Nanoengineered Silica-Based Biomaterials for Regenerative Medicine.

The paradigm of regenerative medicine is undergoing a transformative shift with the emergence of nanoengineered silica-based biomaterials. Their unique confluence of biocompatibility, precisely tunable porosity, and the ability to modulate cellular behavior at the molecular level makes them highly desirable for diverse tissue repair and regeneration applications. Advancements in nanoengineered silica synthesis and functionalization techniques have yielded a new generation of versatile biomaterials with tailored functionalities for targeted drug delivery, biomimetic scaffolds, and integration with stem cell therapy. These functionalities hold the potential to optimize therapeutic efficacy, promote enhanced regeneration, and modulate stem cell behavior for improved regenerative outcomes. Furthermore, the unique properties of silica facilitate non-invasive diagnostics and treatment monitoring through advanced biomedical imaging techniques, enabling a more holistic approach to regenerative medicine. This review comprehensively examines the utilization of nanoengineered silica biomaterials for diverse applications in regenerative medicine. By critically appraising the fabrication and design strategies that govern engineered silica biomaterials, this review underscores their groundbreaking potential to bridge the gap between the vision of regenerative medicine and clinical reality.

Biomineral-Based Composite Materials in Regenerative Medicine.

Regenerative medicine aims to address substantial defects by amplifying the body's natural regenerative abilities and preserving the health of tissues and organs. To achieve these goals, materials that can provide the spatial and biological support for cell proliferation and differentiation, as well as the micro-environment essential for the intended tissue, are needed. Scaffolds such as polymers and metallic materials provide three-dimensional structures for cells to attach to and grow in defects. These materials have limitations in terms of mechanical properties or biocompatibility. In contrast, biominerals are formed by living organisms through biomineralization, which also includes minerals created by replicating this process. Incorporating biominerals into conventional materials allows for enhanced strength, durability, and biocompatibility. Specifically, biominerals can improve the bond between the implant and tissue by mimicking the micro-environment. This enhances cell differentiation and tissue regeneration. Furthermore, biomineral composites have wound healing and antimicrobial properties, which can aid in wound repair. Additionally, biominerals can be engineered as drug carriers, which can efficiently deliver drugs to their intended targets, minimizing side effects and increasing therapeutic efficacy. This article examines the role of biominerals and their composite materials in regenerative medicine applications and discusses their properties, synthesis methods, and potential uses.

Biominerals and Bioinspired Materials in Biosensing: Recent Advancements and Applications.

Inspired by nature's remarkable ability to form intricate minerals, researchers have unlocked transformative strategies for creating next-generation biosensors with exceptional sensitivity, selectivity, and biocompatibility. By mimicking how organisms orchestrate mineral growth, biomimetic and bioinspired materials are significantly impacting biosensor design. Engineered bioinspired materials offer distinct advantages over their natural counterparts, boasting superior tunability, precise controllability, and the ability to integrate specific functionalities for enhanced sensing capabilities. This remarkable versatility enables the construction of various biosensing platforms, including optical sensors, electrochemical sensors, magnetic biosensors, and nucleic acid detection platforms, for diverse applications. Additionally, bioinspired materials facilitate the development of smartphone-assisted biosensing platforms, offering user-friendly and portable diagnostic tools for point-of-care applications. This review comprehensively explores the utilization of naturally occurring and engineered biominerals and materials for diverse biosensing applications. We highlight the fabrication and design strategies that tailor their functionalities to address specific biosensing needs. This in-depth exploration underscores the transformative potential of biominerals and materials in revolutionizing biosensing, paving the way for advancements in healthcare, environmental monitoring, and other critical fields.

Self-Entrapment of Antimicrobial Peptides in Silica Particles for Stable and Effective Antimicrobial Peptide Delivery System.

