Development of clobetasol-loaded biodegradable nanoparticles as an endodontic intracanal medicament.

AIM: The aim of current study is the development and optimization of biodegradable polymeric nanoparticles (NPs) to be used in the field of Endodontics as intracanal medication in cases of avulsed teeth with extended extra-oral time, utilizing PLGA polymers loaded with the anti-inflammatory drug clobetasol propionate (CP). METHODOLOGY: CP-loaded nanoparticles (CP-NPs) were prepared using the solvent displacement method. CP release profile from CP-NPs was assessed for 48 h against free CP. Using extracted human teeth, the degree of infiltration inside the dentinal tubules was studied for both CP-NPs and CP. The anti-inflammatory capacity of CP-NPs was evaluated in vitro measuring their response and reaction against inflammatory cells, in particular against macrophages. The enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokine release of IL-1ß and TNF-α. RESULTS: Optimized CP-NPs displayed an average size below 200 nm and a monomodal population. Additionally, spherical morphology and non-aggregation of CP-NPs were confirmed by transmission electron microscopy. Interaction studies showed that CP was encapsulated inside the NPs and no covalent bonds were formed. Moreover, CP-NPs exhibited a prolonged and steady release with only 21% of the encapsulated CP released after 48 h. Using confocal laser scanning microscopy, it was observed that CP-NPs were able to display enhanced penetration into the dentinal tubules. Neither the release of TNF-α nor IL-1ß increased in CP-NPs compared to the LPS control, displaying results similar and even less than the TCP after 48 h. Moreover, IL-1ß release in LPS-stimulated cells, decreased when macrophages were treated with CP-NPs. CONCLUSIONS: In the present work, CP-NPs were prepared, optimized and characterized displaying significant increase in the degree of infiltration inside the dentinal tubules against CP and were able to significantly reduce TNF-α release. Therefore, CP-NPs constitute a promising therapy for the treatment of avulsed teeth with extended extra-oral time.

Biofabrication of Collagen Tissue-Engineered Blood Vessels with Direct Co-Axial Extrusion.

Cardiovascular diseases are considered one of the worldwide causes of death, with atherosclerosis being the most predominant. Nowadays, the gold standard treatment is blood vessel replacement by bypass surgery; however, autologous source is not always possible. Thereby, tissue-engineered blood vessels (TEBVs) are emerging as a potential alternative source. In terms of composition, collagen has been selected in many occasions to develop TEBVs as it is one of the main extracellular matrix components of arteries. However, it requires specific support or additional processing to maintain the tubular structure and appropriate mechanical properties. Here, we present a method to develop support-free collagen TEBVs with co-axial extrusion in a one-step procedure with high concentrated collagen. The highest concentration of collagen of 20 mg/mL presented a burst pressure of 619.55 ± 48.77 mmHg, being able to withstand perfusion of 10 dynes/cm2. Viability results showed a high percentage of viability (86.1 and 85.8% with 10 and 20 mg/mL, respectively) of human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVEC) after 24 h extrusion. Additionally, HUVEC and HASMCs were mainly localized in their respective layers, mimicking the native distribution. All in all, this approach allows the direct extrusion of collagen TEBVs in a one-step procedure with enough mechanical properties to be perfused.

In vitro assessment of PEEK and titanium implant abutments: Screw loosening and microleakage evaluations under dynamic mechanical testing.

