Silk proteins in reconstructive surgery: Do they possess an inherent antibacterial activity? A systematic review.

The field of reconstructive surgery encompasses a wide range of surgical procedures and regenerative approaches to treat various tissue types. Every surgical procedure is associated with the risk of surgical site infections, which are not only a financial burden but also increase patient morbidity. The surgical armamentarium in this area are biomaterials, particularly natural, biodegradable, biocompatible polymers, including the silk proteins fibroin (SF) and sericin (SS). Silk is known to be derived from silkworms and is mainly composed of 60-80% fibroin, which provides the structural form, and 15-35% sericin, which acts as a glue-like substance for the SF threads. Silk proteins possess most of the desired properties for biomedical applications, including biocompatibility, biodegradability, minimal immunogenicity, and tunable biomechanical behaviour. In an effort to alleviate or even prevent infections associated with the use of biomaterials in surgery, antibacterial/antimicrobial properties have been investigated in numerous studies. In this systematic review, the following question was addressed: Do silk proteins, SF and SS, possess an intrinsic antibacterial property and how could these materials be tailored to achieve such a property?

Do Titanium Dioxide Particles Stimulate Macrophages to Release Proinflammatory Cytokines and Increase the Risk for Peri-implantitis?

PURPOSE: Titanium dioxide (TiO2) particles detached from titanium dental implants by tribocorrosion can be phagocytosed by macrophages, releasing various proinflammatory cytokines at the implant sites that may trigger peri-implantitis. The study objective was to measure the association between peri-implantitis and the presence of non-allergy-related proinflammatory cytokines associated with TiO2 particles. METHODS: The investigators implemented a retrospective cross-sectional study and enrolled a sample of 60 subjects from a dental practice. Subjects were excluded if the plaque index was grade 3 (Silness and Löe). The predictor variable was a positive or negative TiO2 stimulation test, an in vitro macrophage proinflammatory response test. The outcome variable was peri-implantitis status defined as present or absent. Three groups were considered: control group with 20 patients without dental implants (group 1), 2 groups of patients with titanium dental implants, one without peri-implantitis (group 2), and the other with peri-implantitis (group 3) (n = 20 each). For patients with implants, depth of the gingival pockets of the implants were measured, and existing bleeding and suppuration were determined to assess peri-implantitis. Radiographs were taken if one or more factors applied to confirm the diagnosis of peri-implantitis. Further covariates were age, sex, duration of implant wear, and number of implants which were analyzed descriptively. Inferential analyses were undertaken using χ2 test, Kruskal-Wallis-, Wilcoxon-two-sample tests, and logistic regressions. RESULTS: The sample was composed of 35 female and 25 male patients with a mean age of 54.2 years (standard deviation = 14.76). The overall TiO2 stimulation test positivity frequency was 28.3% and were 30.0%, 5.0%, and 50.0% in the control, implants without peri-implantitis, and implants with peri-implantitis groups. No statistically significant differences could be seen in the frequencies of the TiO2 stimulation test results between control group and combined groups 2 and 3 (P-value = .84). The risk for positive TiO2 patients with a titanium implant of developing peri-implantitis was statistically significant and higher compared to negative TiO2 patients (odds ratio, 19.0 with 95% confidence interval [2.12,170.38]; P-value< .01). CONCLUSIONS: The data in this study showed a statistically significant relationship between a positive TiO2 stimulation test and peri-implantitis. Further studies with larger numbers of subjects are recommended to confirm this result.

Tissue Adhesives in Reconstructive and Aesthetic Surgery-Application of Silk Fibroin-Based Biomaterials.

Tissue adhesives have been successfully used in various kind of surgeries such as oral and maxillofacial surgery for some time. They serve as a substitute for suturing of tissues and shorten treatment time. Besides synthetic-based adhesives, a number of biological-based formulations are finding their way into research and clinical application. In natural adhesives, proteins play a crucial role, mediating adhesion and cohesion at the same time. Silk fibroin, as a natural biomaterial, represents an interesting alternative to conventional medical adhesives. Here, the most commonly used bioadhesives as well as the potential of silk fibroin as natural adhesives will be discussed.

Toward Tailoring the Degradation Rate of Magnesium-Based Biomaterials for Various Medical Applications: Assessing Corrosion, Cytocompatibility and Immunological Effects.

