Efficient AAV9 Purification Using a Single-Step AAV9 Magnetic Affinity Beads Isolation.

Adeno-associated viruses (AAVs) have emerged as promising tools for gene therapy due to their safety and efficacy in delivering therapeutic genes or gene editing sequences to various tissues and organs. AAV serotype 9 (AAV9), among AAV serotypes, stands out for its ability to efficiently target multiple tissues, thus holding significant potential for clinical applications. However, existing methods for purifying AAVs are cumbersome, expensive, and often yield inconsistent results. In this study, we explore a novel purification strategy utilizing Dynabeads™ CaptureSelect™ magnetic beads. The AAV9 magnetic beads capture AAV9 with high specificity and recovery between 70 and 90%, whereas the AAVX magnetic beads did not bind to the AAV9. Through continuous interaction with AAVs in solution, these beads offer enhanced clearance of genomic DNA and plasmids even in the absence of endonuclease. The beads could be regenerated at least eight times, and the used beads could be stored for up to six months and reused without a significant reduction in recovery. The potency of the AAV9-purified vectors in vivo was comparable to that of iodixanol purified vectors.

Aerogel-Based Materials in Bone and Cartilage Tissue Engineering-A Review with Future Implications.

Aerogels are fascinating solid materials known for their highly porous nanostructure and exceptional physical, chemical, and mechanical properties. They show great promise in various technological and biomedical applications, including tissue engineering, and bone and cartilage substitution. To evaluate the bioactivity of bone substitutes, researchers typically conduct in vitro tests using simulated body fluids and specific cell lines, while in vivo testing involves the study of materials in different animal species. In this context, our primary focus is to investigate the applications of different types of aerogels, considering their specific materials, microstructure, and porosity in the field of bone and cartilage tissue engineering. From clinically approved materials to experimental aerogels, we present a comprehensive list and summary of various aerogel building blocks and their biological activities. Additionally, we explore how the complexity of aerogel scaffolds influences their in vivo performance, ranging from simple single-component or hybrid aerogels to more intricate and organized structures. We also discuss commonly used formulation and drying methods in aerogel chemistry, including molding, freeze casting, supercritical foaming, freeze drying, subcritical, and supercritical drying techniques. These techniques play a crucial role in shaping aerogels for specific applications. Alongside the progress made, we acknowledge the challenges ahead and assess the near and far future of aerogel-based hard tissue engineering materials, as well as their potential connection with emerging healing techniques.

Evaluation of the anti-inflammatory, antipyretic and antinociceptive activities of the hydroalcoholic extract of Rhynchospora nervosa (Vahl) Boeckeler (Cyperaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Rhynchospora nervosa (Vahl) Boeckeler (Cyperaceae), popularly known as "capim-estrela", is a native species widely distributed in Brazil. The whole plant has been used in local traditional medicine in the form of teas or syrups to treat inflammation, flu, nasal congestion, fever, swelling, and venereal disease. This is the first study to investigate the pharmacological properties of this species. AIM OF THE STUDY: The present study aimed to evaluate the in vivo anti-inflammatory, antipyretic and antinociceptive potential of the lyophilized hydroalcoholic extract of R. nervosa in heterogenic Swiss mice. In addition to pharmacological studies, the total phenol and flavonoid contents of the extract were determined. MATERIAL AND METHODS: The anti-inflammatory effect was evaluated through carrageenan-induced paw edema and peritonitis models. For the antinociceptive assay, the number of acetic acid-induced writhing responses in the animals was counted. Antipyretic activity was tested by yeast-induced pyrexia in mice and evaluated for 4 h. Nitric oxide (NO) concentration and leukocyte migration in the peritoneal fluid were quantified. The acute toxicity of the extract was also calculated. Quantitative analyses of total phenols and flavonoids in the extract were performed by spectrophotometric methods. RESULTS: In short, the lyophilized hydroalcoholic extract of R. nervosa showed low acute toxicity in the preclinical tests (LD50 = 3807 mg/kg). A significant anti-inflammatory effect was observed, with an average reduction of carrageenan-induced paw edema of 96.37%. Comparatively, indomethacin inhibited the development of the carrageenin paw edema by 97.52%. In the peritonitis test, a significant reduction in NO levels was recorded. A reduction in the number of white cells, notably monocytes, was also observed, confirming the anti-inflammatory effect. Writhing was reduced by 86.53%, which indicates antinociceptive activity. As for antipyretic activity, no positive effects of the extract were observed. The lyophilized hydroalcoholic extract of R. nervosa presented a high content of phenolic compounds (322.47 µg GAE/mg) and total flavonoids (440.50 µg QE/mg). CONCLUSION: The lyophilized hydroalcoholic extract of R. nervosa showed significant in vivo anti-inflammatory and antinociceptive activity in mice. These preliminary findings support the indication of the use of this species in folk medicine in Brazil for the treatment of inflammation.