STATEMENT OF PROBLEM: Polyetheretherketone (PEEK) has been advocated to replace metal components in dentistry. Although PEEK is a high-performance polymer with a white color, adequate biological response, and resistance to fracture, data to support PEEK as an alternative material for implant abutments are lacking. PURPOSE: The purpose of this in vitro study was to assess the mechanical and functional properties of PEEK implant abutments as a nonmetallic alternative to titanium abutments, which presents esthetic limitations and greater difficulty to customize clinically. MATERIAL AND METHODS: Implant abutments manufactured by using PEEK (n=24) or titanium grade 5 (n=24) were attached to MIS Implants type M4 3.75×16 mm with an internal screw tightened to 25 Ncm. Screw loosening and microleakage was assessed by submersion in a 2% methylene blue solution for 48 hours at 37 °C. Both groups were compared with and without applying dynamic loading; fatigue testing was performed following the International Organization for Standardization (ISO) 14801:2016 standard. All specimens were observed under a stereomicroscope at ×8 magnification. Statistically significant differences among the PEEK and titanium implant abutments were tested with 2-factor ANOVA and the chi-square analysis for nonpaired and paired data, respectively (α=.05). RESULTS: The implant abutments made of titanium were better in all mechanical tests. The torque loss of titanium abutments was approximately 10%, while PEEK showed a significantly higher (P<.05) torque loss of up to 50%. Moreover, 91.6% of the titanium abutments did not present microleakage, while there was no specimen of PEEK abutments without microleakage, once subjected to dynamic loading (P<.05). CONCLUSIONS: Titanium implant abutments (Ti6Al4V) were better in all tests performed. However, PEEK abutments may be suitable for long-term interim restorations, especially in the anterior area, in patients without parafunction.

In vitro evaluation of a multispecies oral biofilm over antibacterial coated titanium surfaces.

Peri-implantitis is an infectious disease that affects the supporting soft and hard tissues around dental implants and its prevalence is increasing considerably. The development of antibacterial strategies, such as titanium antibacterial-coated surfaces, may be a promising strategy to prevent the onset and progression of peri-implantitis. The aim of this study was to quantify the biofilm adhesion and bacterial cell viability over titanium disc with or without antibacterial surface treatment. Five bacterial strains were used to develop a multispecies oral biofilm. The selected species represent initial (Streptococcus oralis and Actinomyces viscosus), early (Veillonella parvula), secondary (Fusobacterium nucleatum) and late (Porphyromonas gingivalis) colonizers. Bacteria were sequentially inoculated over seven different types of titanium surfaces, combining different roughness level and antibacterial coatings: silver nanoparticles and TESPSA silanization. Biofilm formation, cellular viability and bacterial quantification over each surface were analyzed using scanning electron microscopy, confocal microscopy and real time PCR. Biofilm formation over titanium surfaces with different bacterial morphologies could be observed. TESPSA was able to significantly reduce the cellular viability when compared to all the surfaces (p < 0.05). Silver deposition on titanium surface did not show improved results in terms of biofilm adhesion and cellular viability when compared to its corresponding non-coated surface. The total amount of bacterial biofilm did not significantly differ between groups (p > 0.05). TESPSA was able to reduce biofilm adhesion and cellular viability. However, silver deposition on titanium surface seemed not to confer these antibacterial properties.

Silver deposition on titanium surface by electrochemical anodizing process reduces bacterial adhesion of Streptococcus sanguinis and Lactobacillus salivarius.

OBJECTIVES: The aim of this study was to determine the antibacterial properties of silver-doped titanium surfaces prepared with a novel electrochemical anodizing process. MATERIAL AND METHODS: Titanium samples were anodized with a pulsed process in a solution of silver nitrate and sodium thiosulphate at room temperature with stirring. Samples were processed with different electrolyte concentrations and treatment cycles to improve silver deposition. Physicochemical properties were determined by X-ray photoelectron spectroscopy, contact angle measurements, white-light interferometry, and scanning electron microscopy. Cellular cytotoxicity in human fibroblasts was studied with lactate dehydrogenase assays. The in vitro effect of treated surfaces on two oral bacteria strains (Streptococcus sanguinis and Lactobacillus salivarius) was studied with viable bacterial adhesion measurements and growth curve assays. Nonparametric statistical Kruskal-Wallis and Mann-Whitney U-tests were used for multiple and paired comparisons, respectively. Post hoc Spearman's correlation tests were calculated to check the dependence between bacteria adhesion and surface properties. RESULTS: X-ray photoelectron spectroscopy results confirmed the presence of silver on treated samples and showed that treatments with higher silver nitrate concentration and more cycles increased the silver deposition on titanium surface. No negative effects in fibroblast cell viability were detected and a significant reduction on bacterial adhesion in vitro was achieved in silver-treated samples compared with control titanium. CONCLUSIONS: Silver deposition on titanium with a novel electrochemical anodizing process produced surfaces with significant antibacterial properties in vitro without negative effects on cell viability.