Magnesium (Mg)-based biomaterials hold considerable promise for applications in regenerative medicine. However, the degradation of Mg needs to be reduced to control toxicity caused by its rapid natural corrosion. In the process of developing new Mg alloys with various surface modifications, an efficient assessment of the relevant properties is essential. In the present study, a WE43 Mg alloy with a plasma electrolytic oxidation (PEO)-generated surface was investigated. Surface microstructure, hydrogen gas evolution in immersion tests and cytocompatibility were assessed. In addition, a novel in vitro immunological test using primary human lymphocytes was introduced. On PEO-treated WE43, a larger number of pores and microcracks, as well as increased roughness, were observed compared to untreated WE43. Hydrogen gas evolution after two weeks was reduced by 40.7% through PEO treatment, indicating a significantly reduced corrosion rate. In contrast to untreated WE43, PEO-treated WE43 exhibited excellent cytocompatibility. After incubation for three days, untreated WE43 killed over 90% of lymphocytes while more than 80% of the cells were still vital after incubation with the PEO-treated WE43. PEO-treated WE43 slightly stimulated the activation, proliferation and toxin (perforin and granzyme B) expression of CD8+ T cells. This study demonstrates that the combined assessment of corrosion, cytocompatibility and immunological effects on primary human lymphocytes provide a comprehensive and effective procedure for characterizing Mg variants with tailorable degradation and other features. PEO-treated WE43 is a promising candidate for further development as a degradable biomaterial.

Effects of a Novel Cold Atmospheric Plasma Treatment of Titanium on the Proliferation and Adhesion Behavior of Fibroblasts.

Cold plasma treatment increases the hydrophilicity of the surfaces of implants and may enhance their integration with the surrounding tissues. The implaPrep prototype device from Relyon Plasma generates cold atmospheric plasma via dielectric barrier discharge (DBD). In this study, titanium surfaces were treated with the implaPrep device for 20 s and assessed as a cell culture surface for fibroblasts. One day after seeding, significantly more cells were counted on the surfaces treated with cold plasma than on the untreated control titanium surface. Additionally, the viability assay revealed significantly higher viability on the treated surfaces. Morphological observation of the cells showed certain differences between the treated and untreated titanium surfaces. While conventional plasma devices require compressed gas, such as oxygen or argon, the implaPrep device uses atmospheric air as the gas source. It is, therefore, compact in size and simple to handle, and may provide a safe and convenient tool for treating the surfaces of dental implants, which may further improve the implantation outcome.

Influence of the Casting Concentration on the Mechanical and Optical Properties of FA/CaCl2-Derived Silk Fibroin Membranes.

In this study, we describe the manufacturing and characterization of silk fibroin membranes derived from the silkworm Bombyx mori. To date, the dissolution process used in this study has only been researched to a limited extent, although it entails various potential advantages, such as reduced expenses and the absence of toxic chemicals in comparison to other conventional techniques. Therefore, the aim of this study was to determine the influence of different fibroin concentrations on the process output and resulting membrane properties. Casted membranes were thus characterized with regard to their mechanical, structural and optical assets via tensile testing, SEM, light microscopy and spectrophotometry. Cytotoxicity was evaluated using BrdU, XTT, and LDH assays, followed by live-dead staining. The formic acid (FA) dissolution method was proven to be suitable for the manufacturing of transparent and mechanically stable membranes. The fibroin concentration affects both thickness and transparency of the membranes. The membranes did not exhibit any signs of cytotoxicity. When compared to other current scientific and technical benchmarks, the manufactured membranes displayed promising potential for various biomedical applications. Further research is nevertheless necessary to improve reproducible manufacturing, including a more uniform thickness, less impurity and physiological pH within the membranes.

Biodegradable Silk Fibroin Matrices for Wound Closure in a Human 3D Ex Vivo Approach.

