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Journal of Morphology Sep 2023In viviparous teleosts, intraovarian gestation occurs intrafollicularly, as in poeciliids, or intraluminally, as in goodeids and anablepids. Furthermore, there are two...
In viviparous teleosts, intraovarian gestation occurs intrafollicularly, as in poeciliids, or intraluminally, as in goodeids and anablepids. Furthermore, there are two different forms of embryonic nutrition: lecithotrophy and matrotrophy; depending on the species, these can be exclusive or coexist during gestation. In matrotrophic species, nutrients are transmitted from the mother to the embryo and are especially important in species with intraluminal gestation. Jenynsia lineata is a South American viviparous teleost with intraluminal gestation, characterized by eggs with scarce yolk, which is resorbed when embryos are 6 mm long, thus developing a branchial placenta. Using histological, histochemical, and immunohistochemical techniques, the present study describes the characteristics and changes of the ovarian mucosa in J. lineata during gestational and nongestational phases, and analyzes the embryonic pharyngeal epithelium in the branchial placenta. The ovaries of 30 adult female specimens were processed using histological techniques and stained with hematoxylin-eosin, Masson's trichrome, and Alcian Blue pH 2.5/periodic acid Schiff reagent. To detect cell proliferation, we used antiproliferating cell nuclear antigen antibody. In nonpregnant females, eosinophilic granular cells (EGCs) and lymphocytes were identified in the lamina propria of the tunica mucosa, and melanomacrophage centers (MMCs) and fibroblasts were identified adjacent to tissue debris in the ovarian folds'. In the cellular debris, an embryo in resorption was observed. In pregnant females, the ovarian mucosa has thin vascularization branches entering the opercular chamber of the embryos, in close contact with the forming gill processes, thereby establishing a branchial placenta. Active cell replacement was observed in these ovarian branches. The identification of fibroblasts, lymphocytes, EGCs, and MMCs adjacent to tissue debris could indicate that these cell types are involved in the embryonic resorption process. Considering the new data obtained in this study on the branchial placenta of J. lineata, we conclude that cell proliferation could be involved in the development of maternal-embryonic interaction.
Topics: Female; Animals; Pregnancy; Ovary; Placenta; Cell Nucleus; Cell Proliferation; Cyprinodontiformes
PubMed: 37585233
DOI: 10.1002/jmor.21630 -
Microscopy and Microanalysis : the... Jul 2023
PubMed: 37613754
DOI: 10.1093/micmic/ozad067.505 -
Journal of Orthopaedic Translation Sep 2023Dedifferentiated fat cells (DFATs) are highly homogeneous and multipotent compared with adipose-derived stromal cells (SCs). Infrapatellar fat pad (IFP)-SCs have...
BACKGROUND
Dedifferentiated fat cells (DFATs) are highly homogeneous and multipotent compared with adipose-derived stromal cells (SCs). Infrapatellar fat pad (IFP)-SCs have advanced chondrogenic potency; however, whether IFP-DFATs could serve as better cell material remains unclear. Here, we aimed to examine the influence of age and body mass index (BMI) on the features of IFPs and IFP-derived cells (IFP-SCs and IFP-DFATs) with exploration of the clinical utilization of IFP-DFATs.
METHODS
We collected IFPs with isolation of paired IFP-SCs and IFP-DFATs from individuals aged 65 years and older with distinct body weights who underwent total knee replacement for osteoarthritis (OA). Flow cytometry was used to characterize the cellular immunophenotypes. Adipogenesis and chondrogenesis were performed . Real-time qPCR, western blotting, and Oil Red O or Alcian blue staining were performed to evaluate inflammation, adipogenesis, and chondrogenesis. RNA sequencing and Seahorse analyses were conducted to explore the underlying mechanisms.
RESULTS
We found that IFPs from old or normal-weight individuals with knee OA were pro-inflammatory, and that interleukin-6 (IL-6) signaling was associated with multiple immune-related molecules, whereas IFP-derived cells could escape the inflammatory properties. Aging plays an important role in diminishing the chondrogenic and adipogenic abilities of IFP-SCs; however, this effect was avoided in IFP-DFATs. Generally, IFP-DFATs presented a steady state of chondrogenesis (less influenced by age) and consistently enhanced adipogenesis compared to paired IFP-SCs in different age or BMI groups. RNA sequencing and Seahorse analysis suggested that the downregulation of eukaryotic initiation factor 2 (EIF2) signaling and enhanced mitochondrial function may contribute to the improved cellular biology of IFP-DFATs.