Engineering a microparticle-loaded rough membrane for guided bone regeneration modulating osteoblast response without inducing inflammation.

Guided bone regeneration (GBR) is a widely used procedure that prevents the fast in-growth of soft tissues into bone defect. Among the different types of membranes, the use of collagen membranes is the gold standard. However, these membranes are implanted in tissue location where a severe acute inflammation will occur and can be negatively affected. The aim of this study was to develop a collagen-based membrane for GBR that incorporated alginate-hydroxyapatite microparticles. Membranes were manufactured using collagen type I and gelatin and alginate-hydroxyapatite microparticles. Membranes were assessed in terms of topography by scanning electron microscopy and confocal microscopy; stability by swelling after an overnight incubation in saline and enzymatic degradation against collagenase and mechanical properties by tensile tests. Furthermore, the biological response was assessed with SaOs-2 cells and THP-1 macrophages to determine alkaline phosphatase activity and inflammatory cytokine release. Our results showed that the incorporation of different percentages of these microparticles could induce changes in the surface topography. When the biological response was analyzed, either membranes were not cytotoxic to THP-1 macrophages or to SaOs-2 cells and they did not induce the release of pro-inflammatory cytokines. However, the different surface topographies did not induce changes in the macrophage morphology and the release of pro- and anti-inflammatory cytokines, suggesting that the effect of surface roughness on macrophage behavior could be dependent on other factors such as substrate stiffness and composition. Collagen-gelatin membranes with embedded alginate-hydroxyapatite microparticles increased ALP activity, suggesting a positive effect of them on bone regeneration, remaining unaffected the release of pro- and anti-inflammatory cytokines.

Silver coating on dental implant-abutment connection screws as potential strategy to prevent loosening and minimizing bacteria adhesion.

Introduction: One of the main problems for the long-term behavior of dental implants are loosening of the implant-abutment connection screws and bacterial infiltration. The aim of this work is to increase the screw fixation by silver coating, providing superior mechanical retaining and antibacterial effect. Methods: Eighty dental implants with their abutments and screws have been studied. Twenty screws were not coated and were used as a control while the rest of screws were silver coated by sputtering, with three different thickness: 10, 20 and 40 µm and 20 screws per each thickness. Coating morphology and thickness were determined by scanning electron microscopy using image analysis systems. The screws were tightened for each of the thicknesses and the control with two torques 15 Ncm and 20 Ncm and tested under mechanical fatigue simulating oral stresses up to a maximum of 500,000 cycles. The remaining torques at different cycles were determined with a high-sensitivity torquemeter. Cell viability assays were performed with SaOs-2 osteoblasts and microbiological studies were performed against Streptococcus gordonii and Enterococcus faecalis bacteria strains, determining their metabolic activity and viability using live/dead staining. Results: It was observed a decrease in torque as cycles increase. For a preload of 15 Ncm at 100,000 cycles, the loosening was complete and, for 20 Ncm at 500,000 cycles, 85% of torque was lost. The silver coatings retained the torque, especially the one with a thickness of 40 µm, retaining 90% of the initial torque at 500,000 cycles. It was observed that osteoblastic viability values did not reach 70%, which could indicate a slight cytotoxic effect in contact with cells or tissues; however, the screw should not be in direct contact with tissue or living cells. Silver coating induced a significant reduction of the bacteria metabolic activity for Streptococcus gordonii and Enterococcus faecalis, around 90% and 85% respectively. Discussion: Therefore, this coating may be of interest to prevent loosening of implant systems with a worthy antibacterial response.

Modulation of Macrophage Response by Copper and Magnesium Ions in Combination with Low Concentrations of Dexamethasone.

