Silk Fibroin Hydrogels Coupled with the n16N−β-Chitin Complex: An in Vitro Organic Matrix for Controlling Calcium Carbonate Mineralization

ABSTRACTVaterite is one of the thermodynamically less stable polymorphs of calcium carbonate. Under ambient conditions it transforms into calcite, the most stable form of calcium carbonate. Organisms are able to stabilize minerals such as vaterite by means of organic molecules. The exact mechanisms how biomineralization proteins interact with metastable mineral phases are, however, less well understood. Many in vitro studies were performed using calcite as a model system. A deeper understanding of the interaction of organic molecules with metastable mineral phases would make them useful as a tool to control mineralization processes in vitro. In this study, we report on the co-precipitation of a natively soluble histidine-tagged GFP (green fluorecent protein) with a metastable vaterite phase and the subsequent insolubility of the fluorescent organic matrix in a 30μl calcium carbonate precipitation assay. The intrinsic fluorescence of GFP is conserved during the interaction with the mineral phase, indicating proper folding even in the insoluble state. This experiment can be extended to obtain deeper insights into some mechanistic models of biomineralization proteins by tracking native and modified GFP proteins microscopically during various stages of mineral precipitation and dissolution.

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Lessons from Biomineralisation: the Role of Post-translational Modification

10.26686/wgtn.16968799.v1 ◽

2021 ◽

Author(s):

◽

Benjamin Matthewson

Keyword(s):

Calcium Carbonate ◽

Sea Urchin ◽

Calcium Binding ◽

Keratan Sulfate ◽

Organic Matrix ◽

Active Species ◽

Protein Glycosylation ◽

Post Translational Modification ◽

Spin Filters

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

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Lessons from Biomineralisation: the Role of Post-translational Modification

10.26686/wgtn.16968799 ◽

2021 ◽

Author(s):

◽

Benjamin Matthewson

Keyword(s):

Calcium Carbonate ◽

Sea Urchin ◽

Calcium Binding ◽

Keratan Sulfate ◽

Organic Matrix ◽

Active Species ◽

Protein Glycosylation ◽

Post Translational Modification ◽

Spin Filters

<p>In this thesis we present our findings following analysis of the acidic organic matrix (SMP) occluded in the calcite spines of the New Zealand sea urchin Evechinus chloroticus. The main focus involves correlation of the structure and function of the post-translational modifications (PTMs). The experimental framework developed to achieve this involved mapping the structure of the PTMs throughout SMP based on molecular weight (MW) followed by selective removal of each of the identified PTMs. The functional analysis involved the use of SMP, and its derivatives, as additives in an in vitro calcium carbonate crystallisation assay. The adoption of in vitro methods was considered appropriate as the focus of this work was to develop strategies towards programmable crystal growth in vitro. From analysis of the PTMs we have shown that there is extensive protein glycosylation, sulfation, and phosphorylation; all are involved in rendering the isoelectric point (pI) of the SMP macromolecules. The sulfates are exclusively housed on the glycan framework whereas the phosphate is protein bound. The majority of the SMP glycone is charged with O-glycosylation accounting for 80.0 +/- 4.0 wt%. The structure of the glycans includes sulfated HexNAc oligomers, and potentially mucin-like/keratan sulfate and/or carrageenan structures. Using Stains-All we have shown that the desulfated HexNAc oligomers have the ability to bind calcium which signals relevance in the formation of calcium carbonate. SMP was fractionated by MW across a series of spin-filters. Use of the various fractions in the crystallisation assay showed that the species in the greater than 30 kDa fraction held the ability to increase the number of crystals nucleated. In contrast, the macromolecules in the 10 to 30 kDa range contained the full complement of morphologically active species. The result that these functions can be isolated demonstrates that they are independently controlled. The structure-function relationships determined include: the protein and the acidic glycans are jointly sufficient to generate the nucleating function; deglycosylated SMP holds the complete morphological activity, however, the glycans contribute by increasing reproducibility presumably through regulatory influences; and the sterically hindered phosphate residues make a slight contribution to this morphological activity. These results indicate that analyses which involve characterisation of the morphological function of cloned biomineral proteins may indeed correspond to their native counterparts. The observation that the morphologically active species are phosphorylated identifies them as the calcium-binding phosphoproteins. The morphological activity of SMP stripped of all PTMs is equivalent to the proteins extracted from the aragonitic layer of Haliotis iris. Characterisation of SMP demonstrated similarities with the OMs of other sea urchin species. For example, SMP appears to include SM30. In addition, the overall structure of SMP includes abundant acidic glycosylation with a relatively neutral protein component. This structural make-up is in contrast to the highly acidic proteins which are barely post-translationally modified.</p>

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In vitro Behavior of Silk Fibroin-Based Composite Resorbable Bone Fixation Devices

SSRN Electronic Journal ◽

10.2139/ssrn.3425450 ◽

2019 ◽

Author(s):

Bryant Heimbach ◽

Le Yu ◽

Mei Wei

Keyword(s):

Silk Fibroin ◽

Bone Fixation ◽

Fixation Devices

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Evaluation of Structure-Controlled Silk Fibroin Coatings for Orthopedic Infection and In-Situ Osteogenesis: In Vitro and In Vivo

SSRN Electronic Journal ◽

10.2139/ssrn.3600190 ◽

2020 ◽

Author(s):

Wenhao Zhou ◽

Teng Zhang ◽

Jianglong Yan ◽

QiYao Li ◽

Panpan Xiong ◽

...

