scholarly journals Tet-regulated BMP-2 gene expression in lentivirally transduced primary rabbit chondrocytes for treatment of cartilage defects

2010 ◽  
pp. NA-NA ◽  
Author(s):  
Daniela Wübbenhorst ◽  
Dipl Biol ◽  
Katja Dumler ◽  
Dipl Ing ◽  
Bettina Wagner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cartilage Defects ◽  
Primary Rabbit

Modernizing Storage Conditions for Fresh Osteochondral Allografts by Optimizing Viability at Physiologic Temperatures and Conditions

Cartilage ◽  
2019 ◽  
pp. 194760351988879
Author(s):  
Janet M. Denbeigh ◽  
Mario Hevesi ◽  
Carlo A. Paggi ◽  
Zachary T. Resch ◽  
Leila Bagheri ◽  
...  
Keyword(s):  
Gene Expression ◽  
Clinical Success ◽  
Quantitative Polymerase Chain Reaction ◽  
Viable Cell ◽  
Clinical Results ◽  
Storage Conditions ◽  
Viable Cell Density ◽  
Osteochondral Allograft ◽  
Cartilage Defects ◽  
Chondrocyte Viability

Objective. Osteochondral allograft (OCA) transplantation has demonstrated good long-term outcomes in treatment of cartilage defects. Viability, a key factor in clinical success, decreases with peri-implantation storage at 4°C during pathogen testing, matching logistics, and transportation. Modern, physiologic storage conditions may improve viability and enhance outcomes. Design. Osteochondral specimens from total knee arthroplasty patients (6 males, 5 females, age 56.4 ± 2.2 years) were stored in media and incubated at normoxia (21% O2) at 22°C or 37°C, and hypoxia (2% O2) at 37°C. Histology, live-dead staining, and quantitative polymerase chain reaction (qPCR) was performed 24 hours after harvest and following 7 days of incubation. Tissue architecture, cell viability, and gene expression were analyzed. Results. No significant viability or gene expression deterioration of cartilage was observed 1-week postincubation at 37°C, with or without hypoxia. Baseline viable cell density (VCD) was 94.0% ± 2.7% at day 1. At day 7, VCD was 95.1% (37°C) with normoxic storage and 92.2% (37°C) with hypoxic storage ( P ≥ 0.27). Day 7 VCD (22°C) incubation was significantly lower than both the baseline and 37°C storage values (65.6%; P < 0.01). COL1A1, COL1A2, and ACAN qPCR expression was unchanged from baseline ( P < 0.05) for all storage conditions at day 7, while CD163 expression, indicative of inflammatory macrophages and monocytes, was significantly lower in the 37°C groups ( P < 0.01). Conclusion. Physiologic storage at 37°C demonstrates improved chondrocyte viability and metabolism, and maintained collagen expression compared with storage at 22°C. These novel findings guide development of a method to optimize short-term fresh OCA storage, which may lead to improved clinical results.

scholarly journals New Insights into Xenotransplantation for Cartilage Repair: Porcine Multi-Genetically Modified Chondrocytes as A Promising Cell Source

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2152
Author(s):  
Hanna Tritschler ◽  
Konrad Fischer ◽  
Jochen Seissler ◽  
Jörg Fiedler ◽  
Rebecca Halbgebauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Complement Activation ◽  
Genetically Modified ◽  
Gene Expression Level ◽  
Expression Level ◽  
Cartilage Defects ◽  
Specific Cell ◽  
Human Articular Cartilage ◽  
Xenograft Rejection ◽  
Functional Assays

Transplantation of xenogenic porcine chondrocytes could represent a future strategy for the treatment of human articular cartilage defects. Major obstacles are humoral and cellular rejection processes triggered by xenogenic epitopes like α-1,3-Gal and Neu5Gc. Besides knockout (KO) of genes responsible for the biosynthesis of respective epitopes (GGTA1 and CMAH), transgenic expression of human complement inhibitors and anti-apoptotic as well as anti-inflammatory factors (CD46, CD55, CD59, TNFAIP3 and HMOX1) could synergistically prevent hyperacute xenograft rejection. Therefore, chondrocytes from different strains of single- or multi-genetically modified pigs were characterized concerning their protection from xenogeneic complement activation. Articular chondrocytes were isolated from the knee joints of WT, GalTKO, GalT/CMAH-KO, human CD59/CD55//CD46/TNFAIP3/HMOX1-transgenic (TG), GalTKO/TG and GalT/CMAHKO/TG pigs. The tissue-specific effectiveness of the genetic modifications was tested on gene, protein and epitope expression level or by functional assays. After exposure to 20% and 40% normal human serum (NHS), deposition of C3b/iC3b/C3c and formation of the terminal complement complex (TCC, C5b-9) was quantified by specific cell ELISAs, and generation of the anaphylatoxin C5a by ELISA. Chondrocyte lysis was analyzed by Trypan Blue Exclusion Assay. In all respective KO variants, the absence of α -1,3-Gal and Neu5Gc epitope was verified by FACS analysis. In chondrocytes derived from TG animals, expression of CD55 and CD59 could be confirmed on gene and protein level, TNFAIP3 on gene expression level as well as by functional assays and CD46 only on gene expression level whereas transgenic HMOX1 expression was not evident. Complement activation in the presence of NHS indicated mainly effective although incomplete protection against C3b/iC3b/C3c deposition, C5a-generation and C5b-9 formation being lowest in single GalTKO. Chondrocyte viability under exposure to NHS was significantly improved even by single GalTKO and completely preserved by all other variants including TG chondrocytes without KO of xenoepitopes.