Macrophages have been deemed crucial for correct tissue regeneration, which is a complex process with multiple overlapping phases, including inflammation. Previous studies have suggested that divalent ions are promising cues that can induce an anti-inflammatory response, since they are stable cues that can be released from biomaterials. However, their immunomodulatory potential is limited in a pro-inflammatory environment. Therefore, we investigated whether copper and magnesium ions combined with low concentrations of the anti-inflammatory drug, dexamethasone (dex), could have a synergistic effect in macrophage, with or without pro-inflammatory stimulus, in terms of morphology, metabolic activity and gene expression. Our results showed that the combination of copper and dex strongly decreased the expression of pro-inflammatory markers, while the combination with magnesium upregulated the expression of IL-10. Moreover, in the presence of a pro-inflammatory stimulus, the combination of copper and dex induced a strong TNF-α response, suggesting an impairment of the anti-inflammatory actions of dex. The combination of magnesium and dex in the presence of a pro-inflammatory stimulus did not promote any improvement in comparison to dex alone. The results obtained in this study could be relevant for tissue engineering applications and in the design of platforms with a dual release of divalent ions and small molecules.

Evaluation of the immunomodulatory effects of cobalt, copper and magnesium ions in a pro inflammatory environment.

Biomaterials and scaffolds for Tissue Engineering are widely used for an effective healing and regeneration. However, the implantation of these scaffolds causes an innate immune response in which the macrophage polarization from M1 (pro-inflammatory) to M2 (anti-inflammatory) phenotype is crucial to avoid chronic inflammation. Recent studies have showed that the use of bioactive ions such as cobalt (Co2+), copper (Cu2+) and magnesium (Mg2+) could improve tissue regeneration, although there is limited evidence on their effect on the macrophage response. Therefore, we investigated the immunomodulatory potential of Co2+, Cu2+ and Mg2+ in macrophage polarization. Our results indicate that Mg2+ and concentrations of Cu2+ lower than 10 µM promoted the expression of M2 related genes. However, higher concentrations of Cu2+ and Co2+ (100 µM) stimulated pro-inflammatory marker expression, indicating a concentration dependent effect of these ions. Furthermore, Mg2+ were able to decrease M1 marker expression in presence of a mild pro-inflammatory stimulus, showing that Mg2+ can be used to modulate the inflammatory response, even though their application can be limited in a strong pro-inflammatory environment.

Antibacterial approaches in tissue engineering using metal ions and nanoparticles: From mechanisms to applications.

Bacterial infection of implanted scaffolds may have fatal consequences and, in combination with the emergence of multidrug bacterial resistance, the development of advanced antibacterial biomaterials and constructs is of great interest. Since decades ago, metals and their ions had been used to minimize bacterial infection risk and, more recently, metal-based nanomaterials, with improved antimicrobial properties, have been advocated as a novel and tunable alternative. A comprehensive review is provided on how metal ions and ion nanoparticles have the potential to decrease or eliminate unwanted bacteria. Antibacterial mechanisms such as oxidative stress induction, ion release and disruption of biomolecules are currently well accepted. However, the exact antimicrobial mechanisms of the discussed metal compounds remain poorly understood. The combination of different metal ions and surface decorations of nanoparticles will lead to synergistic effects and improved microbial killing, and allow to mitigate potential side effects to the host. Starting with a general overview of antibacterial mechanisms, we subsequently focus on specific metal ions such as silver, zinc, copper, iron and gold, and outline their distinct modes of action. Finally, we discuss the use of these metal ions and nanoparticles in tissue engineering to prevent implant failure.

Direct extrusion of individually encapsulated endothelial and smooth muscle cells mimicking blood vessel structures and vascular native cell alignment.

