Exosome Structures Supported by Machine Learning Can Be Used as a Promising Diagnostic Tool.

Principal component analysis (PCA) as a machine-learning technique could serve in disease diagnosis and prognosis by evaluating the dynamic morphological features of exosomes via Cryo-TEM-imaging. This hypothesis was investigated after the crude isolation of similarly featured exosomes derived from the extracellular vehicles (EVs) of immature dendritic cells (IDCs) JAWSII. It is possible to identify functional molecular groups by FTIR, but the unique physical and morphological characteristics of exosomes can only be revealed by specialized imaging techniques such as cryo-TEM. On the other hand, PCA has the ability to examine the morphological features of each of these IDC-derived exosomes by considering software parameters such as various membrane projections and differences in Gaussians, Hessian, hue, and class to assess the 3D orientation, shape, size, and brightness of the isolated IDC-derived exosome structures. In addition, Brownian motions from nanoparticle tracking analysis of EV IDC-derived exosomes were also compared with EV IDC-derived exosome images collected by scanning electron microscopy and confocal microscopy. Sodium-Dodecyl-Sulphate-Polyacrylamide-Gel-Electrophoresis (SDS-PAGE) was performed to separate the protein content of the crude isolates showing that no considerable protein contamination occurred during the crude isolation technique of IDC-derived-exosomes. This is an important finding because no additional purification of these exosomes is required, making PCA analysis both valuable and novel.

In vitro testing and efficacy of poly-lactic acid coating incorporating antibiotic loaded coralline bioceramic on Ti6Al4V implant against Staphylococcus aureus.

Biofilm formation on an implant surface is most commonly caused by the human pathogenic bacteria Staphylococcus aureus, which can lead to implant related infections and failure. It is a major problem for both implantable orthopedic and maxillofacial devices. The current antibiotic treatments are typically delivered orally or in an injectable form. They are not highly effective in preventing or removing biofilms, and they increase the risk of antibiotic resistance of bacteria and have a dose-dependent negative biological effect on human cells. Our aim was to improve current treatments via a localized and controlled antibiotic delivery-based implant coating system to deliver the antibiotic, gentamicin (Gm). The coating contains coral skeleton derived hydroxyapatite powders (HAp) that act as antibiotic carrier particles and have a biodegradable poly-lactic acid (PLA) thin film matrix. The system is designed to prevent implant related infections while avoiding the deleterious effects of high concentration antibiotics in implants on local cells including primary human adipose derived stem cells (ADSCs). Testing undertaken in this study measured the rate of S. aureus biofilm formation and determined the growth rate and proliferation of ADSCs. After 24 h, S. aureus biofilm formation and the percentage of live cells found on the surfaces of all 5%-30% (w/w) PLA-Gm-(HAp-Gm) coated Ti6Al4V implants was lower than the control samples. Furthermore, Ti6Al4V implants coated with up to 10% (w/w) PLA-Gm-(HAp-Gm) did not have noticeable Gm related adverse effect on ADSCs, as assessed by morphological and surface attachment analyses. These results support the use and application of the antibacterial PLA-Gm-(HAp-Gm) thin film coating design for implants, as an antibiotic release control mechanism to prevent implant-related infections.

Bio-Activation of HA/ß-TCP Porous Scaffolds by High-Pressure CO2 Surface Remodeling: A Novel "Coating-from" Approach.

Biphasic macroporous Hydroxyapatite/ß-Tricalcium Phosphate (HA/ß-TCP) scaffolds (BCPs) are widely used for bone repair. However, the high-temperature HA and ß-TCP phases exhibit limited bioactivity (low solubility of HA, restricted surface area, low ion release). Strategies were developed to coat such BCPs with biomimetic apatite to enhance bioactivity. However, this can be associated with poor adhesion, and metastable solutions may prove difficult to handle at the industrial scale. Alternative strategies are thus desirable to generate a highly bioactive surface on commercial BCPs. In this work, we developed an innovative "coating from" approach for BCP surface remodeling via hydrothermal treatment under supercritical CO2, used as a reversible pH modifier and with industrial scalability. Based on a set of complementary tools including FEG-SEM, solid state NMR and ion exchange tests, we demonstrate the remodeling of macroporous BCP surface with the occurrence of dissolution-reprecipitation phenomena involving biomimetic CaP phases. The newly precipitated compounds are identified as bone-like nanocrystalline apatite and octacalcium phosphate (OCP), both known for their high bioactivity character, favoring bone healing. We also explored the effects of key process parameters, and showed the possibility to dope the remodeled BCPs with antibacterial Cu2+ ions to convey additional functionality to the scaffolds, which was confirmed by in vitro tests. This new process could enhance the bioactivity of commercial BCP scaffolds via a simple and biocompatible approach.