In this study, the potential of silk fibroin biomaterials for enhancing wound healing is explored, focusing on their integration into a human 3D ex vivo wound model derived from abdominoplasties. For this purpose, cast silk fibroin membranes and electrospun nonwoven matrices from Bombyx mori silk cocoons were compared to untreated controls over 20 days. Keratinocyte behavior and wound healing were analyzed qualitatively and quantitatively by histomorphometric and immune histochemical methods (HE, Ki67, TUNEL). Findings reveal rapid keratinocyte proliferation on both silk fibroin membrane and nonwoven matrices, along with enhanced infiltration in the matrix, suggesting improved early wound closure. Silk fibroin membranes exhibited a significantly improved early regeneration, followed by nonwoven matrices (p < 0.05) compared to untreated wounds, resulting in the formation of multi-layered epidermal structures with complete regeneration. Overall, the materials demonstrated excellent biocompatibility, supporting cell activity with no signs of increased apoptosis or early degradation. These results underscore silk fibroin's potential in clinical wound care, particularly in tissue integration and re-epithelialization, offering valuable insights for advanced and-as a result of the electrospinning technique-individual wound care development. Furthermore, the use of an ex vivo wound model appears to be a viable option for pre-clinical testing.

Doping of casted silk fibroin membranes with extracellular vesicles for regenerative therapy: a proof of concept.

Bioactive material concepts for targeted therapy have been an important research focus in regenerative medicine for years. The aim of this study was to investigate a proof-of-concept composite structure in the form of a membrane made of natural silk fibroin (SF) and extracellular vesicles (EVs) from gingival fibroblasts. EVs have multiple abilities to act on their target cell and can thus play crucial roles in both physiology and regeneration. This study used pH neutral, degradable SF-based membranes, which have excellent cell- and tissue-specific properties, as the carrier material. The characterization of the vesicles showed a size range between 120 and 180 nm and a high expression of the usual EV markers (e.g. CD9, CD63 and CD81), measured by nanoparticle tracking analysis (NTA) and single-EV flow analysis (IFCM). An initial integration of the EVs into the membrane was analyzed using scanning and transmission electron microscopy (SEM and TEM) and vesicles were successfully detected, even if they were not homogeneously distributed in the membrane. Using direct and indirect tests, the cytocompatibility of the membranes with and without EVs could be proven and showed significant differences compared to the toxic control (p < 0.05). Additionally, proliferation of L929 cells was increased on membranes functionalized with EVs (p > 0.05).

Layer-by-Layer Deposition of Regenerated Silk Fibroin─An Approach to the Surface Coating of Biomedical Implant Materials.

Biomaterials and coating techniques unlock major benefits for advanced medical therapies. Here, we explored layer-by-layer (LbL) deposition of silk fibroin (SF) by dip coating to deploy homogeneous films on different materials (titanium, magnesium, and polymers) frequently used for orthopedic and other bone-related implants. Titanium and magnesium specimens underwent preceding plasma electrolytic oxidation (PEO) to increase hydrophilicity. This was determined as surface properties were visualized by scanning electron microscopy and contact angle measurements as well as Fourier transform infrared spectroscopy (FTIR) analysis. Finally, biological in vitro evaluations of hemocompatibility, THP-1 cell culture, and TNF-α assays were conducted. A more hydrophilic surface could be achieved using the PEO surface, and the contact angle for magnesium and titanium showed a reduction from 73 to 18° and from 58 to 17°, respectively. Coating with SF proved successful on all three surfaces, and coating thicknesses of up to 5.14 µm (±SD 0.22 µm) were achieved. Using FTIR analysis, it was shown that the insolubility of the material was achieved by post-treatment with water vapor annealing, although the random coil peak (1640-1649 cm-1) and the α-helix peak (at 1650 cm-1) were still evident. SF did not change hemocompatibility, regardless of the substrate, whereas the PEO-coated materials showed improved hemocompatibility. THP-1 cell culture showed that cells adhered excellently to all of the tested material surfaces. Interestingly, SF coatings induced a significantly higher amount of TNF-α for all materials, indicating an inflammatory response, which plays an important role in a variety of physiological processes, including osteogenesis. LbL coatings of SF are shown to be promising candidates to modulate the body's immune response to implants manufactured from titanium, magnesium, and polymers. They may therefore facilitate future applications for bioactive implant coatings. However, further in vivo studies are needed to confirm the proposed effects on osteogenesis in a physiological environment.

Characterization of Dental Pulp Stem Cell Populations in the Teeth of Patients With Neurofibromatosis Type 1 - Therapeutic Potential for Bone Tissue Engineering.