CONCLUSIONS
Our data indicate that IFP-DFATs are superior cell material compared to IFP-SCs for cartilage differentiation and adipogenesis, particularly in advanced aging patients with knee OA.
THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE
These results provide a novel concept and supportive evidence for the use of IFP-DFATs for cell therapy or tissue engineering in patients with knee OA. Using Ingenuity Pathway Analysis (IPA) of RNA-seq data and Seahorse analysis of mitochondrial metabolic parameters, we highlighted that some molecules, signaling pathways, and mitochondrial functions are likely to be jointly coordinated to determine the enhanced biological function in IFP-DFATs.
PubMed: 37680904
DOI: 10.1016/j.jot.2023.08.001 -
International Journal of Fertility &... Nov 2023Men's infertility and lack of production of healthy and active sperm are concerns of recent years in most countries. Studies on the preparation of extracellular matrix...
BACKGROUND
Men's infertility and lack of production of healthy and active sperm are concerns of recent years in most countries. Studies on the preparation of extracellular matrix (ECM) from decellularization of testis tissue and spermatogenesis could provide proper results to solve some of the men's infertility problems. This study aims to decellularize calf testis by different methods to reach a suitable scaffold and introduce it in spermatogenesis studies.
MATERIALS AND METHODS
In this experimental study, calf testis were decellularized by a freeze-de freeze, 1% sodium deoxycholate (SD), 0.1% sodium dodecyl sulfate (SDS), 0.1% SDS-vacuum, 1% SDS, 1% SDS-vacuum, and Triton- X100 methods. The content of DNA, collagen, and glycosaminoglycan (GAG) was analyzed using the kit and staining with Hematoxylin-Eosin, Masson's trichrome, Alcian blue, and Orcein methods. The morphology of the scaffolds was analyzed with a scanning electron microscope (SEM).
RESULTS
Methods of 1% SDS, 1% SDS-vacuum, and 1% SD completely removed the cells. The preservation of collagen and GAG was confirmed using the staining kit and methods. The use of a vacuum showed greater porosity in the SEM images. Toxicity and hemolysis were not observed in the scaffolds.
CONCLUSION
Testis decellularization with 1% SDS and 1% SD, in addition to cell removal, could maintain the ECM structure to a large extent without having cytotoxic and hemolysis effects.
PubMed: 38041457
DOI: 10.22074/ijfs.2023.1989173.1433 -
The Journal of Surgical Research Jan 2024Chronic inflammation of the intestinal epithelium is an underlying cause of the two main types of inflammatory bowel disease (IBD), ulcerative colitis (UC), and Crohn's...
INTRODUCTION
Chronic inflammation of the intestinal epithelium is an underlying cause of the two main types of inflammatory bowel disease (IBD), ulcerative colitis (UC), and Crohn's disease (CD). Ex vivo organoids derived from the intestinal epithelium are a useful model to study IBD. Whether such cultures can be established from surgically resected diseased IBD intestinal tissues has not been fully explored. In this study, we tested our ability to establish organoids from nondiseased and diseased IBD intestinal tissues.
MATERIALS AND METHODS
From 12 UC patients (n = 54 tissues) and 20 CD patients (n = 49 tissues), tissues were collected from multiple colonic regions, and for CD, the terminal ileum was also surveyed. Organoids were cultured in Matrigel domes using defined media. In primary tissues, we conducted immunohistochemical analysis for mucin 2 (MUC2) and Alcian blue staining for goblet cells. Organoids were stained for Ki67, E-cadherin, and MUC2.
RESULTS
For UC, we were highly successful establishing organoids from nondiseased tissue (n = 12 of 13, 92%). This success rates dropped from tissues with mild (n = 6 of 9, 67%), moderate (n = 2 of 9, 22%), or severe disease (n = 1 of 23, 4%). The rates from nondiseased CD tissues were reduced (n = 11 of 23, 48%) in comparison to such tissues from UC patients. In UC, goblet cells and MUC2 were reduced in diseased tissues and these phenotypes were retained in organoids.
CONCLUSIONS
Organoids can be readily derived from nondiseased surgically resected IBD tissues. While more work is needed to improve their derivation from diseased tissue, our study supports the use of organoids to study IBD pathophysiology.