Antimicrobial peptides (AMPs) have emerged as a promising solution to tackle bacterial infections and combat antibiotic resistance. However, their vulnerability to protease degradation and toxicity towards mammalian cells has hindered their clinical application. To overcome these challenges, our study aims to develop a method to enhance the stability and safety of AMPs applicable to effective drug-device combination products. The KR12 antimicrobial peptide was chosen, and in order to further enhance its delivery and efficacy the human immunodeficiency virus TAT protein-derived cell-penetrating peptide (CPP) was fused to form CPP-KR12. A new product, CPP-KR12@Si, was developed by forming silica particles with self-entrapped CPP-KR12 peptide using biomimetic silica precipitability because of its cationic nature. Peptide delivery from CPP-KR12@Si to bacteria and cells was observed at a slightly delivered rate, with improved stability against trypsin treatment and a reduction in cytotoxicity compared to CPP-KR12. Finally, the antimicrobial potential of the CPP-KR12@Si/bone graft substitute (BGS) combination product was demonstrated. CPP-KR12 is coated in the form of submicron-sized particles on the surface of the BGS. Self-entrapped AMP in silica nanoparticles is a safe and effective AMP delivery method that will be useful for developing a drug-device combination product for tissue regeneration.

Fusion tags to enhance heterologous protein expression.

Escherichia coli is the most widely used heterologous protein expression system. However, this system remains a challenge due to the low solubility of proteins, insufficient yield, and inclusion body formation. Numerous approaches have sought to address these issues. The use of a fusion tag is one of the most powerful strategies for obtaining large amounts of heterologous protein in E. coli expression system. Here, recent advances in fusion tags that increase the expression of proteins are reviewed. In addition, proposed concepts for designing peptide tags to increase protein expression are discussed.

The NT11, a novel fusion tag for enhancing protein expression in Escherichia coli.

The Escherichia coli (E. coli) expression system has been widely used to produce recombinant proteins. However, in some heterologous expressions, there are still difficulties in large-scale production. The use of fusion partners is one of the strategies for improving the expression levels of proteins in E. coli host. Here, we demonstrate a novel fusion element, the NT11-tag, which enhances protein expression. The NT11-tag was derived from the first 11 amino acid residues within the N-terminal N-half domain of a duplicated carbonic anhydrase (dCA) from Dunaliella species. Previously, we have found that the tag improves expression of the C-half domain of dCA when linked to its N-terminus. To verify its use as a protein production enhancer tag, two kinds of CAs derived from Hahella chejuensis (Hc-CA) and Thermovibrio ammonifican (Ta-CA) and the yellow fluorescent protein (YFP) were used as model proteins to measure their increased expression upon fusion with the NT11-tag. The NT11-tag amplified protein expression in E. coli by 6.9- and 7.6-fold for Ta-CA and YFP, respectively. Moreover, the tag also enhanced the soluble expression of Hc-CA, Ta-CA, and YFP by 1.7-, 5.0-, and 3.2-fold, respectively. Furthermore, protein yield was increased without inhibiting protein function. These results indicate that the use of the NT11-tag is a promising method for improving protein production in E. coli.

Analysis of signs and pathology of H5N1-infected ducks from the 2010-2011 Korean highly pathogenic avian influenza outbreak suggests the influence of age and management practices on severity of disease.

We compared the clinical signs, histopathological lesions and distribution of viral antigens among infected young (meat-type) and older (breeder) ducks that were naturally infected with the highly pathogenic avian influenza (HPAI) virus during the 2010-2011 Korean outbreak. The meat-type ducks had a high mortality rate (30%) and showed severe neurological signs such as head tremors and paresis. In contrast, HPAI-infected breeder ducks had minimal clinical signs but a decreased egg production rate. The histopathological characteristics of infected meat-type ducks included necrotic lesions of heart and brain, which may have primarily contributed to the high mortality rate. In contrast, the breeder ducks only presented necrotic splenitis, and viral antigens were only detected in the trachea, lungs and spleen. Younger ducks had a high viral titre in the organs, high levels of viral shedding and a high mortality rate after experimental HPAI virus infection. Compared to the breeder ducks, the meat-type ducks were raised in smaller farms that had poor quarantine and breeding facilities. It is therefore possible that better biosecurity in the breeder farms could have reduced the infection dose and subsequently the severity of the disease. Thus, age and management may be the influencing factors for HPAI susceptibility in ducks.