scholarly journals Decellularized micro-scaffolds as stem cell carriers for regeneration of cartilage defects

2021 ◽  
Author(s):  
Pritiprasanna Maity ◽  
Sumanta Mukherjee ◽  
Mahitosh Mandal ◽  
Shubhayan Das ◽  
Santanu Dhara
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cartilage Defect ◽  
3D Culture ◽  
Size Exclusion ◽  
Cartilage Defects ◽  
Time Interval ◽  
Self Healing ◽  
Block Polymer ◽  
Significant Difference

Cartilage regeneration remains a great challenge in orthopedic treatment owing to their avascular in nature and lack of self-healing ability. Various clinical treatment options are widely used including osteochondral graft transplantation, micro-fracture, blood clot formation and tissue debridement to recover the damaged cartilage. In this circumstance, cartilage defect recovery via tissue engineered functional micro tissue delivery is becoming an emerging trend in musculoskeletal therapeutics. Herein, functional micro tissue particles (MTP) are isolated from Capra ear cartilage and delivered to cartilage defect in hydrogel form. The process involves in size exclusion MTPs formation; top-down approach including mechanical pulverization, decellularization and ethanol based particle separation from mixture. Here we show the 70% ethanol based MTP precipitation from a mixture of 300, 200 and 100 micron size at certain time interval. In addition, various concentration of NaOH is used to decellularize the MTP in which 0.2 M concentration is superior for complete removal of cell. Quantitative sGAG assay indicated that developed decellularization method has slightly reduced the proteoglycan content of tissue in comparison to native tissue. The cell adhesion study indicated that Capra adipose tissue derived mesenchymal stem cells (ADMSCs) adhesion is more in ~100 micron MTPs in comparison to 200-300 micron in size. It may be assumed that the cells are compatible to grow on fibrous surface area (100 micron) in comparison to dense surface (200-300 micron). Further, 100 micron MTPs (1MTPs) was transformed into functional micro tissue (FMT) in presence of high density ADMSCs to our fabricated organoid culture system after 6 days. The FMT has transferred to decellularized cartilage ECM powder and F127 block polymer composed hydrogel for 3D culture. After 21 d cultures, the FMT cluster has utilized for quantitative gene expression study in which COLII and aggrecan genes are expressed strongly in comparison to ADMSCs differentiation culture in chondrogenic media. SOX 9 gene expression does not produce any significant difference between FMT and static ADMSCs differentiation culture. To assess the in vivo cartilage defect regeneration potential, FMT has delivered to rabbit auricular cartilage defect for 15, 30 and 60 d studies. The H&E stained histological analysis showed that the cartilage defect is almost healed in 60 d study in comparison to 15 and 30 d study. Here we conclude that 100 micron MTP has immense potential to ex-vivo micro tissue formation and provides a native cartilage like environment for chondrogenesis of stem cells.

Molecular and Microscopic Analysis of Altered Gene Expression in Transgenic and Gene Targeted Mice

1996 ◽  
Vol 54 ◽  
pp. 12-13
Author(s):  
W. K. Jones ◽  
J. Robbins
Keyword(s):  
Gene Expression ◽  
Pulmonary Veins ◽  
Microscopic Analysis ◽  
Mouse Tissue ◽  
Adult Heart ◽  
Heavy Chains ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Pulmonary Myocardium

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of altered α-MyHC expression in the mouse.We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results of in situ hybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that of α-MyHC (Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding of α-MyHC expression and delimit a third cardiac compartment.

Linking transcription, RNA polymerase II elongation and alternative splicing

2020 ◽  
Vol 477 (16) ◽  
pp. 3091-3104 ◽  
Author(s):  
Luciana E. Giono ◽  
Alberto R. Kornblihtt
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Rna Polymerase Ii ◽  
Environmental Changes ◽  
Transcription Elongation ◽  
Single Gene ◽  
Regulation Of Gene Expression ◽  
Regulation Of Transcription ◽  
Stepping Stones ◽  
Eukaryotic Transcription

Gene expression is an intricately regulated process that is at the basis of cell differentiation, the maintenance of cell identity and the cellular responses to environmental changes. Alternative splicing, the process by which multiple functionally distinct transcripts are generated from a single gene, is one of the main mechanisms that contribute to expand the coding capacity of genomes and help explain the level of complexity achieved by higher organisms. Eukaryotic transcription is subject to multiple layers of regulation both intrinsic — such as promoter structure — and dynamic, allowing the cell to respond to internal and external signals. Similarly, alternative splicing choices are affected by all of these aspects, mainly through the regulation of transcription elongation, making it a regulatory knob on a par with the regulation of gene expression levels. This review aims to recapitulate some of the history and stepping-stones that led to the paradigms held today about transcription and splicing regulation, with major focus on transcription elongation and its effect on alternative splicing.

Role of small nuclear RNAs in eukaryotic gene expression

2013 ◽  
Vol 54 ◽  
pp. 79-90 ◽  
Author(s):  
Saba Valadkhan ◽  
Lalith S. Gunawardane
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Stability ◽  
Splice Sites ◽  
Branch Site ◽  
Small Nuclear Rnas ◽  
Eukaryotic Gene Expression ◽  
Eukaryotic Gene ◽  
Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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