A rabbit osteochondral defect (OCD) model for evaluation of tissue engineered implants on their biosafety and efficacy in osteochondral repair.

Osteochondral defect (OCD) is a common but challenging condition in orthopaedics that imposes huge socioeconomic burdens in our aging society. It is imperative to accelerate the R&D of regenerative scaffolds using osteochondral tissue engineering concepts. Yet, all innovative implant-based treatments require animal testing models to verify their feasibility, biosafety, and efficacy before proceeding to human trials. Rabbit models offer a more clinically relevant platform for studying OCD repair than smaller rodents, while being more cost-effective than large animal models. The core-decompression drilling technique to produce full-thickness distal medial femoral condyle defects in rabbits can mimic one of the trauma-relevant OCD models. This model is commonly used to evaluate the implant's biosafety and efficacy of osteochondral dual-lineage regeneration. In this article, we initially indicate the methodology and describe a minimally-invasive surgical protocol in a step-wise manner to generate a standard and reproducible rabbit OCD for scaffold implantation. Besides, we provide a detailed procedure for sample collection, processing, and evaluation by a series of subsequent standardized biochemical, radiological, biomechanical, and histological assessments. In conclusion, the well-established, easy-handling, reproducible, and reliable rabbit OCD model will play a pivotal role in translational research of osteochondral tissue engineering.

Astragalus Polysaccharide Enhances Voriconazole Metabolism under Inflammatory Conditions through the Gut Microbiota.

Background and Aims: Voriconazole (VRC), a widely used antifungal drug, often causes hepatotoxicity, which presents a significant clinical challenge. Previous studies demonstrated that Astragalus polysaccharide (APS) can regulate VRC metabolism, thereby potentially mitigating its hepatotoxic effects. In this study, we aimed to explore the mechanism by which APS regulates VRC metabolism. Methods: First, we assessed the association of abnormal VRC metabolism with hepatotoxicity using the Roussel Uclaf Causality Assessment Method scale. Second, we conducted a series of basic experiments to verify the promotive effect of APS on VRC metabolism. Various in vitro and in vivo assays, including cytokine profiling, immunohistochemistry, quantitative polymerase chain reaction, metabolite analysis, and drug concentration measurements, were performed using a lipopolysaccharide-induced rat inflammation model. Finally, experiments such as intestinal biodiversity analysis, intestinal clearance assessments, and Bifidobacterium bifidum replenishment were performed to examine the ability of B. bifidum to regulate the expression of the VRC-metabolizing enzyme CYP2C19 through the gut-liver axis. Results: The results indicated that APS does not have a direct effect on hepatocytes. However, the assessment of gut microbiota function revealed that APS significantly increases the abundance of B. bifidum, which could lead to an anti-inflammatory response in the liver and indirectly enhance VRC metabolism. The dual-luciferase reporter gene assay revealed that APS can hinder the secretion of pro-inflammatory mediators and reduce the inhibitory effect on CYP2C19 transcription through the nuclear factor-κB signaling pathway. Conclusions: The study offers valuable insights into the mechanism by which APS alleviates VRC-induced liver damage, highlighting its immunomodulatory influence on hepatic tissues and its indirect regulatory control of VRC-metabolizing enzymes within hepatocytes.

Characterization of the Hemolytic Activity of Mastoparan Family Peptides from Wasp Venoms.

Biologically active peptides have attracted increasing attention in research on the development of new drugs. Mastoparans, a group of wasp venom linear cationic α-helical peptides, have a variety of biological effects, including mast cell degranulation, activation of protein G, and antimicrobial and anticancer activities. However, the potential hemolytic activity of cationic α-helical peptides greatly limits the clinical applications of mastoparans. Here, we systematically and comprehensively studied the hemolytic activity of mastoparans based on our wasp venom mastoparan family peptide library. The results showed that among 55 mastoparans, 18 had strong hemolytic activity (EC50 ≤ 100 µM), 14 had modest hemolytic activity (100 µM < EC50 ≤ 400 µM) and 23 had little hemolytic activity (EC50 > 400 µM), suggesting functional variation in the molecular diversity of mastoparan family peptides from wasp venom. Based on these data, structure-function relationships were further explored, and, hydrophobicity, but not net charge and amphiphilicity, was found to play a critical role in the hemolytic activity of mastoparans. Combining the reported antimicrobial activity with the present hemolytic activity data, we found that four mastoparan peptides, Parapolybia-MP, Mastoparan-like peptide 12b, Dominulin A and Dominulin B, have promise for applications because of their high antimicrobial activity (MIC ≤ 10 µM) and low hemolytic activity (EC50 ≥ 400 µM). Our research not only identified new leads for the antimicrobial application of mastoparans but also provided a large chemical space to support the molecular design and optimization of mastoparan family peptides with low hemolytic activity regardless of net charge or amphiphilicity.

