Tissue engineering. Part C, Methods最新文献

The aim of this study was to analyze the effect of ozone (OZN) therapy on the dynamics of bone tissue in ovariectomized rats treated with zoledronic acid (ZOL). Female Wistar rats aged 6 months (n = 110) were subjected to bilateral ovariectomy (OVX). At month 3 post-OVX, 10 animals were euthanized to characterize the bone tissue architecture using microtomography (micro-CT). The remaining animals were divided into two groups: ZOL group, administered with ZOL (100 μg/kg body weight); saline (SAL) group (0.45 mL of SAL solution), both for 28 days. At month 3 post-treatment, 10 animals from each group were euthanized to characterize the bone architecture using micro-CT. The remaining animals were divided into the following groups: ZOL (n = 20), ZOL + OZN (n = 20); SAL (n = 20), and SAL + OZN (n = 20). The animals in ZOL + OZN and SAL + OZN groups were intraperitoneally administered with OZN (0.7 mg/kg body weight) once every 2 days. On days 30 and 60, six animals from each group were euthanized for analysis and structural characterization of bones in the femoral head and spine. Some samples of the femoral neck were subjected to biomechanical tests, while some samples were analyzed under a laser confocal microscope. The other samples collected from the femoral neck and spine were analyzed for area of neoformed bone and used for performing inflammatory cell and osteocyte counts. Data were submitted to statistical analysis considering a significance level of p < 0.05. Bone volume percentage and osteocyte and inflammatory cell counts were upregulated in the femoral head region of the ZOL + OZN group. Biomechanical analysis of the femoral neck revealed that the modulus of elasticity was similar between the ZOL and ZOL + OZN groups but differed significantly between the SAL and SAL + OZN groups. The positive areas for calcein and alizarin in the ZOL and ZOL + OZN groups were higher than those in the SAL and SAL + OZN groups. This suggested a positive synergistic effect of OZN and ZOL on the maintenance of bone mass and restoration of bone tissue vitality in ovariectomized rats.

Insufficient vascularization is still a challenge that impedes bladder tissue engineering and results in unsatisfied smooth muscle regeneration. Since bladder regeneration is a complex articulated process, the aim of this study is to investigate whether combining multiple pathways by exploiting a combination of biomaterials, cells, and bioactive factors, contributes to the improvements of smooth muscle regeneration and vascularization in tissue-engineered bladder. Autologous endothelial progenitor cells (EPCs) and bladder smooth muscle cells (BSMCs) are cultured and incorporated into our previously prepared porcine bladder acellular matrix (BAM) for bladder augmentation in rabbits. Simultaneously, exogenous vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) mixed with Matrigel were injected around the implanted cells-BAM complex. In the results, compared with control rabbits received bladder augmentation with porcine BAM seeded with BSMCs, the experimental animals showed significantly improved smooth muscle regeneration and vascularization, along with more excellent functional recovery of tissue-engineered bladder, due to the additional combination of autologous EPCs and bioactive factors, including VEGF and PDGF-BB. Furthermore, cell tracking suggested that the seeded EPCs could be directly involved in neovascularization. Therefore, it may be an effective method to combine multiple pathways for tissue-engineering urinary bladder.

In the past, different spheroid-, organotypic-, and three-dimensional (3D) bioprinting lung cancer models were established for in vitro drug testing and personalized medicine. These tissue models cannot depict the tumor microenvironment (TME) and, therefore, research addressing tumor cell-TME interactions is limited. To overcome this hurdle, we applied patient-derived lung tumor samples to establish new in vitro models. To analyze the tissue model properties, we established two-dimensional (2D) and 3D coculture tissue models exposed to static and dynamic culture conditions that afforded tissue culture for up to 28 days. Our tissue models were characterized by hematoxylin eosin staining, M30 enzyme-linked immunosorbent assay, and immunofluorescence staining against specific lung cancer markers (TTF-1 and p40/p63), cancer-associated fibroblast (CAF) markers (α-SMA and MCT4), and fibronectin (FN). The 3D models were generated with higher success rate than the corresponding 2D model. The cell density of the static 3D model increased from 21 to 28 days, whereas the apoptosis decreased. The dynamic 3D model possessed an even higher cell density than the static 3D model. We identified lung cancer cells, CAFs, and FN. Therefore, a novel in vitro 3D lung cancer model was established, which simulated the TME for 28 days and possessed a structural complexity.