Role of vacuolating cytotoxin VacA and cytotoxin-associated antigen CagA of Helicobacter pylori in the progression of gastric cancer.

Helicobacter (H.) pylori strains that express the cagA and s1a vacA genes are associated with an increased risk for gastric cancer. Here, we examined the association between the products of these virulence genes with the development of gastric cancer by immunohistochemical staining of gastric biopsy specimens taken from 208 routine gastroscopies and 43 gastric cancer patients. The correlation was analyzed by multivariate logistic regression. CagA and VacA expressions in gastric mucosa were significantly associated with chronic gastritis (CG) and intestinal metaplasia (IM), respectively, accompanying CG independent of age. The association of CagA expression with IM accompanying CG was increased in patients over 50-year old (p < 0.01) and that of VacA with CG was significant in patients younger than 50 year (p < 0.05). VacA and CagA were associated with mild IM incidence (p = 0.025 and p = 0.076, respectively) but not advanced IM. In the 43 gastric cancer patients, positivity for VacA was significantly higher in cases of CG and IM than carcinoma (p = 0.042), while that for CagA was slightly higher for individuals with carcinoma than those with CG and IM. These results indicate that CagA and VacA are critical factors for inducing CG and the subsequent progression of IM from CG with an increasing age.

ENA-A actimineral resource A extends lifespan associated with antioxidant mechanism in SMP30 knockout mice.

ENA-actimineral resource A (ENA-A) is an alkaline mineral water and has a few biological activities such as antioxidant activity. The aim of this study was to examine the effects of ENA-A on lifespan in mice using senescence marker protein-30 knockout mice. The present study had groups of 18-week-old mice (n = 24), 26-week-old mice (n = 12), and 46-week-old mice (n = 20). Each differently aged mice group was divided into three subgroups: a control group, a 5 % ENA-A-treated group, and a 10 % ENA-A-treated group. Mice in the 18-week-old group were treated with vitamin C drinking water 1.5 g/L. However, the mice in the 26-week-old and 46-week-old groups were not treated with vitamin C. The experiments were done for 18 weeks. All vitamin C-treated mice were alive at week 18 (100% survival rate). In the non-vitamin C group, the 10% ENA-A-treated mice were alive at week 18. The control and 5% ENA-A-treated mice died by week 15. As expected, vitamin C was not detected in the non-vitamin C-treated group. However, vitamin C levels were increased in an ENA-A dose-dependent manner in the vitamin C-treated group. In the TUNEL assay, a number of positive hepatocytes significantly decreased in an ENA-A dose-dependent manner. Periodic acid Schiff positive hepatocytes were significantly increased in an ENA-A dose-dependent manner. In addition, the expression level of CuZnSOD was increased by the ENA-A treatment. These data suggest that the intake of ENA-A has a critical role in the anti-aging mechanism and could be applied toward the lifespans of humans.

Biomimetic Materials for Skin Tissue Regeneration and Electronic Skin.

Biomimetic materials have become a promising alternative in the field of tissue engineering and regenerative medicine to address critical challenges in wound healing and skin regeneration. Skin-mimetic materials have enormous potential to improve wound healing outcomes and enable innovative diagnostic and sensor applications. Human skin, with its complex structure and diverse functions, serves as an excellent model for designing biomaterials. Creating effective wound coverings requires mimicking the unique extracellular matrix composition, mechanical properties, and biochemical cues. Additionally, integrating electronic functionality into these materials presents exciting possibilities for real-time monitoring, diagnostics, and personalized healthcare. This review examines biomimetic skin materials and their role in regenerative wound healing, as well as their integration with electronic skin technologies. It discusses recent advances, challenges, and future directions in this rapidly evolving field.

Use of biosilica to improve loading and delivery of bone morphogenic protein 2.

