Tissue Engineering Part A最新文献

In recent years, there has been a significant expansion in the realm of processing microscopy images, thanks to the advent of machine learning techniques. These techniques offer diverse applications for image processing. Currently, numerous methods are used for processing microscopy images in the field of biology, ranging from conventional machine learning algorithms to sophisticated deep learning artificial neural networks with millions of parameters. However, a comprehensive grasp of the intricacies of these methods usually necessitates proficiency in programming and advanced mathematics. In our comprehensive review, we explore various widely used deep learning approaches tailored for the processing of microscopy images. Our emphasis is on algorithms that have gained popularity in the field of biology and have been adapted to cater to users lacking programming expertise. In essence, our target audience comprises biologists interested in exploring the potential of deep learning algorithms, even without programming skills. Throughout the review, we elucidate each algorithm's fundamental concepts and capabilities without delving into mathematical and programming complexities. Crucially, all the highlighted algorithms are accessible on open platforms without requiring code, and we provide detailed descriptions and links within our review. It's essential to recognize that addressing each specific problem demands an individualized approach. Consequently, our focus is not on comparing algorithms but on delineating the problems they are adept at solving. In practical scenarios, researchers typically select multiple algorithms suited to their tasks and experimentally determine the most effective one. It is worth noting that microscopy extends beyond the realm of biology; its applications span diverse fields such as geology and material science. Although our review predominantly centers on biomedical applications, the algorithms and principles outlined here are equally applicable to other scientific domains. Furthermore, a number of the proposed solutions can be modified for use in entirely distinct computer vision cases.

This study addresses a critical challenge in bioprinting for regenerative medicine, specifically the issue of hypoxia compromising cell viability in engineered tissues. To overcome this hurdle, a novel approach using a microfluidic bioprinter is used to create a two-layer structure resembling the human ovary. This structure incorporates a liposomal oxygen-releasing system to enhance cell viability. The bioprinting technique enables the simultaneous extrusion of two distinct bioinks, namely, bioink A (comprising alginate 1% and 5 mg/mL PEGylated fibrinogen in a 20:1 molar ratio) and bioink B (containing alginate 0.5%). In addition, liposomal catalase and hydrogen peroxide (H₂O₂) are synthesized and incorporated into bioinks A and B, respectively. The liposomes are prepared using thin film hydration with a monodisperse size (140-160 nm) and high encapsulation efficiency. To assess construct functionality, isolated human ovarian cells are added to bioink A. The bioprinted constructs, with or without liposomal oxygen-releasing systems, are cultured under hypoxic and normoxic conditions for 3 days. Live/Dead assay results demonstrate that liposomal oxygen-releasing systems effectively preserve cell viability in hypoxic conditions, resembling viability under normoxic conditions without liposomes. PrestoBlue assay reveals significantly higher mitochondrial activity in constructs with liposomal oxygen delivery systems under both hypoxic and normoxic conditions. The evaluation of apoptosis status through annexin V immunostaining shows that liposomal oxygen-releasing scaffolds successfully protect cells from hypoxic stress, exhibiting a proportion of apoptotic cells similar to normoxic conditions. In contrast, constructs lacking liposomes in hypoxic conditions exhibit a higher incidence of cells in early-stage apoptosis. In conclusion, the study demonstrates the promising potential of bioprinted oxygen-releasing liposomal scaffolds to protect ovarian stromal cells in hypoxic environments. These innovative scaffolds not only offer protection but also recapitulate the mechanical differences between the medulla and the cortex in the normal ovary structure. This opens new avenues for advanced ovary tissue engineering and transplantation strategies.

A mino acids are the essential building blocks for collagen and proteoglycan, which are the main constituents for cartilage extracellular matrix (ECM). Synthesis of ECM proteins requires the uptake of various essential/nonessential amino acids. Analyzing amino acid metabolism during chondrogenesis can help to relate tissue quality to amino acid metabolism under different conditions. In our study, we studied amino acid uptake/secretion using human mesenchymal stem cell (hMSC)-based aggregate chondrogenesis in a serum-free induction medium with a defined chemical formulation. The initial glucose level and medium-change frequency were varied. Our results showed that essential amino acid uptake increased with time during hMSCs chondrogenesis for all initial glucose levels and medium-change frequencies. Essential amino acid uptake rates were initial glucose-level independent. The DNA-normalized glycosaminoglycans and hydroxyproline content of chondrogenic aggregates correlated with cumulative uptake of leucine, valine, and tryptophan regardless of initial glucose levels and medium-change frequencies. Collectively, our results show that amino acid uptake rates during in vitro chondrogenesis were insufficient to produce a tissue with an ECM content similar to that of human neonatal cartilage or adult cartilage. Furthermore, this deficiency was likely related to the downregulation of some key amino acid transporters in the cells. Such deficiency could be partially improved by increasing the amino acid availability in the chondrogenic medium by changing culture conditions.