Background: Chronic kidney disease (CKD) poses a global health challenge, and it needs alternative therapeutic approaches for patients with end-stage renal disease (ESRD). Although organ transplantation is effective, it faces challenges such as declining quality of life, immunological responses, transplant rejection, and donor shortages. Tissue engineering, by using suitable scaffolds, cells, and growth factors, emerges as a promising treatment option for kidney regeneration. Experiment: We precisely decellularized scaffold, derived from rat kidneys while maintaining its native three-dimensional (3D) architecture. The efficiency of decellularization was evaluated through histological examinations, including hematoxylin and eosin, periodic acid-Schiff, and DAPI staining, as well as scanning electron microscopy. The scaffolds were then recellularized with kidney mesenchymal stem cells (kMSCs), and their adhesion, proliferation, and differentiation were assessed over 1, 2, and 3 weeks. The expression of specific renal markers, including Wt-1, ZO-1, AQP-1, and ANG-1, was examined through quantitative reverse transcription-polymerase chain reaction (qRT-PCR) in monolayer and 3D cultures. Results: The infiltration rate of cells into the scaffold increased in a time-dependent manner, and the expression of specific renal markers significantly increased, demonstrating successful differentiation of kMSCs within the scaffold. The application of basic fibroblast growth factor (bFGF) could intensify the expression of kidney-specific genes. Conclusions: The study highlighted the importance of preserving the 3D architecture of the scaffold during decellularization to achieve optimal cellular responses. Moreover, the capacity of mesenchymal stem cells in recellularized scaffolds facilitated tissue regeneration.

Tendon stem/progenitor cell (TSPC) senescence is often associated with age-dependent tendon diseases and greatly reduces the capacities for tendon repair and replacement. Exosomes contain bioactive molecules and have been increasingly used in regenerative medicine. In the present study, we demonstrated the antiaging effects of young exosomes from circPVT1-overexpressing TSPCs at early passages (circPVT1-exo). These exosomes attenuated the phenotypes of aged TSPCs at late passages (L-TSPCs) by enhancing self-renewal and proliferation abilities, suppressing cell senescence, maintaining their tenogenic capacity, and weakening their osteogenic differentiation. Mechanistically, circPVT1-exo inhibited the NF-κB pathway and increased SIRT1 expression in L-TSPCs. Knockdown of SIRT1 reversed these effects as evidenced by increased senescence, decreased proliferation, and tenogenic differentiation. These results suggest that circPVT1-exo may ameliorate aging-impaired TSPC function by modulating the SIRT1/NF-κB pathway, suggesting that circPVT1-exo has therapeutic potential for age-related diseases.

Donor cell-specific tissue-engineered (TE) implants are a promising therapy for personalized treatment of cardiovascular diseases, but current development protocols lack a stable longitudinal assessment of tissue development at subcellular resolution. As a first step toward such an assessment approach, in this study we establish a generalized labeling and imaging protocol to obtain quantified maturation parameters of TE constructs in three dimensions (3D) without the need of histological slicing, thus leaving the tissue intact. Focusing on intracellular matrix (ICM) and extracellular matrix (ECM) networks, multiphoton laser scanning microscopy (MPLSM) was used to investigate TE patches of different conditioning durations of up to 21 days. We show here that with a straightforward labeling procedure of whole-mount samples (so without slicing into thin histological sections), followed by an easy-to-use multiphoton imaging process, we obtained high-quality images of the tissue in 3D at various time points during development. The stacks of images could then be further analyzed to visualize and quantify the volume of cell coverage as well as the volume fraction and network of structural proteins. We showed that collagen and alpha-smooth muscle actin (α-SMA) volume fractions increased as normalized to full tissue volume and proportional to the cell count, with a converging trend to the final density of (4.0% ± 0.6%) and (7.6% ± 0.7%), respectively. The image analysis of ICM and ECM revealed a developing and widely branched interconnected matrix. We are currently working on the second step, that is, to integrate MPLSM endoscopy into a dynamic bioreactor system to monitor the maturation of intact TE constructs over time, thus without the need to take them out.

This work employs nitrogen plasma immersion ion implantation (PIII) to modify electrospinning polylactic acid membranes and immobilizes basic fibroblast growth factors (bFGF) by forming crosslinking bonds. The study investigates the modified membranes' surface characteristics and the stimulatory effects of crosslinked bFGF polylactic acid membranes on osteoblast and fibroblast proliferation. The PIII process occurs under low vacuum conditions and is controlled by processing time and power pulse width. The experimental results indicate that, within a 400-second N₂-PIII treatment, the spun fibers remain undamaged, demonstrating an increase in hydrophilicity (from 117° to 38°/36°) and nitrogen content (from 0% to 7.54%/8.05%). X-ray photoelectron spectroscopy analysis suggests the formation of a C-N-C=O crosslinked bond. Cell culture and activity assessments indicate that the PIII-treated and crosslinked bFGF film exhibits significantly higher cell growth activity (p < 0.05) than the untreated group. These intergroup differences are attributed to the surface crosslinking bond content. In osteogenic induction, the results for each day show that the treated group performs better. However, the intergroup disparities within the crosslinked bFGF group disappear with prolonged culture time due to the rapid osteogenesis prompted by bFGF. The findings suggest that PIII treatment of electrospinning polylactic acid membranes holds promise in promoting osteogenesis in bone tissue scaffolds.