Engineered biochemical cues of regenerative biomaterials to enhance endogenous stem/progenitor cells (ESPCs)-mediated articular cartilage repair.

As a highly specialized shock-absorbing connective tissue, articular cartilage (AC) has very limited self-repair capacity after traumatic injuries, posing a heavy socioeconomic burden. Common clinical therapies for small- to medium-size focal AC defects are well-developed endogenous repair and cell-based strategies, including microfracture, mosaicplasty, autologous chondrocyte implantation (ACI), and matrix-induced ACI (MACI). However, these treatments frequently result in mechanically inferior fibrocartilage, low cost-effectiveness, donor site morbidity, and short-term durability. It prompts an urgent need for innovative approaches to pattern a pro-regenerative microenvironment and yield hyaline-like cartilage with similar biomechanical and biochemical properties as healthy native AC. Acellular regenerative biomaterials can create a favorable local environment for AC repair without causing relevant regulatory and scientific concerns from cell-based treatments. A deeper understanding of the mechanism of endogenous cartilage healing is furthering the (bio)design and application of these scaffolds. Currently, the utilization of regenerative biomaterials to magnify the repairing effect of joint-resident endogenous stem/progenitor cells (ESPCs) presents an evolving improvement for cartilage repair. This review starts by briefly summarizing the current understanding of endogenous AC repair and the vital roles of ESPCs and chemoattractants for cartilage regeneration. Then several intrinsic hurdles for regenerative biomaterials-based AC repair are discussed. The recent advances in novel (bio)design and application regarding regenerative biomaterials with favorable biochemical cues to provide an instructive extracellular microenvironment and to guide the ESPCs (e.g. adhesion, migration, proliferation, differentiation, matrix production, and remodeling) for cartilage repair are summarized. Finally, this review outlines the future directions of engineering the next-generation regenerative biomaterials toward ultimate clinical translation.

Hybrid Therapeutic Device (CUHK-OA-M2) for Relieving Symptoms Induced by Knee Osteoarthritis.

The symptoms of knee osteoarthritis (KOA) severely affect the life quality of the elderly population. Low-level laser therapy, heat therapy, and massage therapy are widely used as independent treatments for joint disorders. However, there are very limited reports of a combination of these therapies into an integrated device for KOA so far. This study aims to develop a novel hybrid therapeutic device that can meet various requirements for knee therapy. Our hybrid therapeutic device (CUHK-OA-M2) integrated with low-level laser therapy, heat therapy, and local massage therapy can effectively provide patients with KOA with relief from their clinical symptoms. A pilot test of 50 community-dwelling elderly volunteers with KOA was performed. Finally, 43 volunteers completed two treatment periods (30 days each) and two post-treatment periods (30 days each). The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores were collected and analyzed after each period. The outputs of the low-level laser, heating, and massage therapies significantly decreased the WOMAC scores in terms of pain, stiffness, function and total WOMAC after two treatment periods (p < 0.05). Although the score increased slightly after the post-treatment period, it was still lower than the baseline, indicating the treatment outcome could last for an extended period. Therefore, our CUHK-OA-M2 device, as an integrated multi-functional hybrid therapeutic device, is therapeutically significant for treating osteoarthritis symptoms on the knee joints of elderly subjects.

[Changes and Related Reflections on the European Union's in Vitro Diagnostic Medical Device Classification Supervision System].

By analyzing and comparing the IVD product classification supervision system in the EU's new IVDR, old IVDD and related guidance documents, concepts of related regulatory reforms were analyzed and explored, to provide references for industrial personnel to understand and master the new EU IVD classification system, and for China's ongoing classification management reform.

[Analysis on Work Idea of Medical Device Classification Dynamic Adjustment Mechanism in the United States].

