograms, the mannose as a monomeric component is substituted for glucose without changing the structure of pullulan.

Production of zoogloea gum by Zoogloea ramigera with glucose analogs

Lee, Jin W.,Yeomans, Walter G.,Allen, Alfred L.,Gross, Richard A.,Kaplan, David L. 東亞大學校附設遺傳工學硏究所 1998 遺傳工學硏究 Vol.- No.5

Zoolgans with altered sugar composition were synthesized by Zoogloea ramigera by varying the glucose concentration and initial medium pH. The relative mol% of the sugar components, glucose and galactose, in the exopolymer made with 2% (w/v) glucose as the carbon source was 66 and 34%, respectively. By varying the glucose concentration and initial medium pH, the mol% ratios of glucose to galactose in zooglan ranged from 70:30 to 58:42. Also, glucose analogs, 3-O-methyl-D-glucose, 2-amino-2-deoxy-D-glucose, and 2-acetamido-2-deoxy-D-glucose, were used as a co-substrate with glucose to produce to produce modified zooglans. The mol% ratios of glucose to galactose in exopolymers produced by co-feeding glucose analogs ranged from 70:30 to 9:91.

Processing Windows for Forming Silk Fibroin Biomaterials into a 3D Porous Matrix

Kim, Hyeon Joo,Kim, Hyun Suk,Matsumoto, Akira,Chin, In-Joo,Jin, Hyoung-Joon,Kaplan, David L. Commonwealth Scientific and Industrial Research Or 2005 Australian journal of chemistry Vol.58 No.10

<P> In the present study we clarify phase diagrams related to silk fibroin processing into three-dimensional porous structures useful for biomaterials and for scaffolds in tissue engineering. All-aqueous and organic solvent (hexafluoroisopropanol) modes of processing are compared relative to solution concentration of silk protein polymer and size of porogen (NaCl particles). The results clarify the range of conditions under which these biomaterial matrices can be formed, with a broader range of pore sizes and smoother surface morphology generated from the organic solvent process. These structures are directly applicable to fundamental studies of protein-based biomaterial assembly as well as cell interactions and tissue formation with these systems. </P>

NF-κB signaling is key in the wound healing processes of silk fibroin

Park, Ye Ri,Sultan, Md. Tipu,Park, Hyun Jung,Lee, Jung Min,Ju, Hyung Woo,Lee, Ok Joo,Lee, Dong Jin,Kaplan, David L.,Park, Chan Hum Elsevier Science B.V. Amsterdam 2018 ACTA BIOMATERIALIA Vol. No.

<P><B>Abstract</B></P> <P>Silk fibroin (SF) is a well-studied biomaterial for tissue engineering applications including wound healing. However, the signaling mechanisms underlying the impact of SF on this phenomenon have not been determined. In this study, through microarray analysis, regulatory genes of NF-ĸB signaling were activated in SF-treated NIH3T3 cells along with other genes. Immunoblot analysis confirmed the activation of the NF-ĸB signaling pathway as SF induced protein expression levels of IKKα, IKKβ, p65, and the degradation of IκBα. The treatment of NIH3T3 cells with SF also increased the expression of cyclin D1, vimentin, fibronectin, and vascular endothelial growth factor (VEGF). The expression of these factors by SF treatment was abrogated when NF-ĸB was inhibited by a pharmacological inhibitor Bay 11-7082. Knockdown of NF-ĸB using siRNA of IKKα and IKKβ also inhibited the SF-induced wound healing response of the NIH3T3 cells in a wound scratch assay. Collectively, these results indicated that SF-induced wound healing through the canonical NF-κB signaling pathway via regulation of the expression of cyclin D1, vimentin, fibronectin, and VEGF by NIH3T3 cells. Using an <I>in vivo</I> study with a partial-thickness excision wound in rats we demonstrated that SF-induced wound healing via NF-κB regulated proteins including cyclin D1, fibronectin, and VEGF. The <I>in vitro</I> and <I>in vivo</I> data suggested that SF induced wound healing via modulation of NF-ĸB signaling regulated proteins.</P> <P><B>Statement of Significance</B></P> <P>Silk fibroin has been effectively used as a dressing for wound treatment for more than a century. However, mechanistic insight into the basis for wound healing via silk fibroin has not been elucidated. Here we report a key mechanism involved in silk fibroin induced wound healing both <I>in vitro</I> and <I>in vivo.</I> Using genetic- and protein-level analyses, NF-κB signaling was found to regulate silk fibroin-induced wound healing by modulating target proteins. Thus, the NF-κB signaling pathway may be utilized as a therapeutic target during the formulation of silk fibroin-based biomaterials for wound healing and tissue engineering.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>