Injectable cartilage microtissues based on 3D culture using porous gelatin microcarriers for cartilage defect treatment.

Cartilage tissues possess an extremely limited capacity for self-repair, and current clinical surgical approaches for treating articular cartilage defects can only provide short-term relief. Despite significant advances in the field of cartilage tissue engineering, avoiding secondary damage caused by invasive surgical procedures remains a challenge. In this study, injectable cartilage microtissues were developed through 3D culture of rat bone marrow mesenchymal stem cells (BMSCs) within porous gelatin microcarriers (GMs) and induced differentiation. These microtissues were then injected for the purpose of treating cartilage defects in vivo, via a minimally invasive approach. GMs were found to be noncytotoxic and favorable for cell attachment, proliferation and migration evaluated with BMSCs. Moreover, cartilage microtissues with a considerable number of cells and abundant extracellular matrix components were obtained from BMSC-laden GMs after induction differentiation culture for 28 days. Notably, ATDC5 cells were complementally tested to verify that the GMs were conducive to cell attachment, proliferation, migration and chondrogenic differentiation. The microtissues obtained from BMSC-laden GMs were then injected into articular cartilage defect areas in rats and achieved superior performance in alleviating inflammation and repairing cartilage. These findings suggest that the use of injectable cartilage microtissues in this study may hold promise for enhancing the long-term outcomes of cartilage defect treatments while minimizing the risk of secondary damage associated with traditional surgical techniques.

Biofabrication of Tissue-Engineered Cartilage Constructs Through Faraday Wave Bioassembly of Cell-Laden Gelatin Microcarriers.

Acoustic biofabrication is an emerging strategy in tissue engineering due to its mild and fast manufacturing process. Herein, tissue-engineered cartilage constructs with high cell viability are fabricated from cell-laden gelatin microcarriers (GMs) through Faraday wave bioassembly, a typical acoustic "bottom-up" manufacturing process. Assembly modules are first prepared by incorporating cartilage precursor cells, the chondrogenic cell line ATDC5, or bone marrow-derived mesenchymal stem cells (BMSCs), into GMs. Patterned structures are formed by Faraday wave bioassembly of the cell-laden GMs. Due to the gentle and efficient assembly process and the protective effects of microcarriers, cells in the patterned structures maintain high activity. Subsequently, tissue-engineered cartilage constructs are obtained by inducing cell differentiation of the patterned structures. Comprehensive evaluations are conducted to verify chondrocyte differentiation and the formation of cartilage tissue constructs in terms of cell viability, morphological analysis, gene expression, and matrix production. Finally, implantation studies with a rat cartilage defect model demonstrate that these tissue-engineered cartilage constructs are beneficial for the repair of articular cartilage damage in vivo. This study provides the first biofabrication of cartilage tissue constructs using Faraday wave bioassembly, extending its application to engineering tissues with a low cell density.

Supramolecular Responsive Chitosan Microcarriers for Cell Detachment Triggered by Adamantane.

Supramolecular responsive microcarriers based on chitosan microspheres were prepared and applied for nonenzymatic cell detachment. Briefly, chitosan microspheres (CSMs) were first prepared by an emulsion crosslinking approach, the surface of which was then modified with ß-cyclodextrin (ß-CD) by chemical grafting. Subsequently, gelatin was attached onto the surface of the CSMs via the host-guest interaction between ß-CD groups and aromatic residues in gelatin. The resultant microspheres were denoted CSM-g-CD-Gel. Due to their superior biocompatibility and gelatin niches, CSM-g-CD-Gel microspheres can be used as effective microcarriers for cell attachment and expansion. L-02, a human fetal hepatocyte line, was used to evaluate cell attachment and expansion with these microcarriers. After incubation for 48 h, the cells attached and expanded to cover the entire surface of microcarriers. Moreover, with the addition of adamantane (AD), cells can be detached from the microcarriers together with gelatin because of the competitive binding between ß-CD and AD. Overall, these supramolecular responsive microcarriers could effectively support cell expansion and achieve nonenzymatic cell detachment and may be potentially reusable with a new cycle of gelatin attachment and detachment.