OBJECTIVE: Discuss the working ideas of the dynamic adjustment mechanism of medical device classification in the United States, and provide reference for the construction of medical device related mechanisms in China. METHODS: Collect and interpret the documents of regulatory background, procedures and orders of the dynamic adjustment mechanism of the medical device classification in the United States, and summarize the overall situation and specific cases of the medical device classification adjustment under this mechanism in recent years. RESULTS: The US work idea of the medical device classification dynamic adjustment mechanism is based on the latest valid scientific evidence, conducting risk analysis and identification, and determining the corresponding measures. CONCLUSIONS: During the adjustment process, industry stakeholders have repeatedly discussed and achieved final agreement. Its procedures and working ideas can be used as a reference for China's work.

Biodegradable magnesium combined with distraction osteogenesis synergistically stimulates bone tissue regeneration via CGRP-FAK-VEGF signaling axis.

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union. Here, we demonstrated that magnesium (Mg) nail implantation into the marrow cavity degraded gradually accompanied with about 4-fold increase of new bone formation and over 5-fold of new vessel formation as compared with DO alone group in the 5 mm femoral segmental defect rat model at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. CGRP concentration-dependently promoted endothelial cell migration and tube formation. Meanwhile, CGRP promoted the phosphorylation of focal adhesion kinase (FAK) at Y397 site and elevated the expression of vascular endothelial growth factor A (VEGFA). Moreover, inhibitor/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation. We revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications.

Innovative Tissue-Engineered Strategies for Osteochondral Defect Repair and Regeneration: Current Progress and Challenges.

Clinical treatments for the repair of osteochondral defects (OCD) are merely palliative, not completely curative, and thus enormously unfulfilled challenges. With the in-depth studies of biology, medicine, materials, and engineering technology, the conception of OCD repair and regeneration should be renewed. During the past decades, many innovative tissue-engineered approaches for repairing and regenerating damaged osteochondral units have been widely explored. Various scaffold-free and scaffold-based strategies, such as monophasic, biphasic, and currently fabricated multiphasic and gradient architectures have been proposed and evaluated. Meanwhile, progenitor cells and tissue-specific cells have also been intensively investigated in vivo as well as ex vivo. Concerning bioactive factors and drugs, they have been combined with scaffolds and/or living cells, and even released in a spatiotemporally controlled manner. Although tremendous progress has been achieved, further research and development (R&D) is needed to convert preclinical outcomes into clinical applications. Here, the osteochondral unit structure, its defect classifications, and diagnosis are summarized. Commonly used clinical reparative techniques, tissue-engineered strategies, emerging 3D-bioprinting technologies, and the status of their clinical applications are discussed. Existing challenges to translation are also discussed and potential solutions for future R&D directions are proposed.

[Analysis of Changes in Medical Device Classification Concept of the New EU Regulations].

By comparing new medical device regulations with existing directives in the European Union, the revising ideas of new EU medical device regulations, especially the changes of the classification management concepts, were analyzed to provide clues for the industry to interpret the new EU regulations, and references for the classification management innovation in our country.

Chemoenzymatic synthesis of two new halogenated coumarin glycosides as potential antifungal agents.

Two new potential antifungal coumarin glycosides, 6-chlorocoumarin 7-O-beta-D-glucopyranoside (1) and 7-hydroxy-4-trifluoromethyl-coumarin 5-O-beta-D-glucopyranoside (2), were synthesized via enzyme-mediated glycosylation of the respective aglycone, 6-chloro-7-hydroxycoumarin and 5,7-dihydroxy-4-trifluoromethylcoumarin, using transgenic hairy roots of Polygonum multiflorum. Instead of application of the isolated enzyme and exogenous sugar donors, hairy roots of P. multiflorum were successfully adapted as a whole-cell biocatalyst.

Biotransformation of hydroxycoumarin derivatives by cultured suspension cells of Catharanthus roseus.

Using 7-hydroxy-4-methylcoumarin (1), 7-hydroxy-4-phenylcoumarin (2), 5,7-dihydroxy-4-methylcoumarin (3), 7,8-dihydroxycoumarin (4) as substrates, three new compounds, 4-methylcoumarin-7-O-beta-D-xylopyranosyl (1 --> 6) beta-D-glucopyranoside (1b), 4-phenylcoumarin-7-O-beta-D-glucopyranosyl (1 --> 6) beta-D-glucopyranoside (2b), 4-methylcoumarin-5,7-O-beta-D-diglucoside (3b), along with four known compounds (1a, 2a, 3a and 4a) were biosynthesized by the cell suspension cultures of Catharanthus roseus as biocatalyst. Their structures were elucidated by analysis of the physicochemical properties and 1H NMR, 13C NMR, HMBC and ESI-MS spectra. Besides, the results also showed that this novel biocatalyst system of C. roseus cell cultures has a great capacity of regio-selective glycosylation to the hydroxyl group of the exogenous substrates. Co-culture time curves on conversion were also established.

