Encapsulated bacteriophages in alginate-nanohydroxyapatite hydrogel as a novel delivery system to prevent orthopedic implant-associated infections.

An innovative delivery system based on bacteriophages-loaded alginate-nanohydroxyapatite hydrogel was developed as a multifunctional approach for local tissue regeneration and infection prevention and control. Bacteriophages were efficiently encapsulated, without jeopardizing phage viability and functionality, nor affecting hydrogel morphology and chemical composition. Bacteriophage delivery occurred by swelling-disintegration-degradation process of the alginate structure and was influenced by environmental pH. Good tissue response was observed following the implantation of bacteriophages-loaded hydrogels, sustaining their biosafety profile. Bacteriophages-loaded hydrogels did not affect osteoblastic cells' proliferation and morphology. A strong osteogenic and mineralization response was promoted through the implantation of hydrogels system with nanohydroxyapatite. Lastly, bacteriophages-loaded hydrogel showed excellent antimicrobial activity inhibiting the attachment and colonization of multidrug-resistant E. faecalis surrounding and within femoral tissues. This new local delivery approach could be a promising approach to prevent and control bacterial contamination during implantation and bone integration.

Antimicrobial Biomaterials for the Healing of Infected Bone Tissue: A Systematic Review of Microtomographic Data on Experimental Animal Models.

Bone tissue infection is a major clinical challenge with high morbidity and a significant healthcare burden. Therapeutic approaches are usually based on systemic antibacterial therapies, despite the potential adverse effects associated with antibiotic resistance, persistent and opportunistic infections, hypersensitivity, and toxicity issues. Most recently, tissue engineering strategies, embracing local delivery systems and antibacterial biomaterials, have emerged as a promising alternative to systemic treatments. Despite the reported efficacy in managing bacterial infection, little is known regarding the outcomes of these devices on the bone healing process. Accordingly, this systematic review aims, for the first time, to characterize the efficacy of antibacterial biomaterials/tissue engineering constructs on the healing process of the infected bone within experimental animal models and upon microtomographic characterization. Briefly, a systematic evaluation of pre-clinical studies was performed according to the PRISMA guidelines, further complemented with bias analysis and methodological quality assessments. Data reported a significant improvement in the healing of the infected bone when an antibacterial construct was implanted, compared with the control-construct devoid of antibacterial activity, particularly at longer time points. Furthermore, considering the assessment of bias, most included studies revealed an inadequate reporting methodology, which may lead to an unclear or high risk of bias and directly hinder future studies.

Pharmacogenetic and Pharmacokinetic Assays from Saliva Samples Can Guarantee Personalized Drug Prescription.

Saliva is widely used for clinical and laboratory analysis. This study proposed to use DNA extracted from saliva for genotyping and pharmacokinetics of piroxicam. A fast and efficient genotyping method was used to determine relevant allelic variants of CYP2C9 (*2 and *3), since genetic factors can influence in non-steroidal anti-inflammatory drugs (NSAIDs) metabolization. DNA Extract All Reagents Kit® was used for DNA extraction and genotyping was performed using TaqMan® GTXpress™ Master Mix, SNP genotyping assays and a Viia7 Real-Time PCR system. Volunteers performed sequential collections of saliva samples before and after taking a single dose of piroxicam (0.25 to 72 h) which were used for pharmacokinetics assays. Piroxicam concentrations were analyzed using LC-MS/MS. Sixty-six percent of volunteers were ancestral homozygous (CYP2C9*1/*1), and 34% showed one or both polymorphisms. Of these 34%, 22 individuals showed CYP2C9*2 polymorphism, 8 CYP2C9*3, and 4 CYP2C9*2/*3. Piroxicam pharmacokinetics were performed in 5 subjects. Areas under the curve (AUC0-t(h*ng/mL)) for CYP2C9*1/*1, *1/*2 and *1/*3 were, respectively, 194.33±70.93, 166 and 303. Maximum concentrations (Cmax(ng/mL)) for these genotypes were respectively 6.46±2.56, 4.3 and 10.2. Saliva sampling was a very effective matrix for both pharmacogenetic and pharmacokinetic tests, ensuring the speed of the procedure and the well-being and agreement of the participants. Once having the knowledge about the slow and fast metabolizers, it is possible to make an adequate prescription in order to avoid the adverse effects of the medication and to guarantee greater analgesic comfort to the patients respectively.

Quantification of piroxicam and 5'-hydroxypiroxicam in human plasma and saliva using liquid chromatography-tandem mass spectrometry following oral administration.