The clinical utility of bone morphogenetic protein 2 (BMP2) is limited because of the poor attraction between BMP2 and carriers, resulting in low loading efficiency and initial burst release. Here, the high binding affinity of BMP2 to the biosilica surface was utilized to overcome this limitation. Atomic force microscopy revealed that BMP2 bound nearly 8- and 2-fold more strongly to biosilica-coated hydroxyapatite than to uncoated and plain silica-coated hydroxyapatite, respectively. To achieve controlled release, collagen was introduced between the silica layers on hydroxyapatite, which was optimized by adjusting the collagen concentration and number of layers. The optimal biosilica/collagen formulation induced sustained BMP2 release without compromising loading efficiency. BMP2 combined with the mentioned formulation led to an increase in osteogenesis, as compared to the combination of BMP2 with either biosilica-coated or non-coated hydroxyapatite in vitro. In rat calvarial defect models, the biosilica/collagen-coated hydroxyapatite with 1 µg BMP2 showed 26 % more bone regeneration than the same dose of BMP2-loaded hydroxyapatite and 10.6 % more than hydroxyapatite with 2.5-fold dose of BMP2. Using BMP2 affinity carriers coated with biosilica/collagen allows for more efficacious in situ loading and delivery of BMP2, making them suitable for the clinical application of growth factors through a soaking method.

Antimicrobial effects of coprisin on wounds infected with Staphylococcus aureus in rats.

Antimicrobial peptides (AMPs) are naturally produced antibiotics that play important roles in host defense mechanisms. These proteins are found in variety of animal and plant species. The antibiotic effects of AMPs are gaining attention for use in human medicine. In this study, the antimicrobial effects of coprisin, a novel AMP isolated from the dung beetle (Copris tripartitus), were evaluated. The peptide was used to treat rats with wounds infected with Staphylococcus aureus. Coprisin accelerated wound closure both grossly and microscopically compared with the untreated group. Additionally, treatment with this peptide decreased phosphorylated-Smad2/3 (p-Smad2/3) levels, a downstream factor of the transforming growth factor-ß signaling pathway which is believed to inhibit reepithelization, in the nucleus and cytoplasm of regenerating cells. Moreover, increased cell populations and angiogenesis were observed in lesions treated with coprisin, suggesting that this peptide promotes wound healing via its antimicrobial activity against S. aureus. Our results demonstrated that coprisin is a potential therapeutic agent that can possibly replace traditional antibiotics and overcome microbial resistance.

JNK1 and JNK2 regulate α-SMA in hepatic stellate cells during CCl4 -induced fibrosis in the rat liver.

Following liver injuries, hepatic stellate cells (HSCs) express α-SMA. Mitogen activated protein kinase (MAPK) signaling pathways mediate α-SMA expression in distinct cell types. However, the regulation of α-SMA expression by MAPKs in HSCs has been rarely studied. We aimed to study the role of MAPKs in the activation of HSCs during liver fibrosis. Liver fibrosis of rats was induced by carbon tetrachloride. HSC-T6 cells, murine embryonic fibroblasts, JNK1(-/-) and JNK2(-/-) cells were used for in vitro studies. Immunohistochemistry and immunoblot analysis were used. We have found that the expression of JNK and α-SMA co-localized in HSCs during liver fibrosis, but ERK and p38 expressed in macrophages. The expression of α-SMA was up-regulated by JNK1 and JNK2 in non-stress condition. Under TGF-ß stimulation, however, the level α-SMA expression was increased by only JNK1, but not significantly changed by JNK2. We suggest that JNKs are responsible for α-SMA regulation, and especially JNK1 has a major role in up-regulation of α-SMA expression in HSCs under stress condition induced by TGF-ß during liver fibrosis.

Surface immobilization of protein via biosilification catalyzed by silicatein fused to glutathione S-transferase (GST).

Silicatein from Suberites domuncula was known to catalyze silica deposition in vitro under near neutral pH and ambient temperature conditions. In this study, we employed GST-glutathione (GSH) interaction system to increase the production of silicatein and develop an efficient protein immobilization method. Recombinant silicatein fused with GST (GST-SIL) was produced in E. coli and the GST-SIL protein was employed on GSH-coated glass plate. GST-SIL bound surface or matrix can catalyze the formation of silica layer in the presence of tetraethyl orthosilicate as a substrate at an ambient temperature and neutral pH. During silicatein-mediated silicification, green fluorescent protein (GFP) or horseradish peroxidase (HRP) can be efficiently immobilized on the silica surface. Immobilized GFP or HRP retained their activity and were released gradually. This biocompatible silica coating technique can be employed to prepare biomolecule-immobilized surfaces or matrixes, which are useful for the development of biocatalytic, diagnostic and biosensing system, or tissue culture scaffolds.