Synthetic tissue engineering with smart, cytomimetic protocells.

Synthetic protocells are rudimentary origin-of-life versions of natural cell counterparts. Protocells are widely engineered to advance efforts and useful accepted outcomes in synthetic biology, soft matter chemistry and bioinspired materials chemistry. Protocells in collective symbiosis generate synthetic proto-tissues that display unprecedented autonomy and yield advanced materials with desirable life-like features for smart multi-drug delivery, micro bioreactors, renewable fuel production, environmental clean-up, and medicine. Current levels of protocell and proto-tissue functionality and adaptivity are just sufficient to apply them in tissue engineering and regenerative medicine, where they animate biomaterials and increase therapeutic cell productivity. As of now, structural biomaterials for tissue engineering lack the properties of living biomaterials such as self-repair, stochasticity, cell synergy and the sequencing of molecular and cellular events. Future protocell-based biomaterials provide these core properties of living organisms, but excluding evolution. Most importantly, protocells are programmable for a broad array of cell functions and behaviors and collectively in consortia are tunable for multivariate functions. Inspired by upcoming designs of smart protocells, we review their developmental background and cover the most recently reported developments in this promising field of synthetic proto-biology. Our emphasis is on manufacturing proto-tissues for tissue engineering of organoids, stem cell niches and reprogramming and tissue formation through stages of embryonic development. We also highlight the exciting reported developments arising from fusing living cells and tissues, in a valuable hybrid symbiosis, with synthetic counterparts to bring about novel functions, and living tissue products for a new synthetic tissue engineering discipline.

Comparative Analysis of NF-κB in the MyD88-Mediated Pathway After Implantation of Titanium Alloy and Stainless Steel and the Role of Regulatory T Cells.

OBJECTIVE: Development of immunologically smart implants, integrated to biological systems, is a key aim to minimize the inflammatory response of the host to biomaterial implants. METHODS: The aim of this study is to investigate the influence of titanium alloy and stainless steel implants on immunological responses in rats by comparative analysis of nuclear factor kappa B (NF-κB) profiles in the activation of inflammatory signaling pathways and the role of CD4+CD25+Foxp3+. RESULTS: Both Ti alloy and stainless steel alloy group implantation affect Toll-like receptors-4 pathways and CD4+CD25+ regulatory T cells in different ways. CONCLUSIONS: Results show that NF-κB/p65 and NF-κB1/p50 possess potential as a therapeutic target in the prevention of adverse reactions to metal, especially for controlling inflammation after the implantation.

Specifiable biomimetic microsponges for timed release of crystal entrapped biomolecules useful in bone repair.

Most marine materials, by nature, contain crystals of inorganic matter with specific structures that allow the loading, release, and delivery of biomolecules that can be utilized in clinical applications. These structures can be biomimetically synthesized. Aggregates of inorganic particles generated by biomimetic microsponges may provide surfaces and structures for cell attachment, organization, and promotion of matrix synthesis. Biomimetic microsponges have been developed with tunable release profiles differing by the rate (speed over distance), velocity (rate of change in direction), and the quantity discharged over time, according to biomolecular species. Specifically, the types of proteins involved guide and regulate cells in physical contact with the microsponges, for instance, reprogramming somatic cells, the switching phenotypes, or specifying stem cell differentiation. Applications for these microsponges include gene transfection of localized cells and promotion of bone matrix synthesis by the externalized display of RGD cell adhesive peptides and the release of crystal entrapped, occluded, adsorbed and infused rhBMP-2 and plasmid. A requirement for de novo bone formation is a solid structure to enable osteocytes to lay new bone tissue. In this study, biomimetic microsponges highlight tremendous potential as osteoconductive packing material in bone repair with parallel influence on regeneration. Majorly, microsponges offer pronounced osteoinductivity, unlike many other bone particulates, by solid-state integration of active regenerative biological molecules through the prism of the biomineral crystalline structure.