Role of alkali metal oxide type on the degradation and in vivo biocompatibility of soda-lime-borate bioactive glass.

In this work, it is the first time to study the effect of replacing of Na2 O by a fixed amount of Li2 O or K2 O in soda-lime-borate glass on its in vivo biocompatibility. The glass composition was based on xM2 O-20x Na2 O20 CaO60 B2 O3 , (wt %), where, M2 OLi2 O and K2 O, and consequently, samples encoded BN100, BK50, and BL50. The degradation test was carried out in 0.25 M K2 HPO4 solution. The in vivo test was performed in the femoral bone defect of Sprague-Dawley adult male rat. Following up bone formation was conducted by the histological analyses and bone formation markers (alkaline phosphatase [ALP] and osteocalcin [OCN]). Furthermore, the glass effect on the liver and kidney functions was addressed in this study using (alanine transaminase [ALT] and aspartate transaminase [AST]) and (urea and creatinine), respectively. The results of the degradation test showed that the glass dissolution rate was increased by incorporating of K2 O, and its ion release was occurred by a diffusion-controlled process. Moreover, in vivo bioactivity test showed that serum activity of ALP, OCN level, and the newly formed bone was higher in BL50-implanted group than that of BN100 andBK50at 3 w and 6 w post-surgery. As well as, implantation of all glass samples in the femoral bone defect did not alter the liver and kidney functions. In conclusion, the synthesized borate glass was well served as a controlled delivery system for Li+ ion release, which enhanced bone formation as shown from the bone formation markers (ALP and OCN).

Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination.

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and ß-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Relationship between in vitro apatite-forming ability measured using simulated body fluid and in vivo bioactivity of biomaterials.

In a large number of studies, it has been assumed that the in vitro apatite-forming ability measured by simulated body fluid (SBF) test is a predictor of in vivo bioactivity. Several researchers have argued in favor and against this assumption; but the actual experimental evidence is not yet fully examined. The purpose of this study is to review the currently available evidence that supports or rejects the above-mentioned assumption. Ultimately, it is important that SBF tests could simulate the actual physiological conditions experienced by biomaterials within the human body. Given that in vivo animal experiments provide the best pre-clinical test conditions, all studies in which both in vitro apatite forming ability and in vivo performance of two or more biomaterials are compared were found by searching the literature. From all studies that satisfied the inclusion criteria (33), in 25 studies in vitro apatite-forming ability could predict the relative performance of the tested biomaterials in vivo. In 8 studies, in vitro performance did not correctly predict the relative in vivo performance. In majority of failure cases (i.e. 5/8), none of the compared biomaterials formed apatite, while all compared biomaterials showed bioactive behavior in vivo. It is therefore concluded that, in majority of cases, the SBF immersion test has been successful in predicting the relative performance of biomaterials in vivo. However, the details of the test protocols and the (expected) mechanisms of bioactivity of tested biomaterials should be carefully considered in the design of SBF immersion tests and in interpretation of their results. Certain guidelines are devised based on the results of this review for the design of SBF immersion test protocols and interpretation of the test results. These guidelines could help in designing better SBF test protocols that have better chances of predicting the bioactivity of biomaterials for potential application in clinical orthopedics.