[Impact of 2, 3-dihydrocyclopentacoumarins to growth of transgenic hairy roots of Polygonum multiflorum and accumulation of stilbene glucosides].

OBJECTIVE: To investigate the influence of 2,3-dihydrocyclopenta coumarins to the growth of transgenic hairy root of Polygonum multiflorum and the accumulation of active ingredient stilbene glucoside in the culture. METHOD: In different culture time, 2,3-dihydrocyclopenta coumarin compounds were administrated to the hairy roots. The yield of stilbene glucoside and the growth of the hairy roots were determined after some period of co-culture. Through the investigation of the different concentrations of the 2,3-dihydrocyclopentacoumarin, and sub-culture times, the optimal experimental conditions were established. RESULT: The optimal administration time of the 2,3-dihydrocyclopentacoumarin was on the fourth day, the optimal concentration of 2,3-dihydrocyclopentacoumarin was 0.025 g x L(-1). Under this condition, the growth of the hairy root greatly increased. Comparing with control group, the accumulation of stilbene glucoside was increased about four times. CONCLUSION: 2, 3-dihydrocyclopentacoumarins could promote the hairy hair root growth and stimulate the stilbene glucoside accumulation.

Biosynthesis of coumarin glycosides by transgenic hairy roots of Polygonum multiflorum.

To investigate the substrate specificity and regio-selectivity of coumarin glycosyltransferases in transgenic hairy roots of Polygonum multiflorum, esculetin (1) and eight hydroxycoumarins (2-9) were employed as substrates. Nine corresponding glycosides (10-18) involving four new compounds, 6-chloro-4-methylcoumarin 7-O-ß-D-glucopyranoside (15), 6-chloro-4-phenylcoumarin 7-O-ß-D-glucopyranoside (16), 8-hydroxy-4-methylcoumarin 7-O-ß-D-glucopyranoside (17), and 8-allyl-4-methylcoumarin 7-O-ß-D-glucopyranoside (18), were biosynthesized by the hairy roots.

[Biotransformation of 4-methyl-7-allyloxy coumarin by transgenic hairy roots of Polygonum multiflorum].

OBJECTIVE: To synthesis 4-methyl-7-allyloxy coumarin by Williamson etherification from 4-methyl-7-hydroxy coumarin and apply as a substrate in hairy roots of Polygonum multiflorum. METHODS: The synthesis reaction of 4-methyl-7-allyloxy coumarin used the allyl bromide and potassium carbonate as catalysts, and acetone as solvent reacted for 17 hours, then the product was isolated. 4-methyl-7-allyloxy coumarin was added into the media of supension transgenic hairy roots of Polygonum multiflorum which had been precultured for 8 d, and then co-cultured for another 7 d. The biotransformation products were detected by TLC and HPLC and isolated by various chromatographic methods. RESULTS: Two biotransformation products, 4-methyl-7-hydroxy coumarin and 4-methyl-coumarin-7-O-beta-D-glucoside were isolated and identified. CONCLUSION: Hairy roots of Polygonum multiflorum contains not only glycosyltransferase but also hydrolysis enzymes.

[Biotransformation of daphnetin by suspension transgenic hairy roots of Polygonum multiflorum].

OBJECTIVE: To investigate the biotransformation of daphnetin by suspension transgenic hairy root of Polygonum multiflorum and provide a biotechnological method for large-scale production of the daphnetin-8-O-beta-D-glucoside using this new culture system. METHOD: Daphnetin was added into the media of suspension to culture 36 h. The biotransformation product was detected with TLC and HPLC, and isolated by various chromatographic methods. The influence of co-cultured time on conversion ratio, content of degradation product and the reason for the degradation of product II were investigated using HPLC. RESULT: One biotransformation product, daphnetin-8-O-beta-D-glucoside, was obtained, the optimal co-cultured time in suspension hairy root of P. multiflorum was 36 h with the highest biotransformation molar ratio of 32.11%, the sucrose medium (sucrose-only) can increase the biotransformation molar ratio to 72.44%. The result demonstrated that the degradation products of the product II was induced by the MS medium. CONCLUSION: The potential application of suspension transgenic hairy root of P. multflorum in the sucrose-only medium on generating daphnetin-8-Obeta-D- glucoside could be prospective.