Mechanical testing of antimicrobial biocomposite coating on metallic medical implants as drug delivery system.

Post-operative infection often occurs following orthopedic and dental implant placement requiring systemically administered antibiotics. However, this does not provide long-term protection. Over the last few decades, alternative methods involving slow drug delivery systems based on biodegradable poly-lactic acid and antibiotic loaded hydroxyapatite microspheres were developed to prevent post-operative infection. In this study, thermally anodised and untreated Ti6Al4V discs were coated with Poly-Lactic Acid (PLA) containing Gentamicin (Gm) antibiotic-loaded coralline Hydroxyapatite (HAp) are investigated. Following chemical characterization, mechanical properties of the coated samples were measured using nanoindentation and scratch tests to determine the elastic modulus, hardness and bonding adhesion between film and substrate. It was found that PLA biocomposite multilayered films were around 400nm thick and the influence and effect of the substrate were clearly observed during the nanoindentation studies with heavier loads. Scratch tests of PLA coated samples conducted at ~160nm depth showed the minimal difference in the measured friction between Gm and non Gm containing films. It is also observed that the hardness values of PLA film coated anodised samples ranged from 0.45 to 1.9GPa (dependent on the applied loads) against untreated coated samples which ranged from 0.28 to 0.8GPa.

Drug Delivery From Polymer-Based Nanopharmaceuticals-An Experimental Study Complemented by Simulations of Selected Diffusion Processes.

The success of medical therapy depends on the correct amount and the appropriate delivery of the required drugs for treatment. By using biodegradable polymers a drug delivery over a time span of weeks or even months is made possible. This opens up a variety of strategies for better medication. The drug is embedded in a biodegradable polymer (the "carrier") and injected in a particular position of the human body. As a consequence of the interplay between the diffusion process and the degrading polymer the drug is released in a controlled manner. In this work we study the controlled release of medication experimentally by measuring the delivered amount of drug within a cylindrical shell over a long time interval into the body fluid. Moreover, a simple continuum model of the Fickean type is initially proposed and solved in closed-form. It is used for simulating some of the observed release processes for this type of carrier and takes the geometry of the drug container explicitly into account. By comparing the measurement data and the model predictions diffusion coefficients are obtained. It turns out that within this simple model the coefficients change over time. This contradicts the idea that diffusion coefficients are constants independent of the considered geometry. The model is therefore extended by taking an additional absorption term into account leading to a concentration dependent diffusion coefficient. This could now be used for further predictions of drug release in carriers of different shape. For a better understanding of the complex diffusion and degradation phenomena the underlying physics is discussed in detail and even more sophisticated models involving different degradation and mass transport phenomena are proposed for future work and study.

Marine Skeletons: Towards Hard Tissue Repair and Regeneration.

Musculoskeletal disorders in the elderly have significantly increased due to the increase in an ageing population. The treatment of these diseases necessitates surgical procedures, including total joint replacements such as hip and knee joints. Over the years a number of treatment options have been specifically established which are either permanent or use temporary natural materials such as marine skeletons that possess unique architectural structure and chemical composition for the repair and regeneration of bone tissue. This review paper will give an overview of presently used materials and marine structures for hard tissue repair and regeneration, drugs of marine origin and other marine products which show potential for musculoskeletal treatment.

Functionalisation of Ti6Al4V and hydroxyapatite surfaces with combined peptides based on KKLPDA and EEEEEEEE peptides.