Negative pressure therapy (NPT) has been shown to facilitate wound healing and promote hair growth in a porcine model. However, there is a paucity of research on the impact of negative pressure on hair growth in murine models. Despite the ability of nude mice to develop hair follicles, the hair they produce is often flawed towing to genetically induced keratin disorders, rendering them a pertinent animal model for assessing hair regeneration. Therefore, this study aims to investigate the effects of negative pressure on hair follicle growth in a nude mouse model. To achieve this, a customized external tissue expansion device was developed to apply negative pressure to the dorsum of nude mice. The mice were subjected to several treatment courses consisting of 15 and 30 min of continuous negative pressure at 10 mmHg, which were repeated 5 and 10 times every other day until sacrifice. Dorsal skin samples were subsequently extracted from the suction and nonsuction areas. The sections were stained with various antibodies to assess the expression of SOX-9, LHX-2, Keratin-15, β-catenin, CD31, and vascular endothelial growth factor-A, and a TUNEL assay was used to analyze cell apoptosis. The results showed that the number of hair follicles and angiogenesis were significantly higher in the suction area than in the nonsuction area in all groups. Moreover, mice that received NPT for 15 min for 10 times had a higher hair follicle density than the other three groups. Immunofluorescence staining for LHX-2 and Keratin 15 further validated the results of these findings. In conclusion, this study demonstrated that negative pressure effectively promotes hair follicle growth and angiogenesis in nude mice through SOX-9- and LHX-2-mediated follicular regeneration and β-catenin-mediated hair follicle morphogenesis.

Current treatment options for craniofacial volumetric muscle loss (VML) have disadvantages and cannot fully restore normal function. Bio-inspired semisynthetic acrylated hyaluronic acid (AcHyA) hydrogel, which fills irregularly shaped defects, resembles an extracellular matrix, and induces a minimal inflammatory response, has shown promise in experimental studies of extremity VML. We therefore sought to study AcHyA hydrogel in the treatment of craniofacial VML. For this, we used a novel model of masseter VML in the rat. Following the creation of a 5 mm × 5 mm injury to the superficial masseter and administration of AcHyA to the wound, masseters were explanted between 2 and 16 weeks postoperatively and were analyzed for evidence of muscle regeneration including fibrosis, defect size, and fiber cross-sectional area (FCSA). At 8 and 16 weeks, masseters treated with AcHyA showed significantly less fibrosis than nonrepaired controls and a smaller decrease in defect size. The mean FCSA among fibers near the defect was significantly greater among hydrogel-repaired than control masseters at 8 weeks, 12 weeks, and 16 weeks. These results show that the hydrogel mitigates the fibrotic healing response and wound contracture. Our findings also suggest that hydrogel-based treatments have potential use as a treatment for the regeneration of craniofacial VML and demonstrate a system for evaluating subsequent iterations of materials in VML injuries.

Cerebral cavitation is usual following acute brain injuries, such as stroke and traumatic brain injuries, as well as after tumor resection. Minimally invasive implantation of an injectable scaffold in the cavity is a promising approach for potential regeneration of tissue loss. This study aimed at designing an in situ-gelling conductive hydrogel containing silk fibroin (SF), brain decellularized extracellular matrix (dECM), and carbon nanotubes (CNT) for potential use in brain tissue regeneration. Two percent w/v SF hydrogels with different concentrations of dECM (0.1%, 0.2%, or 0.3% w/v) and CNTs (0.05%, 0.1%, or 0.25% w/v) were fabricated and characterized. It was observed that with the addition of dECM, the porosity decreased, whereas swelling and electrical conductivity tended to increase. The addition of dECM also led to a faster resorption rate, but no significant change in compressive modulus. Addition of CNTs, on the other hand, led to a denser, stronger, and more regular porous structure, higher swelling ratio, faster gelation time, slower degradation rate, and a significant increase in electrical conductivity. dECM and CNTs combined together resulted in superior porosity, swelling, resorption rate, mechanical properties, and electrical conductivity compared with SF scaffolds containing only dECM or CNTs. Hydrogel samples containing 2% SF, 0.3% dECM, and 0.1% CNTs had a high porosity (58.9%), low swelling ratio (15.9%), high conductivity (2.35 × 10^-4 S/m), and moderate degradation rate (37.3% after 21 days), appropriate for neural tissue engineering applications. Cell evaluation studies also showed that the hydrogel systems support the cell adhesion and growth, with no sign of significant cytotoxicity. Impact statement Tissue loss and formation of a fluid-filled cavity following stroke, traumatic brain injury, or brain tumor resection lead to sensorimotor and/or cognitive deficits. The lack of a healthy extracellular matrix in the cavity avoids the endogenous cell migration and axonal sprouting and may also worsen the secondary injuries to peri-lesional tissue. Due to the brain anatomy, simple implantation of tissue engineering scaffolds to the injured site is not possible in many cases. Therefore, the development of injectable scaffolds that support neural growth and differentiation is crucial for tissue repair or limiting the expansion of damage region.