Cardiovascular diseases (CVDs) are considered the principal cause of worldwide death, being atherosclerosis the main etiology. Up to now, the predominant treatment for CVDs has been bypass surgery from autologous source. However, due to previous harvest or the type of disease, this is not always an option. For this reason, tissue engineered blood vessels (TEBV) emerged as an alternative graft source for blood vessel replacement. In order to develop a TEBV, it should mimic the architecture of a native blood vessel encapsulating the specific vascular cells in their respective layers with native alignment, and with appropriate mechanical stability. Here, we propose the extrusion of two different cell encapsulating hydrogels, mainly alginate and collagen, and a sacrificial polymer, through a triple coaxial nozzle, which in contact with a crosslinking solution allows the formation of bilayered hollow fibers, mimicking the architecture of native blood vessels. Prior to extrusion, the innermost cell encapsulating hydrogel was loaded with human umbilical vein endothelial cells (HUVECs), whereas the outer hydrogel was loaded with human aortic smooth muscle cells (HASMCs). The size of the TEVB could be controlled by changing the injection speed, presenting homogeneity between the constructs. The obtained structures were robust, allowing its manipulation as well as the perfusion of liquids. Both cell types presented high rates of survival after the extrusion process as well as after 20 d in culture (over 90%). Additionally, a high percentage of HASMC and HUVEC were aligned perpendicular and parallel to the TEBV, respectively, in their own layers, resembling the physiological arrangement foundin vivo. Our approach enables the rapid formation of TEBV-like structures presenting high cell viability and allowing proliferation and natural alignment of vascular cells.

Biological Roles and Delivery Strategies for Ions to Promote Osteogenic Induction.

Bone is the most studied tissue in the field of tissue regeneration. Even though it has intrinsic capability to regenerate upon injury, several pathologies and injuries could hamper the highly orchestrated bone formation and resorption process. Bone tissue engineering seeks to mimic the extracellular matrix of the tissue and the different biochemical pathways that lead to successful regeneration. For many years, the use of extrinsic factors (i.e., growth factors and drugs) to modulate these biological processes have been the preferred choice in the field. Even though it has been successful in some instances, this approach presents several drawbacks, such as safety-concerns, short release profile and half-time life of the compounds. On the other hand, the use of inorganic ions has attracted significant attention due to their therapeutic effects, stability and lower biological risks. Biomaterials play a key role in such strategies where they serve as a substrate for the incorporation and release of the ions. In this review, the methodologies used to incorporate ions in biomaterials is presented, highlighting the osteogenic properties of such ions and the roles of biomaterials in controlling their release.

Development macro-porous electro-spun meshes with clinically relevant mechanical properties-a technical note.

The nano-fibrous architecture of electro-spun meshes favours their use in biomedicine, but their low mechanical properties prohibit their wide use in clinical practice. Introduction of porosity, essential of tissue integration, decreases further mechanical integrity. Herein, we hypothesised that macro-porous electro-spun meshes with adequate mechanical properties can be fabricated through layering and subsequent compression. Two and three layers electro-spun poly-ε-caprolactone scaffolds were fabricated, compressed and subsequently 30% circular porosity was introduced through laser cutting. Three-layered porous electro-spun meshes exhibited mechanical properties similar to commercially available scaffolds without any structural or cytotoxic effect. This study brings electro-spun materials closer to clinical translation and commercialisation.

In vitro and preclinical characterisation of compressed, macro-porous and collagen coated poly-ε-caprolactone electro-spun scaffolds.

Low in macro-porosity electro-spun scaffolds are often associated with foreign body response, whilst macro-porous electro-spun scaffolds have low mechanical integrity. Herein, compressed, macro-porous and collagen (bovine Achilles tendon and human recombinant) coated electro-spun poly-ε-caprolactone scaffolds were developed and their biomechanical, in vitro and in vivo properties were assessed. Collagen coating, independently of the source, did not significantly affect the biomechanical properties of the scaffolds. Although no significant difference in cell viability was observed between the groups, collagen coated scaffolds induced significantly higher DNA concentration. In vivo, no signs of adverse tissue effect were observed in any of the groups and all groups appeared to equally integrate into the subcutaneous tissue. It is evidenced that macro-porous poly-ε-caprolactone electro-spun meshes with adequate mechanical properties and acceptable host response can be developed for biomedical applications.

The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development.