Surface modifications are usually performed on titanium alloys to improve osteo-integration and surface bioactivity. Modifications such as alkaline and acid etching, or coating with bioactive materials such as hydroxyapatite, have previously been demonstrated. The aim of this work is to develop a peptide with combined titanium oxide and hydroxyapatite binders in order to achieve a biomimetic hydroxyapatite coating on titanium surfaces. The technology would also be applicable for the functionalisation of titanium and hydroxyapatite surfaces for selective protein adsorption, conjugation of antimicrobial peptides, and adsorption of specialised drugs for drug delivery. In this work, functionalisation of Ti6Al4V and hydroxyapatite surfaces was achieved using combined titanium-hydroxyapatite (Ti-Hap) peptides based on titanium peptide binder (KKLPDA) and hydroxyapatite peptide binder (EEEEEEEE). Homogeneous peptide coatings on Ti6Al4V surfaces were obtained after surface chemical treatments with a 30wt% aqueous solution of H2O2 for 24 and 48h. The treated titanium surfaces presented an average roughness of Sa=197nm (24h) and Sa=128nm (48h); an untreated mirror polished sample exhibited an Sa of 13nm. The advancing water contact angle of the titanium oxide layer after 1h of exposure to 30wt% aqueous solution of H2O2 was around 65°, decreasing gradually with time until it reached 35° after a 48h exposure, suggesting that the surface hydrophilicity increased over etching time. The presence of a lysine (L) amino acid in the sequence of the titanium binder resulted in fluorescence intensity roughly 16% higher compared with the arginine (R) amino acid analogue and therefore the lysine containing titanium peptide binder was used in this work. The Ti-Hap peptide KKLPDAEEEEEEEE (Ti-Hap1) was not adsorbed by the treated Ti6Al4V surfaces and therefore was modified. The modifications involved the inclusion of a glycine spacer between the binding terminals (Ti-Hap2) and the addition of a second titanium binder (KKLPDA) (Ti-Hap3 and Ti-Hap4). The combined Ti-Hap peptide which exhibited the strongest intensity after the titanium surface dip coating was KKLPDAKKLPDAEEEEEEEE (Ti-HAp4). On the other hand, hydroxyapatite surfaces, exhibiting an average roughness of Sa=1.42µm, showed a higher fluorescence for peptides with a higher negative net charge.

Three-Dimensional Implant Positioning with a Piezosurgery Implant Site Preparation Technique and an Intraoral Surgical Navigation System: Case Report.

This case report describes new implant site preparation techniques joining the benefits of using an intraoral navigation system to optimize three-dimensional implant site positioning in combination with an ultrasonic osteotomy. A report of five patients is presented, and the implant positions as planned in the navigation software with the postoperative scan image were compared. The preliminary results are useful, although further clinical studies with larger populations are needed to confirm these findings.

Development and dissolution studies of bisphosphonate (clodronate)-containing hydroxyapatite-polylactic acid biocomposites for slow drug delivery.

An increase in clinical demand on the controlled release of bisphosphonates (BPs) due to complications associated with systemic administration, has been the current driving force on the development of BP drug-release systems. Bisphosphonates have the ability to bind to divalent metal ions, such as Ca2+ , in bone mineral and prevent bone resorption by influencing the apoptosis of osteoclasts. Localized delivery using biodegradable materials, such as polylactic acid (PLA) and hydroxyapatite (HAp), which are ideal in this approach, have been used in this study to investigate the dissolution of clodronate (non-nitrogen-containing bisphosphonate) in a new release system. The effects of coral structure-derived HAp and the release kinetics of the composites were evaluated. The release kinetics of clodronate from PLA-BP and PLA-HAp-BP systems seemed to follow the power law model described by Korsmeyer-Peppas. Drug release was quantified by 31 P-NMR with detection and quantification limits of 9.2 and 30.7 mM, respectively. The results suggest that these biocomposite systems could be tuned to release clodronate for both relatively short and prolonged period of time. In addition to drug delivery, the degradation of HAp supplies both Ca2+ and phosphate ions that can help in bone mineralization. Copyright © 2015 John Wiley & Sons, Ltd.

Natural and Synthetic Coral Biomineralization for Human Bone Revitalization.