Saliva sampling used to quantify piroxicam and 5'-hydroxypiroxicam is a noninvasive and painless method when compared to sequential blood sampling. For that, a rapid, selective and sensitive liquid chromatography-tandem mass spectrometric method for simultaneous determination of piroxicam and 5'-hydroxypiroxicam in saliva and human plasma was developed and validated. Piroxicam and its major metabolite were separated using a LiChroCART 125-4 RP Select-B Sorbent C18 column using a mixture of methanol and 2% phosphoric acid (pH 2.7) (70:30, v/v) for the mobile phase with a flow injection of 1mL/min. The run time was 4min. Volunteers had saliva and blood sampled before, 1, 2, 3, 4, 5, 6, 8, 11, 24, 48 and 72h after taking a 20mg oral dose of piroxicam. The pharmacokinetic parameters of piroxicam in plasma samples were as follows: AUC0-72 (64819hng/mL), predicted clearance (0.2L/h), distribution volume (14.8L), elimination half-life (50.7h) and saliva/plasma concentration ratio (0.003). The estimation of all pharmacokinetic parameters for 5'-hydroxypiroxicam would require collections beyond 72h; however, it was possible to quantify the mean maximum concentration (133ng/mL), time to peak concentration (53.6h), mean AUC0-72 (6213hng/mL), predicted clearance (110.3L/h) and saliva/plasma concentration ratio (0.04). The developed methods proved effective and sensitive for determining the lower quantification limit of piroxicam in plasma (6.1ng/mL) and saliva (0.15ng/mL) and of 5'-hydroxypiroxicam in plasma (1.2ng/mL) and saliva (0.15ng/mL).

Pharmacogenetic and pharmacokinetic assays from saliva samples can guarantee personalized drug prescription

Abstract Saliva is widely used for clinical and laboratory analysis. This study proposed to use DNA extracted from saliva for genotyping and pharmacokinetics of piroxicam. A fast and efficient genotyping method was used to determine relevant allelic variants of CYP2C9 (*2 and *3), since genetic factors can influence in non-steroidal anti-inflammatory drugs (NSAIDs) metabolization. DNA Extract All Reagents Kit® was used for DNA extraction and genotyping was performed using TaqMan® GTXpress™ Master Mix, SNP genotyping assays and a Viia7 Real-Time PCR system. Volunteers performed sequential collections of saliva samples before and after taking a single dose of piroxicam (0.25 to 72 h) which were used for pharmacokinetics assays. Piroxicam concentrations were analyzed using LC-MS/MS. Sixty-six percent of volunteers were ancestral homozygous (CYP2C9*1/*1), and 34% showed one or both polymorphisms. Of these 34%, 22 individuals showed CYP2C9*2 polymorphism, 8 CYP2C9*3, and 4 CYP2C9*2/*3. Piroxicam pharmacokinetics were performed in 5 subjects. Areas under the curve (AUC0-t(h*ng/mL)) for CYP2C9*1/*1, *1/*2 and *1/*3 were, respectively, 194.33±70.93, 166 and 303. Maximum concentrations (Cmax(ng/mL)) for these genotypes were respectively 6.46±2.56, 4.3 and 10.2. Saliva sampling was a very effective matrix for both pharmacogenetic and pharmacokinetic tests, ensuring the speed of the procedure and the well-being and agreement of the participants. Once having the knowledge about the slow and fast metabolizers, it is possible to make an adequate prescription in order to avoid the adverse effects of the medication and to guarantee greater analgesic comfort to the patients respectively.

Resumo Saliva é amplamente utilizada para análises clínicas e laboratoriais. Este estudo propôs o uso de DNA extraído da saliva para genotipagem e farmacocinética do piroxicam. Um método de genotipagem rápido e eficiente foi usado para determinar as variantes alélicas clinicamente relevantes de CYP2C9 (* 2 e * 3), uma vez que fatores genéticos podem influenciar nas respostas metabólicas individuais a medicamentos como anti-inflamatórios não esteroides (AINEs). DNA Extract All Reagents Kit® foi usado para extração de DNA e a genotipagem foi realizada usando TaqMan® GTXpress ™ Master Mix, ensaios de genotipagem SNP e um sistema Viia7 Real-Time PCR. Os voluntários realizaram coletas sequenciais de amostras de saliva antes e após a ingestão de uma única dose de piroxicam (0,25 a 72 h) que foram utilizadas para ensaios farmacocinéticos. As concentrações de piroxicam foram analisadas usando LC - MS / MS. Sessenta e seis por cento dos voluntários eram homozigotos ancestrais (CYP2C9 * 1 / * 1) e 34% apresentaram um ou ambos os polimorfismos. Destes 34%, 22 indivíduos apresentaram polimorfismo CYP2C9 * 2, 8 CYP2C9 * 3 e 4 CYP2C9 * 2 / * 3. A farmacocinética do piroxicam foi realizada em 5 indivíduos. As áreas sob a curva (AUC0-t (h * ng / mL)) para CYP2C9 * 1 / * 1, * 1 / * 2 e * 1 / * 3 foram, respectivamente, 194,33±70,93, 166 e 303. Concentrações máximas (Cmax (ng / mL)) para esses genótipos foram, respectivamente, 6,46±2,56, 4,3 e 10,2. A amostra de saliva foi uma matriz muito eficaz tanto para os testes farmacogenéticos quanto para os farmacocinéticos, garantindo a agilidade do procedimento e o bem-estar e concordância dos participantes. Com o conhecimento dos metabolizadores lentos e rápidos, é possível fazer uma prescrição adequada para evitar os efeitos adversos da medicação e garantir maior conforto analgésico aos pacientes respectivamente.