Topics: Humans; Inflammatory Bowel Diseases; Colitis, Ulcerative; Crohn Disease; Intestinal Mucosa; Patient Acuity; Organoids
PubMed: 37776721
DOI: 10.1016/j.jss.2023.08.027 -
Journal of Visualized Experiments : JoVE Dec 2023The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells' natural environment in vitro....
The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells' natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.
Topics: Animals; Cattle; Hydrogels; Extracellular Matrix; Collagen; Tissue Engineering; Lung; Tissue Scaffolds
PubMed: 38145381
DOI: 10.3791/65768 -
Journal of Orthopaedic Surgery and... Oct 2023Temporomandibular joint osteoarthritis (TMJOA) is a common disease that negatively affects the life quality of human beings. Circadian rhythm acts an important role in...
PURPOSE
Temporomandibular joint osteoarthritis (TMJOA) is a common disease that negatively affects the life quality of human beings. Circadian rhythm acts an important role in life activities. However, whether the clock genes are rhythmic expressed in mandibular condylar chondrocytes, or the clock genes have an effect on the progression of TMJOA remains unknown. In this study, we aim to explore expression of clock genes and regulatory mechanism of TMJOA in rat mandibular condylar chondrocytes.
METHODS
After synchronized by dexamethasone, the expression of core clock genes Per1, Per2, Clock, Cry1, Cry2 and Bmal1 and cartilage matrix degrading factor gene Mmp13 were analyzed in mandibular condylar chondrocytes every 4 h with RT-qPCR. The mandibular condylar chondrocytes were stimulated with IL-1β, and expression of Per1, Mmp13, P65 and p-P65 was assessed by RT-qPCR and Western blot. Sh-Per1 lentivirus was used to assess the effect of clock gene Per1 in IL-1β-induced chondrocytes, and expression of Mmp13, P65 and p-P65 was measured. After establishing a rat TMJOA model using unilateral anterior crossbite (UAC), micro-CT, H & E, Alcian Blue & Nuclear Fast Red and Safranin O & Fast Green, cartilage thickness was utilized to assess the damage of cartilage and subchondral bone. Immunohistochemistry of PER1, MMP13 and P65 was performed in condylar sections.
RESULTS
All core clock genes and Mmp13 were rhythmically expressed. And Mmp13 expression curve was closed in phase and amplitude with Per1. After stimulation with IL-1β, the expression of MMP13, PER1 and P65 and ratio of p-P65/P65 increased in condylar chondrocytes. After Per1 was down-regulated in condylar chondrocytes, the expression of MMP13 and P65 and ratio of p-P65/P65 decreased. Compared with the condyles of Sham group, the bony parameters of UAC group were significantly worse. The thickness of cartilage in UAC group significantly reduced. The modified Mankin scores and the expression of PER1, MMP13 and P65 in cartilage of UAC group significantly increased compared with Sham group.
CONCLUSION
Core clock genes and Mmp13 are rhythmic expressed in rat mandibular condylar chondrocytes. PER1 can regulate the expression of MMP13 through NF-κB pathway in IL-1β-induced mandibular condylar chondrocytes.
Topics: Animals; Rats; Chondrocytes; Mandibular Condyle; Matrix Metalloproteinase 13; NF-kappa B; Osteoarthritis; Period Circadian Proteins; Temporomandibular Joint
PubMed: 37907921
DOI: 10.1186/s13018-023-04301-7 -
Methods in Molecular Biology (Clifton,... 2024Mucins are often stained with the basic dye Alcian blue, but mucins with a low acidic glycan content cannot be stained with it. Succinylation-Alcian blue staining is a...
Mucins are often stained with the basic dye Alcian blue, but mucins with a low acidic glycan content cannot be stained with it. Succinylation-Alcian blue staining is a method that temporarily modifies glycans with succinic acid to visualize mucins with low acidic glycan content. This method can be used to stain mucins on polyvinylidene difluoride (PVDF) membranes separated via supported molecular matrix electrophoresis (SMME) and mucins blotted onto PVDF membranes from gel electrophoreses. The succinyl groups of the modified glycans can be easily and completely removed by releasing O-glycan from the stained mucin bands. Therefore, the glycans can be analyzed using the same methods as those used for mucins with a high acidic glycan content.