Coral skeletons can regenerate replacement human bone in nonload-bearing excavated skeletal locations. A combination of multiscale, interconnected pores and channels and highly bioactive surface chemistry has established corals as an important alternative to using healthy host bone replacements. Here, we highlight how coral skeletal systems are being remolded into new calcified structures or synthetic corals by biomimetic processes, as places for the organized permeation of bone tissue cells and blood vessels. Progressive technologies in coral aquaculture and self-organization inorganic chemistry are helping to modify natural corals and create synthetic coral architectures able to accelerate bone regeneration with proper host integration at more skeletal locations, adapted to recent surgical techniques and used to treat intrinsic skeletal deformities and metabolic conditions.

Calcium phosphate nanocoatings and nanocomposites, part 2: thin films for slow drug delivery and osteomyelitis.

During the last two decades although many calcium phosphate based nanomaterials have been proposed for both drug delivery, and bone regeneration, their coating applications have been somehow slow due to the problems related to their complicated synthesis methods. In order to control the efficiency of local drug delivery of a biomaterial the critical pore sizes as well as good control of the chemical composition is pertinent. A variety of calcium phosphate based nanocoated composite drug delivery systems are currently being investigated. This review aims to give an update into the advancements of calcium phosphate nanocoatings and thin film nanolaminates. In particular recent research on PLA/hydroxyapatite composite thin films and coatings into the slow drug delivery for the possible treatment of osteomyelitis is covered.

Bioresorbable zinc hydroxyapatite guided bone regeneration membrane for bone regeneration.

OBJECTIVES: The aim of this study was to investigate the bone regenerative properties of a heat treated cross-linked GBR membrane with zinc hydroxyapatite powders in the rat calvarial defect model over a 6-week period. MATERIAL AND METHODS: In vitro physio-chemical characterization involved X-ray diffraction analysis, surface topology by scanning electron microscopy, and zinc release studies in physiological buffers. Bilateral rat calvarial defects were used to compare the Zn-HAp membranes against the commercially available collagen membranes and the unfilled defect group through radiological and histological evaluation. RESULTS: The synthesized Zn-MEM (100 µm thick) showed no zinc ions released in the phosphate buffer solution (PBS) buffer, but zinc was observed under acidic conditions. At 6 weeks, both the micro-CT and histological analyses revealed that the Zn-MEM group yielded significantly greater bone formation with 80 ± 2% of bone filled, as compared with 60 ± 5% in the collagen membrane and 40 ± 2% in the unfilled control group. CONCLUSION: This study demonstrated the use of heat treatment as an alternative method to cross-linking the Zn-MEM to be applied as a GBR membrane. Its synthesis and production are relatively simple to fabricate, and the membrane had rough surface features on one side, which might be beneficial for cellular activities. In a rat calvarial defect model, it was shown that new bone formation was accelerated in comparison with the collagen membrane and the unfilled defect groups. These results would suggest that Zn-MEM has the potential for further development in dental applications.

The effectiveness of the controlled release of simvastatin from ß-TCP macrosphere in the treatment of OVX mice.

Simvastatin, a cholesterol treatment drug, has been shown to stimulate bone regeneration. As such, there has been an increase interest in the development of suitable materials and systems for the delivery of simvastatin. Without the appropriate dosage of simvastatin, the therapeutic effects on bone growth will be significantly reduced. Furthermore, similar to many pharmaceutical compounds, at high concentration simvastatin can cause various adverse side-effects. Given the associated side-effects with the usage of simvastatin, the development of suitable controlled drug release system is pertinent. Calcium phosphate in particularly beta-tricalcium phosphate (ß-TCP) has been extensively studied and used as a carrier material for drug delivery system. In this study, Foraminifera exoskeletons were used as calcium carbonate precursor materials, which were hydrothermally converted to ß-TCP as a carrier material for simvastatin. Natural marine exoskeletons posses interconnected and uniformly porous network capable of improving drug loading and release rate. To prolong the release of simvastatin, an apatite coating was made around the ß-TCP sample and in vitro release studies in simulated body fluid (SBF) showed a significant decrease in release rate. Osteoporotic mice were used to examine the compare therapeutic effectiveness of ß-TCP, ß-TCP with simvastatin, apatite-coated ß-TCP with simvastatin and direct injection of simvastatin near the right femur of the mice. Localized and systemic effect were compared with the femur of the non-implanted side (left) and showed that ß-TCP with or without simvastatin was able to induce significant bone formation over 6 weeks. Mechanical analysis showed that apatite-coated ß-TCP with simvastatin produced significantly stronger bones compared with other experimental groups. This study shows that natural exoskeletons with the appropriate structure can be successfully used as a drug delivery system for simvastatin and can its release can be prolonged with an apatite coating to significantly promote relevant bone formation.