Keyword(s):

Silk Fibroin ◽

Orthopedic Infection

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Cytotoxicity Studies of Curcumin Loaded-Cockle Shell-Derived Calcium Carbonate Nanoparticles

Nanoscience &Nanotechnology-Asia ◽

10.2174/2210681209666191128155819 ◽

2019 ◽

Vol 09 ◽

Cited By ~ 1

Author(s):

Maryam Muhammad Mailafiya ◽

Mohamad Aris Mohd Moklas ◽

Kabeer Abubakar ◽

Abubakar Danmaigoro ◽

Samaila Musa Chiroma ◽

...

Keyword(s):

Calcium Carbonate ◽

Safety Margin ◽

Bone Diseases ◽

In Vivo Studies ◽

Normal Cells ◽

Cytotoxicity Studies ◽

Standard Techniques ◽

Cockle Shell

Background: Cockle shell-derived calcium carbonate nanoparticles (CSCaCO3NP) are natural biogenic inorganic material that is used in drug delivery mainly as a bone-remodeling agent as well as a delivery agent for various therapeutics against bone diseases. Curcumin possess wide safety margin and yet puzzled with the problem of poor bioavailability due to insolubility. Propounding in vitro and in vivo studies on toxicity assessments of newly synthesized nanoparticles are ongoing to overcome some crucial challenges regarding their safety administration. Nanotoxicology has paved ways for concise test protocols to monitor sequential events with regards to possible toxicity of newly synthesized nanomaterials. The development of nanoparticle with no or less toxic effect has gained tremendous attentions. Objective: This study aimed at evaluating the in vitro cytotoxic effect of curcumin-loaded cockle shell-derived calcium carbonate nanoparticles (Cur-CSCaCO3NP) and assessing its biocompatibility on normal cells using standard techniques of WST’s assay. Method: Standard techniques of WST’s assay was used for the evaluation of the biocompatibility and cytotoxicity. Result: The result showed that CSCaCO3NP and Cur-CSCaCO3NP possess minimal toxicity and high biocompatibility on normal cells even at higher dose of 500 µg/ml and 40 µg/ml respectively. Conclusion: CSCaCO3NP can be termed an excellent non-toxic nanocarrier for curcumin delivery. Hence, curcumin loaded cockle shell derived calcium carbonate nanoparticles (Cur-CSCaCO3NP) could further be assessed for various in vivo and in vitro therapeutic applications against various bone related ailments.

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Anti-Inflammatory Properties of Injectable Betamethasone-Loaded Tyramine-Modified Gellan Gum/Silk Fibroin Hydrogels

Biomolecules ◽

10.3390/biom10101456 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1456

Author(s):

Isabel Matos Oliveira ◽

Cristiana Gonçalves ◽

Myeong Eun Shin ◽

Sumi Lee ◽

Rui Luis Reis ◽

...

Keyword(s):

Rheumatoid Arthritis ◽

Mechanical Properties ◽

Silk Fibroin ◽

Therapeutic Efficacy ◽

Composite Structures ◽

Gelation Time ◽

Polymeric Materials ◽

Gellan Gum ◽

Traditional Use

Rheumatoid arthritis is a rheumatic disease for which a healing treatment does not presently exist. Silk fibroin has been extensively studied for use in drug delivery systems due to its uniqueness, versatility and strong clinical track record in medicine. However, in general, natural polymeric materials are not mechanically stable enough, and have high rates of biodegradation. Thus, synthetic materials such as gellan gum can be used to produce composite structures with biological signals to promote tissue-specific interactions while providing the desired mechanical properties. In this work, we aimed to produce hydrogels of tyramine-modified gellan gum with silk fibroin (Ty–GG/SF) via horseradish peroxidase (HRP), with encapsulated betamethasone, to improve the biocompatibility and mechanical properties, and further increase therapeutic efficacy to treat rheumatoid arthritis (RA). The Ty–GG/SF hydrogels presented a β-sheet secondary structure, with gelation time around 2–5 min, good resistance to enzymatic degradation, a suitable injectability profile, viscoelastic capacity with a significant solid component and a betamethasone-controlled release profile over time. In vitro studies showed that Ty–GG/SF hydrogels did not produce a deleterious effect on cellular metabolic activity, morphology or proliferation. Furthermore, Ty–GG/SF hydrogels with encapsulated betamethasone revealed greater therapeutic efficacy than the drug applied alone. Therefore, this strategy can provide an improvement in therapeutic efficacy when compared to the traditional use of drugs for the treatment of rheumatoid arthritis.