Biomimetic cell culture for cell adhesive propagation for tissue engineering strategies.

Biomimetic cell culture, which involves creating a biomimetic microenvironment for cells in vitro by engineering approaches, has aroused increasing interest given that it maintains the normal cellular phenotype, genotype and functions displayed in vivo. Therefore, it can provide a more precise platform for disease modelling, drug development and regenerative medicine than the conventional plate cell culture. In this review, initially, we discuss the principle of biomimetic cell culture in terms of the spatial microenvironment, chemical microenvironment, and physical microenvironment. Then, the main strategies of biomimetic cell culture and their state-of-the-art progress are summarized. To create a biomimetic microenvironment for cells, a variety of strategies has been developed, ranging from conventional scaffold strategies, such as macroscopic scaffolds, microcarriers, and microgels, to emerging scaffold-free strategies, such as spheroids, organoids, and assembloids, to simulate the native cellular microenvironment. Recently, 3D bioprinting and microfluidic chip technology have been applied as integrative platforms to obtain more complex biomimetic structures. Finally, the challenges in this area are discussed and future directions are discussed to shed some light on the community.

Resistance to Azoxystrobin and Thiophanate-Methyl Is Widespread in Colletotrichum spp. Isolates From the Mid-Atlantic Strawberry Fields.

Multiple Colletotrichum species have been found to be responsible for strawberry anthracnose, and prevalence of each species seems to vary by regions and/or host tissues. In this study, a total of 200 Colletotrichum isolates were obtained from different strawberry cultivars displaying anthracnose symptoms in the mid-Atlantic fields. Analysis of g3pdh, tub2, and/or internal transcribed spacer sequences revealed four Colletotrichum species, including C. nymphaeae, C. fioriniae, C. siamense, and C. lineola. C. nymphaeae was the predominant species, representing 90% of all isolates collected. This species was found from all strawberry organs/tissues examined, whereas C. siamense and C. fioriniae were limited to the crown and fruit, respectively. Further, all Colletotrichum isolates were screened for resistance to azoxystrobin in vitro, and all C. siamense isolates were additionally screened for resistance to thiophanate-methyl. The overall frequency of resistance to azoxystrobin and thiophanate-methyl was 48 and 67%, respectively. G143A in the cytochrome b gene was found in all C. nymphaeae and C. siamense isolates with high level of resistance, with EC50 >100 µg/ml, while F129L was found in two of the five C. nymphaeae isolates with moderate resistance, with EC50 values ranging from 2.6 to 7.8 µg/ml. All C. fioriniae isolates tested were found to be less sensitive to azoxystrobin, with EC50 values ranging from 9.7 to 14.4 µg/ml, despite no mutations detected in the cytochrome b gene. Moreover, E198A in tub2 was linked with C. siamense isolates resistant to thiophanate-methyl (EC50 >100 µg/ml). These results revealed that resistance in Colletotrichum spp. to primary fungicides is widespread in the mid-Atlantic strawberry fields.

Comparison of three sequential extraction procedures for arsenic fractionation in highly polluted sites.

Three sequential extraction procedures (SEPs) including Tessier, Rauret, and Shiowatana SEPs, were compared for arsenic fractionation using highly polluted soils. In the definition context of exchangeable, reducible, oxidizable and residual fractions, with similar arsenic recovery and reproducibility, Tessier and Rauret SEPs were comparable to each other, whereas Shiowatana SEP showed higher extraction efficiency in all the first three arsenic fractions, although it might overestimate the reducible arsenic. Pot experiment indicated three SEPs all could provide an estimation of the most bioavailable arsenic fraction, and the application of Shiowatana SEP should be preferred. Accordingly, a case study with Shiowatana SEP for a site near a realgar mine area is conducted. The results show that although arsenic in this area presents predominantly in the stable fractions, the sum of most bioavailable fractions was accounted around 11% of total arsenic, and moreover, about another 10% of the total arsenic, the fourth fraction in Shiowatana SEP is likely to be transferred into bioavailable species under suitable conditions, such as strong acid impact, revealing a real major risk source being formed. The study indicated that Shiowatana should be more suitable for arsenic fractionation to provide valuable information in the framework of risk assessment.