Expression, reconstruction and characterization of codon-optimized carbonic anhydrase from Hahella chejuensis for CO2 sequestration application.

The high production of functional carbonic anhydrase (CA) is required for practical CO2 sequestration application mediated by CA. Here, the synthetic gene based on Escherichia coli codon usage of new α-type CA (HC-aCA) of Hahella chejuensis, a Korea marine microorganism, was highly expressed in E. coli. We obtained a high yield of functional HC-aCA by denaturing/refolding process and incorporating zinc ion into its active site. The refolded HC-aCA displayed a half-deactivation temperature of 60 °C with maximal activity at 50 °C, and had high pH stability in alkali condition with maximal activity at pH 10.0. The esterase activity of HC-aCA almost doubled at high salt concentration ranging from 0.67 to 2.0 M NaCl. HC-aCA catalyzed the conversion of CO2 to CaCO3 as calcites form in the presence of Ca(2+). The refolded HC-aCA could be a promising candidate for the development of efficient CA-based CO2 sequestration processes.

Smad3 deficiency ameliorates hepatic fibrogenesis through the expression of senescence marker protein-30, an antioxidant-related protein.

Smad3 is a key mediator of the transforming growth factor (TGF)-ß1 signaling pathway that plays central role in inflammation and fibrosis. In present study, we evaluated the effect of Smad3 deficiency in Smad3-/- mice with carbon tetrachloride (CCl4)-induced liver fibrosis. The animals were received CCl4 or olive oil three times a week for 4 weeks. Histopathological analyses were performed to evaluate the fibrosis development in the mice. Alteration of protein expression controlled by Smad3 was examined using a proteomic analysis. CCl4-induced liver fibrosis was rarely detected in Smad3-/- mice compared to Smad3+/+. Proteomic analysis revealed that proteins related to antioxidant activities such as senescence marker protein-30 (SMP30), selenium-binding proteins (SP56) and glutathione S-transferases (GSTs) were up-regulated in Smad3-/- mice. Western blot analysis confirmed that SMP30 protein expression was increased in Smad3-/- mice. And SMP30 levels were decreased in CCl4-treated Smad3+/+ and Smad3-/- mice. These results indicate that Smad3 deficiency influences the proteins level related to antioxidant activities during early liver fibrosis. Thus, we suggest that Smad3 deteriorate hepatic injury by inhibitor of antioxidant proteins as well as mediator of TGF-ß1 signaling.

Advanced Delivery System of Polyphenols for Effective Cancer Prevention and Therapy.

Polyphenols from plants such as fruits and vegetables are phytochemicals with physiological and pharmacological activity as potential drugs to modulate oxidative stress and inflammation associated with cardiovascular disease, chronic disease, and cancer. However, due to the limited water solubility and bioavailability of many natural compounds, their pharmacological applications have been limited. Researchers have made progress in the development of nano- and micro-carriers that can address these issues and facilitate effective drug delivery. The currently developed drug delivery systems maximize the fundamental effects in various aspects such as absorption rate, stability, cellular absorption, and bioactivity of polyphenols. This review focuses on the antioxidant and anti-inflammatory effects of polyphenols enhanced by the introduction of drug delivery systems, and ultimately discusses the inhibition of cancer cell proliferation, growth, and angiogenesis.

Biomimetic Silica Particles with Self-Loading BMP-2 Knuckle Epitope Peptide and Its Delivery for Bone Regeneration.