BACKGROUND/AIM: Neurofibromas (NF) are the most common benign nerve sheath tumors in the tongue, gingiva, major salivary glands, and jaw bones. Nowadays, tissue engineering is a revolutionary technique for reconstructing tissues. To explore the feasibility of using stem cells derived from NF teeth to treat orofacial bone defects, the differences in cell biological properties between an NF teeth group and Normal teeth group. PATIENTS AND METHODS: The intra-dental pulp tissues from each tooth were extracted. The cell survival rates, morphology, proliferation rates, cell activity, and differentiation abilities were contrastively analyzed between the NF teeth group and Normal teeth group. RESULTS: Between the two groups, there were no differences in the primary generation (P0) cells (p>0.05), the cell yield, and the time required for the cells to grow out of the pulp tissue and attach to the culture plate. Furthermore, no differences were found at the first generation (passage) between the two groups in colony formation rate and cell survival rate. The proliferation capacity, cell growth curve, and surface marker expression of dental pulp cells was not altered in the third generation (p>0.05). CONCLUSION: Dental pulp stem cells from NF teeth were successfully obtained and were not different from normal dental pulp stem cells. Although, clinical research using tissue-engineered bone to repair bone defects is still in its infancy, it will eventually enter the clinic and become a routine means of bone defect reconstruction treatment as related disciplines and technologies develop.

Tolerance of Human Fibroblasts to Benfo-Oxythiamine In Vitro.

OBJECTIVES: To explore the potential application of B-OT in the aspiration tract. MATERIALS AND METHODS: We conceived and optimized an in vitro model simulating the mouth-washing process to assess tolerance to B-OT on primary human gingival fibroblasts. Cells derived from 4 unrelated donors were flushed with medium containing drugs of various concentration for one minute twice daily for 3 days. RESULTS: No effect was seen on the cells up to 1000 µM B-OT. In addition, we treated the cells with B-OT permanently in medium, corresponding to a systemic treatment. No effect was seen by 10 µM B-OT and only a slight reduction (approximately 10%) was seen by 100 µM B-OT. CONCLUSIONS: Our results suggest good tolerance of oral cells for B-OT, favoring the further development of this antiviral reagent as a mouth-washing solution and nasal spray.

New and innovative biomaterials, techniques and therapy concepts: Biologization in maxillofacial surgery, oral surgery and dentistry is in full swing. PRF, PRGF, PRP, blood plasma-stabilized augmentations, supplementation of micronutrients and vitamins - what opportunities do such "biological" approaches actually offer? We introduce them here.

Biomaterials of natural origin have recently gained increasing attention in the field of dental implantology. The requirements for such materials, however, are very high. In addition to high clinical efficiency in tissue regeneration, wound healing should be demonstrably positively influenced. The translational division for regenerative orofacial medicine of the Clinic and Polyclinic for Oral and Maxillofacial Surgery of the University Medical Center Hamburg-Eppendorf (UKE) is examining this research topic by investigating which innovative treatment methods for the reconstruction of bone defects or for augmentative procedures can be applied in the future or are already being applied in the field of oral and maxillofacial surgery.

Antibacterial properties of functionalized silk fibroin and sericin membranes for wound healing applications in oral and maxillofacial surgery.

Oral wounds are among the most troublesome injuries which easily affect the patients' quality of life. To date, the development of functional antibacterial dressings for oral wound healing remains a challenge. In this regard, we investigated antibacterial silk protein-based membranes for the application as wound dressings in oral and maxillofacial surgery. The present study includes five variants of casted membranes, i.e., i) membranes-silver nanoparticles (CM-Ag), ii) membranes-gentamicin (CM-G), iii) membranes-control (without functionalization) (CM-C), iv) membranes-silk sericin control (CM-SSC), and v) membranes-silk fibroin/silk sericin (CM-SF/SS), and three variants of nonwovens, i.e., i) silver nanoparticles (NW-Ag), ii) gentamicin (NW-G), iii) control (without functionalization) (NW-C). The surface structure of the samples was visualized with scanning electron microscopy. In addition, antibacterial testing was accomplished using agar diffusion assay, colony forming unit (CFU) analysis, and qrt-PCR. Following antibacterial assays, biocompatibility was evaluated by cell proliferation assay (XTT), cytotoxicity assay (LDH), and live-dead assay on L929 mouse fibroblasts. Findings indicated significantly lower bacterial colony growth and DNA counts for CM-Ag with a reduction of bacterial counts by 3log levels (99.9% reduction) in CFU and qrt-PCR assay compared to untreated control membranes (CM-C and CM-SSC) and membranes functionalized with gentamicin (CM-G and NW-G) (p < 0.001). Similarly, NW-G yielded significantly lower DNA and colony growth counts compared to NW-Ag and NW-C (p < 0.001). In conclusion, CM-Ag represented 1log level better antibacterial activity compared to NW-G, whereas NW-G showed better cytocompatibility for L929 cells. As data suggest, these two membranes have the potential of application in the field of bacteria-free oral wound healing. However, provided that loading strategy and cytocompatibility are adjusted according to the antibacterial agents' characteristic and fabrication technique of the membranes.