Collagen is the oldest and most abundant extracellular matrix protein that has found many applications in food, cosmetic, pharmaceutical, and biomedical industries. First, an overview of the family of collagens and their respective structures, conformation, and biosynthesis is provided. The advances and shortfalls of various collagen preparations (e.g., mammalian/marine extracted collagen, cell-produced collagens, recombinant collagens, and collagen-like peptides) and crosslinking technologies (e.g., chemical, physical, and biological) are then critically discussed. Subsequently, an array of structural, thermal, mechanical, biochemical, and biological assays is examined, which are developed to analyze and characterize collagenous structures. Lastly, a comprehensive review is provided on how advances in engineering, chemistry, and biology have enabled the development of bioactive, 3D structures (e.g., tissue grafts, biomaterials, cell-assembled tissue equivalents) that closely imitate native supramolecular assemblies and have the capacity to deliver in a localized and sustained manner viable cell populations and/or bioactive/therapeutic molecules. Clearly, collagens have a long history in both evolution and biotechnology and continue to offer both challenges and exciting opportunities in regenerative medicine as nature's biomaterial of choice.

Influence of Cross-Linking Method and Disinfection/Sterilization Treatment on the Structural, Biophysical, Biochemical, and Biological Properties of Collagen-Based Devices.

Disinfection/sterilization is an essential step during the manufacturing process of any implantable medical device. Cross-linking is also required for biopolymers to control resistance to degradation and enhance mechanical integrity. To date, there is still no single disinfection/sterilization treatment and cross-linking method that can be used universally for collagen-based devices. Herein, we assessed the influence of ethylene oxide, ethanol, gamma irradiation, and gas plasma disinfection/sterilization on the structural, biophysical, biochemical, and biological properties of self-assembled collagen films cross-linked with 4-arms polyethylene glycol succinimidyl glutarate and genipin. Microscopy analysis revealed that gas plasma treatment induced the most profound differences in the non-cross-linked and 4-arms polyethylene glycol succinimidyl glutarate cross-linked collagen films. Gas plasma also significantly increased the swelling ratio of the non-cross-linked and the 4-arms polyethylene glycol succinimidyl glutarate cross-linked films. Non-cross-linked and gas plasma treated 4-arms polyethylene glycol succinimidyl glutarate collagen films exhibited the lowest resistance to collagenase degradation and denaturation temperature. Between the non-cross-linked groups, the gas plasma treatment resulted in the collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Within the 4-arms polyethylene glycol succinimidyl glutarate groups, the ethylene oxide treatment resulted in collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Within the genipin groups, the gas plasma treatment resulted in collagen films with the lowest stress at break, strain at break, force at break, and Young's modulus values. Proliferation, metabolic activity, and viability of human skin fibroblasts were not affected as a function of cross-linking method and disinfection/sterilization treatment. However, proliferation, metabolic activity, and viability of THP1 cells were significantly reduced as a function of the cross-linking method, but they were not affected as a function of the disinfection/sterilization treatment. Overall, our data illustrate that the cross-linking method and the disinfection/sterilization treatment differentially affect the structural, biophysical, biochemical, and biological properties of collagen-based devices, and thus, they should be optimized according to the clinical indication.

Double acid etching treatment of dental implants for enhanced biological properties.

BACKGROUND: The topographical features on the surface of dental implants have been considered as a critical parameter for enhancing the osseointegration of implants. In this work, we proposed a surface obtained by a combination of shot blasting and double acid etching. The double acid etching was hypothesized to increase the submicron topography and hence further stimulate the biological properties of the titanium implant. METHODS: The topographical features (surface roughness and real surface area), wettability and surface chemical composition were analyzed. RESULTS: The results showed that the proposed method produced a dual roughness, mainly composed of randomly distributed peaks and valleys with a superimposed nanoroughness, and hence with an increased specific surface area. Despite the fact that the proposed method does not introduce significant chemical changes, this treatment combination slightly increased the amount of titanium available on the surface, reducing potential surface contaminants. Furthermore, the surface showed increased contact angle values demonstrating an enhanced hydrophobicity on the surface. The biological behavior of the implants was then assessed by culturing osteoblast-like cells on the surface, showing enhanced osteoblast adhesion, proliferation and differentiation on the novel surface. CONCLUSIONS: Based on these results, the described surface with dual roughness obtained by double acid etching may be a novel route to obtain key features on the surface to enhance the osseointegration of the implant. Our approach is a simple method to obtain a dual roughness that mimics the bone structure modified by osteoclasts and increases surface area, which enhances osseointegration of dental implants.