Topics: Mucins; Alcian Blue; Staining and Labeling; Polysaccharides; Fluorocarbon Polymers; Polyvinyls
PubMed: 38347404
DOI: 10.1007/978-1-0716-3670-1_9 -
Pain Practice : the Official Journal of... Sep 2023An abnormal increase in spontaneous neurotransmission can induce subsynaptic knots in the myocyte called myofascial trigger points. The treatment of choice is to destroy...
INTRODUCTION
An abnormal increase in spontaneous neurotransmission can induce subsynaptic knots in the myocyte called myofascial trigger points. The treatment of choice is to destroy these trigger points by inserting needles. However, 10% of the population has a phobia of needles, blood, or injuries. Therefore, the objective of this study is to verify the usefulness of shock waves in the treatment of myofascial trigger points.
METHODS
Two groups of mice have been developed for this: healthy muscles treated with shock waves; trigger points affected muscles artificially generated with neostigmine and subsequently treated with shock waves. Muscles were stained with methylene blue, PAS-Alcian Blue, and labeling the axons with fluorescein and the acetylcholine receptors with rhodamine. Using intracellular recording the frequency of miniature endplate potentials (mEPPs) was recorded and endplate noise was recorded with electromyography.
RESULTS
No healthy muscles treated with shock waves showed injury. Twitch knots in mice previously treated with neostigmine disappeared after shock wave treatment. Several motor axonal branches were retracted. On the other hand, shock wave treatment reduces the frequency of mEPPs and the number of areas with endplate noise.
DISCUSSION
Shock waves seem to be a suitable treatment for myofascial trigger points. In the present study, with a single session of shock waves, very relevant results have been obtained, both functional (normalization of spontaneous neurotransmission) and morphological (disappearance of myofascial trigger points). Patients with a phobia of needles, blood, or injuries who cannot benefit from dry needling may turn to noninvasive radial shock wave treatment.
Topics: Mice; Animals; Trigger Points; Myofascial Pain Syndromes; Neostigmine; Muscle, Skeletal; Electromyography
PubMed: 37102243
DOI: 10.1111/papr.13237 -
Tissue Engineering. Part C, Methods Jul 2023Regenerative medicine approaches to restore the mandibular condyle of the temporomandibular joint (TMJ) may fill an unmet patient need. In this study, a method to...
Regenerative medicine approaches to restore the mandibular condyle of the temporomandibular joint (TMJ) may fill an unmet patient need. In this study, a method to implant an acellular regenerative TMJ prosthesis was developed for orthotopic implantation in a pilot goat study. The scaffold incorporated a porous, polycaprolactone-hydroxyapatite (PCL-HAp, 20wt% HAp) 3D printed condyle with a cartilage-matrix-containing hydrogel. A series of material characterizations was used to determine the structure, fluid transport, and mechanical properties of 3D printed PCL-HAp. To promote marrow uptake for cell seeding, a scaffold pore size of 152 ± 68 μm resulted in a whole blood transport initial velocity of 3.7 ± 1.2 mm·s transported to the full 1 cm height. The Young's modulus of PCL was increased by 67% with the addition of HAp, resulting in a stiffness of 269 ± 20 MPa for etched PCL-HAp. In addition, the bending modulus increased by 2.06-fold with the addition of HAp to 470 MPa for PCL-HAp. The prosthesis design with an integrated hydrogel was compared with unoperated contralateral control and no-hydrogel group in a goat model for 6 months. A guide was used to make the condylectomy cut, and the TMJ disc was preserved. MicroCT assessment of bone suggested variable tissue responses with some regions of bone growth and loss, although more loss may have been exhibited by the hydrogel group than the no-hydrogel group. A benchtop load transmission test suggested that the prosthesis was not shielding load to the underlying bone. Although variable, signs of neocartilage formation were exhibited by Alcian blue and collagen II staining on the anterior, functional surface of the condyle. Overall, this study demonstrated signs of functional TMJ restoration with an acellular prosthesis. There were apparent limitations to continuous, reproducible bone formation, and stratified zonal cartilage regeneration. Future work may refine the prosthesis design for a regenerative TMJ prosthesis amenable to clinical translation.
Topics: Animals; Tissue Scaffolds; Temporomandibular Joint; Bone and Bones; Temporomandibular Joint Disc; Goats; Tissue Engineering
PubMed: 37335050
DOI: 10.1089/ten.TEC.2023.0093