Juvenile idiopathic arthritis is an inflammatory disease that can affect the temporomandibular joint (TMJ) and lower jaw growth. Better treatment options are needed, so this study investigated the effect of low-intensity pulsed ultrasound (LIPUS) on TMJ arthritis. Seventy-two 3-week-old male Wistar rats were in vivo microcomputed tomography (micro-CT) scanned and divided into eight groups (n = 9). These groups were Group 1-TMJ arthritis and immediate LIPUS treatment (20 min/day, 4 weeks); Group 2-immediate LIPUS treatment and no TMJ arthritis; Group 3-TMJ arthritis and no LIPUS; Group 4-no TMJ arthritis and no LIPUS; Group 5-TMJ arthritis and LIPUS treatment with a delayed start by 4 weeks; Group 6-Delayed LIPUS and no TMJ arthritis; Group 7-TMJ arthritis and no (delayed) LIPUS; and Group 8-no TMJ arthritis and no (delayed) LIPUS. Ex vivo micro-CT scanning was completed, and samples were prepared for tissue analysis. Synovitis was observed in the TMJ arthritis (collagen-induced arthritis [CIA]) groups, but the severity appeared greater in the groups without LIPUS treatment. Fibrocartilage and hypertrophic cell layer thicknesses in the CIA group without LIPUS treatment were significantly greater (p < 0.05). Proteoglycan staining appeared greater in the LIPUS groups. Immediate LIPUS treatment increased the expression of type II collagen, type X collagen, and transforming growth factor-beta 1 (TGF-β1) immunostaining, and CIA (no LIPUS) increased MMP-13, vascular endothelial growth factor, and interleukin-1 beta (IL-1β) immunostaining. LIPUS treatment prevented growth disturbances observed in the CIA groups (no LIPUS) (p < 0.005). Our results have contributed to the understanding of the uses and limitations of the CIA juvenile rat model and have demonstrated the effects of LIPUS on the TMJ and mandibular growth. This information will help in designing future studies for investigating LIPUS and TMJ arthritis, leading to the development of new treatment options for children with juvenile arthritis in their TMJs.

In the realm of in situ cartilage engineering, the targeted delivery of both cells and hydrogel materials to the site of a defect serves to directly stimulate chondral repair. Although the in situ application of stem cell-laden soft hydrogels to tissue defects holds great promise for cartilage regeneration, a significant challenge lies in overcoming the inherent limitation of these soft hydrogels, which must attain mechanical properties akin to the native tissue to withstand physiological loading. We therefore developed a system where a gelatin methacryloyl hydrogel laden with human adipose-derived mesenchymal stem cells is combined with a secondary structure to provide bulk mechanical reinforcement. In this study, we used the negative embodied sacrificial template 3D printing technique to generate eight different lattice-based reinforcement structures made of polycaprolactone, which ranged in porosity from 80% to 90% with stiffnesses from 28 ± 5 kPa to 2853 ± 236 kPa. The most promising of these designs, the hex prism edge, was combined with the cellular hydrogel and retained a stable stiffness over 41 days of chondrogenic differentiation. There was no significant difference between the hydrogel-only and hydrogel scaffold group in the sulfated glycosaminoglycan production (340.46 ± 13.32 µg and 338.92 ± 47.33 µg, respectively) or Type II Collagen gene expression. As such, the use of negative printing represents a promising solution for the integration of bulk reinforcement without losing the ability to produce new chondrogenic matrix.