Nanobiomaterial Coatings in Dentistry.

During the last decade, there has been a major increase in the interest of nanostructured materials in advanced technologies for biomedical and dental clinical applications. Nanostructured materials are associated with a variety of applications within the dental and biomedical field, for example nanoparticles in drug delivery systems and nanostructured scaffolds in tissue engineering. More importantly, nanotechnology has also been linked with the modification of surface properties of synthetic implants in an attempt to improve their bioactivity, reliability and protection from the release of harmful or unnecessary metal ions. This is achieved through the use of nanocoatings and nanocomposite coatings. These new-generation coatings based on inorganic materials and biological materials such as proteins and peptides are currently investigated and applied. This chapter aims to give an overview of the recent advances in nanocoatings and their composites being investigated or used in dentistry.

Calcium phosphate nanocoatings and nanocomposites, part I: recent developments and advancements in tissue engineering and bioimaging.

A number of materials have been applied as implant coatings and as tissue regeneration materials. Calcium phosphate holds a special consideration, due to its chemical similarity to human bone and, most importantly, its dissolution characteristics, which allow for bone growth and regeneration. The applications of molecular and nanoscale-based biological materials have been and will continue to play an ever increasing role in enhancing and improving the osseointegration of dental and orthopedic implants. More recently, extensive research efforts have been focused on the development and applications of fluorescent nanoparticles and nanocoatings for in vivo imaging and diagnostics as well as devising methods of adding luminescent or fluorescent capabilities to enhance the in vivo functionality of calcium phosphate-based biomedical materials.

Marine structure derived calcium phosphate-polymer biocomposites for local antibiotic delivery.

Hydrothermally converted coralline hydroxyapatite (HAp) particles loaded with medically active substances were used to develop polylactic acid (PLA) thin film composites for slow drug delivery systems. The effects of HAp particles within PLA matrix on the gentamicin (GM) release and release kinetics were studied. The gentamicin release kinetics seemed to follow Power law Korsmeyer Peppas model with mainly diffusional process with a number of different drug transport mechanisms. Statistical analysis shows very significant difference on the release of gentamicin between GM containing PLA (PLAGM) and GM containing HAp microspheres within PLA matrix (PLAHApGM) devices, which PLAHApGM displays lower release rates. The use of HAp particles improved drug stabilization and higher drug encapsulation efficiency of the carrier. HAp is also the source of Ca2+ for the regeneration and repair of diseased bone tissue. The release profiles, exhibited a steady state release rate with significant antimicrobial activity against Staphylococcus aureus (S. aureus) (SH1000) even at high concentration of bacteria. The devices also indicated significant ability to control the growth of bacterial even after four weeks of drug release. Clinical release profiles can be easily tuned from drug-HAp physicochemical interactions and degradation kinetics of polymer matrix. The developed systems could be applied to prevent microbial adhesion to medical implant surfaces and to treat infections mainly caused by S. aureus in surgery.

Biomechanics and functional distortion of the human mandible.

The reaction to the use of finite element analysis (FEA) in the study of the human body has been particularly enthusiastic. Of equal and challenging complexity is the investigation of load/stress distribution and morphological distortion of the human mandible under functional loads. Furthermore, the mandible also impacts directly on body function and esthetics, playing a vital role, such as mastication and speech. The application of FEA to the biomechanical investigation of the oral systems, such as human teeth and mandibular bone remodeling, began in the early 1970s. The clinical significance of jaw deformation is unknown. The primary concern is that deformation might result in an ill-fitting superstructure or the creation of harmful strains in the patient-implant complex. Although mandibular implant treatment has a high success rate, the possibility of failure caused by these dimensional changes and the related micromotion cannot be ignored.