Fecal incontinence, although not life-threatening, has a high impact on the economy and patient quality of life. So far, available treatments are based on both surgical and nonsurgical approaches. These can range from changes in diet, to bowel training, or sacral nerve stimulation, but none of which provides a long-term solution. New regenerative medicine-based therapies are emerging, which aim at regenerating the sphincter muscle and restoring continence. Usually, these consist of the administration of a suspension of expanded skeletal-derived muscle cells (SkMDCs) to the damaged site. However, this strategy often results in a reduced cell viability due to the need for cell harvesting from the expansion platform, as well as the non-native use of a cell suspension to deliver the anchorage-dependent cells. In this study, we propose the proof-of-concept for the bioprocessing of a new cell delivery method for the treatment of fecal incontinence, obtained by a scalable two-step process. First, patient-isolated SkMDCs were expanded using planar static culture systems. Second, by using a single-use PBS-MINI Vertical-Wheel^® bioreactor, the expanded SkMDCs were combined with biocompatible and biodegradable (i.e., directly implantable) poly(lactic-co-glycolic acid) microcarriers prepared by thermally induced phase separation. This process allowed for up to 80% efficiency of SkMDCs to attach to the microcarriers. Importantly, SkMDCs were viable during all the process and maintained their myogenic features (e.g., expression of the CD56 marker) after adhesion and culture on the microcarriers. When SkMDC-containing microcarriers were placed on a culture dish, cells were able to migrate from the microcarriers onto the culture surface and differentiate into multinucleated myotubes, which highlights their potential to regenerate the damaged sphincter muscle after administration into the patient. Overall, this study proposes an innovative method to attach SkMDCs to biodegradable microcarriers, which can provide a new treatment for fecal incontinence.

Traumatic injuries may result in the formation of soft tissue adhesions between peripheral nerves and surrounding soft tissue. These soft tissue adhesions lead to compression and ischemic stress within fascicles due to nonpliability of adhered scar tissue, and nerve tension due to loss of nerve gliding from scar tethering. These changes in the soft tissue bed surrounding the nerve may result in axon degeneration and neuroma-in-continuity. Preclinical models that simulate clinically relevant levels of scar in the nerve environment may be impactful to the development of surgical techniques and treatments to prevent adhesions. This study presents the results of a rodent model with an induced indirect nerve injury by (1) thermal insult to the soft tissue bed surrounding the nerve and (2) air-drying the surrounding soft tissue bed of the nerve. Our findings suggest that inducing an injury of the soft tissue bed results in increased intraneural scar and extraneural adhesions to the nerve compared to a sham procedure. Thermal induced injuries showed more macrophages and changes in nerve health compared to air-dried induced injuries. The changes in the nerves of the induced injury groups, specifically the thermal injury group, may be meaningful for evaluating treatments for nontransected nerve injuries.

Cardiac fibrosis is characterized by pathological proliferation and activation of cardiac fibroblasts to myofibroblasts. Inhibition and reverse of transdifferentiation of cardiac fibroblasts to myofibroblasts is a potential strategy for cardiac fibrosis. Despite substantial progress, more effort is needed to discover effective drugs to improve and reverse cardiac fibrosis. The main reason for the slow development of antifibrotic drugs is that the traditional polystyrene culture platform does not recapitulate the microenvironment where cells reside in tissues. In this study, we propose an in vitro cardiac fibrotic model by seeding electrospun yarn scaffolds with cardiac fibroblasts. Our results show that yarn scaffolds allow three-dimensional growth of cardiac fibroblasts, promote extracellular matrix (ECM) deposition, and induce the transdifferentiation of cardiac fibroblasts to myofibroblasts. Exogenous transforming growth factor-β1 further promotes cardiac fibroblast activation and ECM deposition, which makes it a suitable fibrotic model to predict the antifibrotic potential of drugs. By using this platform, we demonstrate that both Honokiol (HKL) and Pirfenidone (PFD) show potential in antifibrosis to some extent. HKL is more efficient in antifibrosis than PFD as revealed by biochemical composition, gene, and molecular analyses as well as histological and biomechanical analysis. The electrospun yarn scaffold provides a novel platform for constructing in vitro fibrotic models to study cardiac fibrosis and to predict the antifibrotic efficacy of novel drugs.