Effect of process parameters on additive-free electrospinning of regenerated silk fibroin nonwovens.

Silk fibroin is a biomaterial with multiple beneficial properties for use in regenerative medicine and tissue engineering. When dissolving and processing the reconstituted silk fibroin solution by electrospinning, the arrangement and size of fibers can be manifold varied and according fiber diameters reduced to the nanometer range. Such nonwovens show high porosity as well as potential biocompatibility. Usually, electrospinning of most biomaterials demands for the application of additives, which enable stable electrospinning by adjusting viscosity, and are intended to evaporate during processing or to be washed out afterwards. However, the use of such additives increases costs and has to be taken into account in terms of biological risks when used for biomedical applications. In this study, we explored the possibilities of additive-free electrospinning of pure fibroin nonwovens and tried to optimize process parameters to enable stable processing. We used natural silk derived from the mulberry silkworm Bombyx mori. After degumming, the silk fibroin was dissolved and the viscosity of the spinning solution was controlled by partial evaporation of the initial solving agent. This way, we were able to completely avoid the use of additives and manufacture nonwovens, which potentially offer higher biocompatibility and reduced immunogenicity. Temperature and relative humidity during electrospinning were systematically varied (25-35 °C, 25-30% RH). In a second step, the nonwovens optionally underwent methanol treatment to initiate beta-sheet formation in order to increase structural integrity and strength. Comprehensive surface analysis on the different nonwovens was performed using scanning electron microscopy and supplemented by additional mechanical testing. Cytotoxicity was evaluated using BrdU-assay, XTT-assay, LDH-assay and live-dead staining. Our findings were, that an increase of temperature and relative humidity led to unequal fiber diameters and defective nonwovens. Resistance to penetration decreased accordingly. The most uniform fiber diameters of 998 ± 63 nm were obtained at 30 °C and 25% relative humidity, also showing the highest value for resistance to penetration (0.20 N). The according pure fibroin nonwoven also showed no signs of cytotoxicity. However, while the biological response showed statistical evidence, the material characteristics showed no statistically significant correlation to changes of the ambient conditions within the investigated ranges. We suggest that further experiments should explore additional ranges for temperature and humidity and further focus on the repeatability of material properties in dependency of suitable process windows.

Production and Characterization of Porous Fibroin Scaffolds for Regenerative Medical Application.

BACKGROUND/AIM: Silk is a natural biomaterial with several superior features for applications in regenerative medicine. In the present study an optimized process for manufacturing porous scaffolds out of the silk protein fibroin was developed. MATERIALS AND METHODS: The silk protein fibroin was dissolved in Ajisawa's reagent and the resulting fibroin solution was used to produce scaffolds by means of freeze-thawing cycling. Porosity, pressure and stab resistance as well as degradation behavior were assessed in order to characterize the physical properties of the resulting scaffolds. RESULTS: The resulting sponge-like fibroin scaffolds were highly porous while the porosity correlated inversely with the concentration of the starting fibroin solution. Increased initial fibroin concentrations of the scaffolds resulted in increased compressive and cannulation resistance. The majority of the fibroin scaffolds were digested by 1 mg/ml protease XIV in 3 weeks, indicating their biodegradability. CONCLUSION: The production of scaffolds made of varying fibroin concentrations by means of freeze-thawing, following dissolution using Ajisawa's reagent, provides a simple and straightforward strategy for adjusting the physical and chemical properties of fibroin scaffolds for various medical applications.