Subchondral drilling (SD), a bone marrow stimulation technique, is used to repair cartilage lesions that lack regenerative potential. Cartilage repair outcomes upon SD are typically fibrocartilaginous in nature with inferior functionality. The lack of cues to foster the chondrogenic differentiation of egressed mesenchymal stromal cells upon SD can be attributed for the poor outcomes. Continuous low-intensity ultrasound (cLIUS) at 3.8 MHz is proposed as a treatment modality for improving cartilage repair outcomes upon marrow stimulation. Bilateral defects were created by SD on the femoral medial condyle of female New Zealand white rabbits (n = 12), and the left joint received cLIUS treatment (3.8 MHz, 3.5 Vpp, 8 min/application/day) and the contralateral right joint served as the control. On day 7 postsurgery, synovial fluid was aspirated, and the cytokine levels were assessed by Quantibody™ assay. Rabbits were euthanized at 8 weeks and outcomes were assessed macroscopically and histologically. Defect areas in the right joints exhibited boundaries, incomplete fill, irregular cartilage surfaces, loss of glycosaminoglycan (GAG), and absence of chondrocytes. In contrast, the repaired defect area in the joints that received cLIUS showed complete fill, positive staining for GAG with rounded chondrocyte morphology, COL2A1 staining, and columnar organization. Synovial fluid collected from cLIUS-treated left knee joints had lower levels of IL1, TNFα, and IFNγ when compared to untreated right knee joints, alluding to the potential of cLIUS to mitigate early inflammation. Further at 8 weeks, left knee joints (n = 12) consistently scored higher on the O'Driscoll scale, with a higher percent hyaline cartilage score. No adverse impact on bone or change in the joint space was noted. Upon a single exposure of cLIUS to TNFα-treated cells, nuclear localization of pNFκB and SOX9 was visualized by double immunofluorescence and the expression of markers associated with the NFκB pathway was assayed by quantitative real-time polymerase chain reaction. cLIUS extends its chondroprotective effects by titrating pNFκB levels, preventing its nuclear translocation, while maintaining the expression of SOX9, the collagen II transcription factor. Our combined results demonstrate that healing of chondral defects treated with marrow stimulation by SD can be accelerated by employing cLIUS regimen that possesses chondroinductive and chondroprotective properties. Impact statement Repair of cartilage represents an unsolved biomedical burden. In vitro, continuous low-intensity ultrasound (cLIUS) has been demonstrated to possess chondroinductive and chondroprotective potential. To our best knowledge, the use of cLIUS to improve cartilage repair outcomes upon marrow stimulation, in vivo, has not been reported and our work reported here fills that gap. Our results demonstrated enhanced cartilage repair outcomes under cLIUS (3.8 MHz) in a rabbit model of s

In this study, we develop a bio-based and bioactive nanofibrous patch based on bacterial cellulose (BC) and chitin nanofibrils (CNs) using an ionic liquid as a solvent for BC, aimed at tympanic membrane (TM) repair. Electrospun BC nanofiber meshes were produced via electrospinning, and surface-modified with CNs using electrospray. The rheology of the BC/ionic liquid system was investigated. The obtained CN/BC meshes underwent comprehensive morphological, physicochemical, and mechanical characterization. Cytotoxicity tests were conducted using L929 mouse fibroblasts, revealing a cell viability of 97.8%. In vivo tests on rabbit skin demonstrated that the patches were nonirritating. Furthermore, the CN/BC fiber meshes were tested in vitro using human dermal keratinocytes (HaCaT cells) and human umbilical vein endothelial cells as model cells for TM perforation healing. Both cell types demonstrated successful growth on these scaffolds. The presence of CNs resulted in improved indirect antimicrobial activity of the electrospun fiber meshes. HaCaT cells exhibited an upregulated mRNA expression at 6 and 24 h of key proinflammatory cytokines crucial for the wound healing process, indicating the potential benefits of CNs in the healing response. Overall, this study presents a natural and eco-sustainable fiber mesh with great promise for applications in TM repair, leveraging the synergistic effects of BC and CNs to possibly enhance tissue regeneration and healing. Impact statement Repair of tympanic membrane perforations following chronic otitis media is a main clinical issue in otologic surgery, where the underlying infection obstacles self-healing. To address this challenge, our study proposes a bio-based patch made of nanoscale carbohydrate materials (i.e., bacterial cellulose electrospun fibers and chitin nanofibrils) processed via green solvents. The scaffold is nonirritating in vivo, and cytocompatible with fibroblasts, endothelial cells, and keratinocytes. In epithelial cells, it stimulates the expression of the antimicrobial peptide human beta defensin 2, with a pathway of cytokine expression compatible with the wound healing process. Therefore, it could be applied with unsolved infective pathology.