Saturday, January 29, 2011

Comparison of Fibrocartilage to LSJL gene expression

Fibrocartilage is a mixture of fibrous tissue and cartilage.  Formation of fibrocartilage could be a sign of preliminary ectopic growth plate formation via LSJL.

Discrimination of meniscal cell phenotypes using gene expression profiles.

"The purpose of this study was to identify quantifiable characteristics of meniscal cells and thereby find phenotypical markers that could effectively categorize cells based on their tissue of origin (cartilage, inner, middle, and outer meniscus). The combination of gene expression ratios collagen VI/collagen II, ADAMTS-5/collagen II, and collagen I/collagen II was the most effective indicator of variation among different tissue regions."

The meniscus is a fibrocartilaginous structure.

"Although the meniscus is primarily composed of collagen I, the inner region shows a higher content of collagen II, the major component and indicator of hyaline cartilage"<-Both collagen types are upregulated by LSJL.

Genes upregulated in the meniscus versus articular cartilage also upregulated in LSJL:


Neutral(no obvious trends):

"Cilp2 is expressed differentially by articular chondrocytes. CILP-2 is highly homologous to CILP-1 (cartilage intermediate layer protein 1), which is expressed in the intermediate zone of articular cartilage. Cilp2 has a restricted mRNA distribution at the surface of the mouse articular cartilage during development, becoming localized to the intermediate zone of articular cartilage and meniscal cartilage with maturity. Although the extracellular CILP-2 protein localization is broadly similar to CILP-1, CILP-2 appears to be more localized in the deeper intermediate zone of the articular cartilage extracellular matrix at maturity. CILP-2 was shown to be proteolytically processed, N-glycosylated, and present in human articular cartilage. In surgically induced osteoarthritis in mice, Cilp1 and Cilp2 gene expression was dysregulated. However, whereas Cilp1 expression was increased, Cilp2 gene expression was down-regulated demonstrating a differential response to mechanically induced joint destabilization. CILP-2 protein was reduced in the mouse osteoarthritic cartilage. Ultrastructural analysis also suggested that CILP-2 may be associated with collagen VI microfibrils and thus may mediate interactions between matrix components in the territorial and inter-territorial articular cartilage matrix."

"The N-terminal of CILP-1 has been shown to bind to and inhibit TGFβ1 in vitro, and CILP1 mRNA is induced by TGFβ1. CILP-1 levels increase with age."

Cilp2 is not expressed in growth plate cartilage.

"The absence of Cilp2 mRNA and protein in growth plate cartilage suggests that CILP-2 is a component of permanent cartilage, rather than transient cartilage that will undergo ossification, and further defining the specialized composition of the extracellular matrix synthesized by articular chondrocytes."

"we employ scaffolds composed of co-aligned nanofibers that direct mesenchymal stem cell (MSC) orientation and the formation of organized extracellular matrix (ECM). Concomitant with ECM synthesis, the mechanical properties of constructs increase with free-swelling culture, but ultimately failed to achieve equivalence with meniscal fibrocartilage.  This work examined the effect of cyclic tensile loading on MSC-laden nanofibrous constructs. Fiber-aligned scaffolds were seeded with MSCs and dynamically loaded daily in tension or maintained as nonloaded controls for 4 weeks. With mechanical stimulation, fibrous gene expression increased, collagen deposition increased, and the tensile modulus increased by 16% relative to controls."

"Four weeks of loading resulted in a two-fold increase in collagen I expression compared to nonloaded controls, with no effect on collagen II expression"

"Fibronectin, which is responsible for cell binding to the surrounding ECM and is a precursor to collagen deposition, increased by two-fold with conditioning. Likewise, lysyl oxidase{up in LSJL}, an enzyme that cross-links collagen fibrils, was significantly upregulated in loaded constructs compared to nonloaded controls"

"Fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis{up} and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. Yhe fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues."

In vitro study of stem cell communication via gap junctions for fibrocartilage regeneration at entheses.

"Entheses are fibrocartilaginous organs that bridge ligament with bone at their interface and add significant insertional strength. T
Rabbit bone/ligament fibroblasts were dual-stained with DiI-Red and calcein (gap-junction permeable dye), and cocultured with unlabeled BMSCs at fixed ratio (1:10). The functional gap junction was demonstrated by the transfer of calcein from donor to recipient cells that was confirmed and quantified by flow cytometry. Type 2 collagen (cartilage extracellular matrix-specific protein) expressed by the mixed cell lines in the cocultures were estimated by real-time reverse transcription PCR and compared with that of the ligament-bone coculture (control).
Significant transfer of calcein into BMSCs was observed and flow cytometry analyses showed a gradual increase in the percentage of BMSCs acquiring calcein with time. Cocultures that included BMSCs expressed significantly more type 2 collagen compared with the control.
The current study, for the first time, reported the expression of gap-junctional communication of BMSCs with two adherent cell lines of musculoskeletal system in vitro and also confirmed that incorporation of stem cells augments fibrocartilage regeneration."

couldn't get full study.

Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles.

"Human synovium, meniscus, intraarticular ligament, muscle, adipose tissue, and bone marrow were harvested, and colony-forming cells were analyzed. All these cells showed multipotentiality and surface markers typical of MSCs. Gene profiles of intraarticular tissue MSCs and chondrocytes were closer to each other than those of extraarticular tissues MSCs. Among three characteristic genes specific for intraarticular tissue MSCs, we focused on proline arginine-rich end leucine-rich repeat protein (PRELP). Higher expression of PRELP was confirmed in chondrocytes and intraarticular tissue MSCs among three elderly and three young donors. Synovium MSCs stably expressed PRELP, contrarily, bone marrow MSCs increased PRELP expression during in vitro chondrogenesis. In conclusion, MSCs could be isolated from various intraarticular tissues including meniscus and ligament, gene expression profiles of intraarticular tissue MSCs closely resembled each other, and the higher expression of PRELP was characteristic of intraarticular tissue MSCs."

Pellets from the synovium and bone marrow were over 1mg indicative of chondrogenic potential.

PRELP was expressed higher in chondrocytes and intra-articular MSCs than other tissues.

"OGN{down} and ECRG4 expressions were higher in synovium MSCs than in bone marrow MSCs"

DSP is expressed lowever in cartilage than bone marrow and is downregulated in LSJL.

"Synovium MSCs expressed PRELP stably during the chondrogenesis, contrarily, bone marrow MSCs increased PRELP expression along with the chondrocyte differentiation"

Expression profiles of two types of human knee-joint cartilage.

"We have performed a comprehensive analysis of gene-expression profiles in human articular cartilage (hyaline cartilage) and meniscus (fibrocartilage) by means of a cDNA microarray consisting of 23,040 human genes. Comparing the profiles of the two types of cartilage with those of 29 other normal human tissues identified 24 genes that were specifically expressed in both cartilaginous tissues; these genes might be involved in maintaining phenotypes common to cartilage. We also compared the cartilage profiles with gene expression in human mesenchymal stem cells (hMSC), and detected 22 genes that were differentially expressed in cells representing the two cartilaginous lineages, 11 specific to each type, which could serve as markers for predicting the direction of chondrocyte differentiation."

Genes expressed both in hyaline and fibrocartilage also expressed in LSJL:
Sox9(Sox9 was also expressed in non-cartilagenous samples however)

Genes only in hyaline cartilage and not fibrocartilage versus MSCs also upregulated in LSJL:

Genes only in fibrocartilage:

IGF2 is a marker gene for cartilage over fibrocartilage.

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