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Calcium Carbonate Nanoparticles—Toxicity and Effect of In Ovo Inoculation on Chicken Embryo Development, Broiler Performance and Bone Status

Animals ◽

10.3390/ani11040932 ◽

2021 ◽

Vol 11 (4) ◽

pp. 932

Author(s):

Arkadiusz Matuszewski ◽

Monika Łukasiewicz ◽

Jan Niemiec ◽

Maciej Kamaszewski ◽

Sławomir Jaworski ◽

...

Keyword(s):

Calcium Carbonate ◽

Embryo Development ◽

Bone Quality ◽

Chicken Embryo ◽

Broiler Chickens ◽

Selection Procedure ◽

High Specificity ◽

Control Group ◽

In Ovo

The use of intensive selection procedure in modern broiler chicken lines has led to the development of several skeletal disorders in broiler chickens. Therefore, current research is focused on methods to improve the bone quality in birds. In ovo technology, using nanoparticles with a high specificity to bones, is a potential approach. The present study aimed to evaluate the effect of in ovo inoculation (IOI) of calcium carbonate nanoparticles (CCN) on chicken embryo development, health status, bone characteristics, and on broiler production results and bone quality. After assessing in vitro cell viability, the IOI procedure was performed with an injection of 500 μg/mL CCN. The control group was not inoculated with CCN. Hatchability, weight, and selected bone and serum parameters were measured in embryos. Part of hatchlings were reared under standard conditions until 42 days, and production results, meat quality, and bone quality of broilers were determined. CCN did not show cytotoxicity to cells and chicken embryo and positively influenced bone parameters of the embryos and of broilers later (calcification) without negatively affecting the production results. Thus, the IOI of CCN could modify the molecular responses at the stage of embryogenesis, resulting in better mineralization, and could provide a sustained effect, thereby improving bone quality in adult birds.

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Photocuring Hyaluronic Acid/Silk Fibroin Hydrogel Containing Curcumin Loaded CHITOSAN Nanoparticles for the Treatment of MG-63 Cells and ME3T3-E1 Cells

Polymers ◽

10.3390/polym13142302 ◽

2021 ◽

Vol 13 (14) ◽

pp. 2302

Author(s):

Qingwen Yu ◽

Zhiyuan Meng ◽

Yichao Liu ◽

Zehao Li ◽

Xing Sun ◽

...

Keyword(s):

Hyaluronic Acid ◽

Bone Regeneration ◽

Water Uptake ◽

Silk Fibroin ◽

Bone Repair ◽

Ph Value ◽

Chitosan Nanoparticles ◽

Vitro Assay ◽

Long Term Treatment

After an osteosarcoma excision, recurrence and bone defects are significant challenges for clinicians. In this study, the curcumin (Cur) loaded chitosan (CS) nanoparticles (CCNP) encapsulated silk fibroin (SF)/hyaluronic acid esterified by methacrylate (HAMA) (CCNPs-SF/HAMA) hydrogel for the osteosarcoma therapy and bone regeneration was developed by photocuring and ethanol treatment. The micro or nanofibers networks were observed in the CCNPs-SF/HAMA hydrogel. The FTIR results demonstrated that alcohol vapor treatment caused an increase in β-sheets of SF, resulting in the high compression stress and Young’s modulus of CCNPs-SF/HAMA hydrogel. According to the water uptake analysis, SF caused a slight decrease in water uptake of CCNPs-SF/HAMA hydrogel while CCNPs could enhance the water uptake of it. The swelling kinetic results showed that both the CCNPs and the SF increased the swelling ratio of CCNPs-SF/HAMA hydrogel. The accumulative release profile of CCNPs-SF/HAMA hydrogel showed that the release of Cur from CCNPs-SF/HAMA hydrogel was accelerated when pH value was decreased from 7.4 to 5.5. Besides, compared with CCNPs, the CCNPs-SF/HAMA hydrogel had a more sustainable drug release, which was beneficial for the long-term treatment of osteosarcoma. In vitro assay results indicated that CCNPs-SF/HAMA hydrogel with equivalent Cur concentration of 150 μg/mL possessed both the effect of anti-cancer and promoting the proliferation of osteoblasts. These results suggest that CCNPs-SF/HAMA hydrogel with superior physical properties and the bifunctional osteosarcoma therapy and bone repair may be an excellent candidate for local cancer therapy and bone regeneration.

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