* Collagen Cross-Linking: Biophysical, Biochemical, and Biological Response Analysis.

Extracted forms of collagen are subjected to chemical cross-linking to enhance their stability. However, traditional cross-linking approaches are associated with toxicity and inflammation. This work investigates the stabilization capacity, cytotoxicity and inflammatory response of collagen scaffolds cross-linked with glutaraldehyde (GTA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, 4-arm polyethylene glycol (PEG) succinimidyl glutarate (4SP), genipin (GEN), and oleuropein. Although all cross-linking methods reduced free amine groups, variable data were obtained with respect to denaturation temperature, resistance to collagenase digestion, and mechanical properties. With respect to biological analysis, fibroblast cultures showed no significant difference between the treatments. Although direct cultures with human-derived leukemic monocyte cells (THP-1) clearly demonstrated the cytotoxic effect of GTA, THP-1 cultures supplemented with conditioned medium from the various groups showed no significant difference between the treatments. With respect to cytokine profile, no significant difference in secretion of proinflammatory (e.g., interleukin [IL]-1ß, IL-8, tumor necrosis factor-α) and anti-inflammatory (e.g., vascular endothelial growth factor) cytokines was observed between the noncross-linked and the 4SP and GEN cross-linked groups, suggesting the suitability of these agents as collagen cross-linkers.

Acetic acid and pepsin result in high yield, high purity and low macrophage response collagen for biomedical applications.

Collagen based devices are frequently associated with foreign body response. Although several pre- (e.g. species, state of animal, tissue) and post- (e.g. cross-linking, scaffold architecture) extraction method factors have a profound effect on foreign body response, little is known about which and how during the extraction process factors mediate foreign body response. In this study, we assessed the influence of acetic acid and hydrochloric acid and the utilisation or not of pepsin or salt precipitation during collagen extraction on the yield, purity, free amines, denaturation temperature, resistance to collagenase degradation and macrophage response. Acetic acid/pepsin extracted collagen exhibited the highest yield, purity and free amine content and the lowest denaturation temperature. No differences in resistance to collagenase digestion were detected between the groups. Although all treatments exhibited similar macrophage morphology comprised of round cells (M1 phenotype), elongated cells (M2 phenotype) and cell aggregates (foreign body response), significantly more elongated cells were observed on HC films. Although no differences in metabolic activity were observed between the groups, the DNA concentration was significantly lower for the hydrochloric acid treatments. Further, cytokine analysis revealed that hydrochloric acid treatments induced significantly higher IL-1ß and TNF-α release with respect to acetic acid treatments. Salt precipitation did not influence the parameters assessed. Collectively, these data suggest that during the collagen extraction process variables should also be monitored as, evidently, they affect the physicochemical and biological properties of collagen preparations.

Influence of porosity and pore shape on structural, mechanical and biological properties of poly ϵ-caprolactone electro-spun fibrous scaffolds.

BACKGROUND: Electro-spun scaffolds are utilized in a diverse spectrum of clinical targets, with an ever-increasing quantity of work progressing to clinical studies and commercialization. The limited number of conformations in which the scaffolds can be fabricated hampers their wide acceptance in clinical practice. MATERIALS & METHODS: Herein, we assessed a single-strep fabrication process for predesigned electro-spun scaffold preparation and the ramifications of the introduction of porosity (0, 30, 50, 70%) and pore shape (circle, rhomboid, square) on structural, mechanical (tensile and ball burst) and biological (dermal fibroblast and THP-1) properties. RESULTS: The collector design did not affect the fibrous nature of the scaffold. Modulation of the porosity and pore shape offered control over the mechanical properties of the scaffolds. Neither the porosity nor the pore shape affected cellular (dermal fibroblast and THP-1) response. CONCLUSION: Overall, herein we provide evidence that electro-spun scaffolds of controlled architecture can be fabricated with fibrous fidelity, adequate mechanical properties and acceptable cytocompatibility for a diverse range of clinical targets.