Biomimetic silica deposition is an in-situ immobilization method for bioactive molecules under biocompatible conditions. The osteoinductive P4 peptide derived from the knuckle epitope of bone morphogenetic protein (BMP), which binds to BMP receptor-II (BMPRII), has been newly found to contain silica formation ability. We found that the two lysine residues at the N-terminus of P4 played a vital role in silica deposition. The P4 peptide co-precipitated with silica during P4-mediated silicification, yielding P4/silica hybrid particles (P4@Si) with a high loading efficiency of 87%. P4 was released from P4@Si at a constant rate for over 250 h, representing a zero-order kinetic model. In flow cytometric analysis, P4@Si showed a 1.5-fold increase in the delivery capacity to MC3T3 E1 cells than the free form of P4. Furthermore, P4 was found anchored to hydroxyapatite (HA) through a hexa-glutamate tag, followed by P4-mediated silicification, yielding P4@Si coated HA. This suggested a superior osteoinductive potential compared to silica or P4 alone coated HA in the in vitro study. In conclusion, the co-delivery of the osteoinductive P4 peptide and silica by P4-mediated silica deposition is an efficient method for capturing and delivering its molecules and inducing synergistic osteogenesis.

BMP2-Mediated Silica Deposition: An Effective Strategy for Bone Mineralization.

The combined use of an osteogenic factor, such as bone morphogenetic protein 2 (BMP2), with a bone scaffold was quite functional for the reconstruction of bone defects. Although many studies using BMP2 have been done, there is still a need to develop an efficient way to apply BMP2 in the bone scaffold. Here, we reported an interesting fact that BMP2 has a silica deposition ability in the presence of silicic acid and proposed that such an ability of BMP2 can effectively immobilize and transport itself by a kind of coprecipitation of BMP2 with a silica matrix. The presence of BMP2 in the resulting silica was proved by SEM and EDS and was visualized by FITC-labeled BMP2. The delivery efficacy of BMP2 of silica-entrapped BMP2 on osteoblast differentiation and mineralization using MC3T3 E1 preosteoblast cells was evaluated in vitro. The coprecipitated BMP2 with silica exhibited osteogenesis at a low concentration that was insufficient to give an osteoinductive signal as the free form. Expectedly, the silica-entrapped BMP2 exhibited thermal stability over free BMP2. When applied to bone graft substitution, e.g., hydroxyapatite granules (HA), silica-entrapped BMP 2 laden HA (BMP2@Si/HA) showed sustained BMP2 release, whereas free BMP2 adsorbed HA by a simple dipping method (BMP2/HA) displayed a burst release of BMP2 at an initial time. In the rat critical-size calvarial defect model, BMP2@Si/HA showed better bone regeneration than BMP2/HA by about 10%. The BMP2/silica hybrid deposited on a carrier surface via BMP2-mediated silica precipitation demonstrated an increase in the loading efficiency, a decrease in the burst release of BMP2, and an increase in bone regeneration. Taken together, the coprecipitated BMP2 with a silica matrix has the advantages of not only being able to immobilize BMP2 efficiently without compromising its function but also serving as a stable carrier for BMP2 delivery.

Tailored Functionalized Protein Nanocarriers for Cancer Therapy: Recent Developments and Prospects.

Recently, the potential use of nanoparticles for the targeted delivery of therapeutic and diagnostic agents has garnered increased interest. Several nanoparticle drug delivery systems have been developed for cancer treatment. Typically, protein-based nanocarriers offer several advantages, including biodegradability and biocompatibility. Using genetic engineering or chemical conjugation approaches, well-known naturally occurring protein nanoparticles can be further prepared, engineered, and functionalized in their self-assembly to meet the demands of clinical production efficiency. Accordingly, promising protein nanoparticles have been developed with outstanding tumor-targeting capabilities, ultimately overcoming multidrug resistance issues, in vivo delivery barriers, and mimicking the tumor microenvironment. Bioinspired by natural nanoparticles, advanced computational techniques have been harnessed for the programmable design of highly homogenous protein nanoparticles, which could open new routes for the rational design of vaccines and drug formulations. The current review aims to present several significant advancements made in protein nanoparticle technology, and their use in cancer therapy. Additionally, tailored construction methods and therapeutic applications of engineered protein-based nanoparticles are discussed.