Effect of nitrogen-rich cell culture surfaces on type X collagen expression by bovine growth plate chondrocytes

Background: Recent evidence indicates that osteoarthritis (OA) may be a systemic disease since mesenchymal stem cells (MSCs) from OA patients express type X collagen, a marker of late stage chondrocyte hypertrophy (associated with endochondral ossification). We recently showed that the expression of type X collagen was suppressed when MSCs from OA patients were cultured on nitrogen (N)-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36 atomic percentage (at.%) of N.Methods: In the present study, we examined the expression of type X collagen in fetal bovine growth plate chondrocytes (containing hypertrophic chondrocytes) cultured on PPE:N. We also studied the effect of PPE: N on the expression of matrix molecules such as type II collagen and aggrecan, as well as on proteases (matrix metalloproteinase-13 (MMP-13) and molecules implicated in cell division (cyclin B2). Two other culture surfaces, "hydrophilic" polystyrene (PS, regular culture dishes) and nitrogen-containing cation polystyrene (Primaria (R)), were also investigated for comparison.Results: Results showed that type X collagen mRNA levels were suppressed when cultured for 4 days on PPE: N, suggesting that type X collagen is regulated similarly in hypertrophic chondrocytes and in human MSCs from OA patients. However, the levels of type X collagen mRNA almost returned to control value after 20 days in culture on these surfaces. Culture on the various surfaces had no significant effects on type II collagen, aggrecan, MMP-13, and cyclin B2 mRNA levels.Conclusion: Hypertrophy is diminished by culturing growth plate chondrocytes on nitrogen-rich surfaces, a mechanism that is beneficial for MSC chondrogenesis. Furthermore, one major advantage of such "intelligent surfaces" over recombinant growth factors for tissue engineering and cartilage repair is potentially large cost-saving

The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration

Abstract Introduction Intervertebral disc (IVD) degeneration is associated with proteolytic degradation of the extracellular matrix, and its repair requires both the production of extracellular matrix and the downregulation of proteinase activity. These properties are associated with several growth factors. However, the use of growth factors in clinical practice is limited by their high cost. This cost can be circumvented using synthetic peptides, such as Link N, which can stimulate the synthesis of proteoglycan and collagen by IVD cells in vitro. The purpose of the present study was to evaluate the effect of Link N in vivo in a rabbit model of IVD degeneration. Methods New Zealand white rabbits received annular puncture in two lumbar discs. Two weeks after puncture, both punctured discs of each rabbit were injected with either Link N or saline. After 2 weeks, nine rabbits were euthanized and the annulus fibrosus (AF) and nucleus pulposus (NP) of Link N-injected and saline-injected IVDs were removed and used to prepare total RNA. Following reverse transcription, quantitative PCR was performed for aggrecan, COL2A1, COL1A1, ADAMTS-4, ADAMTS-5 and MMP-3. After 12 weeks, 19 rabbits were euthanized and the injected IVDs were removed for biochemical and histological analysis. Proteinase K digests were analyzed for DNA and sulfated glycosaminoglycan content. Disc height was monitored radiographically biweekly. Results Following needle puncture, disc height decreased by about 25% over 2 weeks, and was partially restored by Link N injection. Puncture of the IVD resulted in a trend towards decreased proteoglycan content in both the NP and AF, and a trend towards partial restoration following Link N injection, although under the time course used this did not achieve statistical significance. Link N did not alter the DNA content of the discs. Link N injection led to a significant increase in aggrecan gene expression and a significant decrease in proteinase gene expression in both the NP and AF, when compared with saline alone. Conclusions When administered to the degenerate disc in vivo, Link N stimulated aggrecan gene expression and downregulated metalloproteinase expression, and there was a trend towards increased proteoglycan content of the disc, in both the NP and AF. These are features needed for any agent designed to stimulate disc repair. In principle, therefore, Link N supplementation could be an option for treating disc degeneration

The Constitutive Expression of Type X Collagen in Mesenchymal Stem Cells from Osteoarthritis Patients Is Reproduced in a Rabbit Model of Osteoarthritis

The expression of type X collagen (COL X), a late-stage chondrocyte hypertrophy marker in human mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients poses a major setback to current cartilage and intervertebral disc tissue engineering efforts. However, it is not yet clear whether COL X is expressed by all human bone marrow stem cells or if it is related to age, gender, site, disease status, or drug therapy. In the current study, we report that COL X expression is upregulated in MSCs from rabbits in a surgical instability model of OA (anterior cruciate ligament transection (ACLT)) when compared to control rabbit MSCs. Thus COL X expression in OA is a common phenomenon that is due to the disease process itself and not to other environmental factors. It is, therefore, critical to understand MSC phenotype in OA patients, as these cells are essential clinically for biological repair of cartilage lesions using autologous stem cells

Enhanced proliferation and growth of non-differentiated and osteoblast-differentiated human stem cells on the surface of HVOF-sprayed nano TiO\u2082-HA coatings

Peer reviewed: YesNRC publication: Ye

Epidemiology of eating disorders part III: Social epidemiology and case definitions revisited.

Accepted manuscript version. Published version at <a href=http://doi.org/10.1080/21662630.2015.1022197>http://doi.org/10.1080/21662630.2015.1022197</a>.The previous papers in this series outlined a historical panorama and presented updated knowledge about putative risk factors and how eating disorders are distributed in various populations. In this final paper, we discuss in what way comorbidity findings and transdiagnostic issues may change our conceptions about ‘an epidemiological case’ from the current definition of eating disorders based on the recent version of the Diagnostic and Statistical Manual of Mental Disorders (i.e. the DSM-5), and to what extent an alternative definition may introduce new perspectives of prevention. The paper also provides an update on issues relevant for treatment dissemination

The effect of novel nitrogen-rich plasma polymer coatings on the phenotypic profile of notochordal cells

Background: The loss of the notochordal cells from the nucleus pulposus is associated with ageing and disc degeneration. However, understanding the mechanisms responsible for the loss of these cells has been hampered in part due to the difficulty of culturing and maintaining their phenotype. Furthermore, little is known about the influence of the substratum on the molecular markers of notochordal cells. Methods: Notochordal cells were isolated from lumbar spine of non-chondrodystrophoid dogs and cultured on N-rich plasma polymer layers, so-called "PPE:N" ( N-doped plasma-polymerised ethylene, containing up to 36% [N]) surfaces, for 3, 7 or 14 days. Gene expression of vimentin ( VIM), pleiotrophin ( PTN), matrix Gla protein ( MGP), cartilage oligomeric matrix protein ( COMP), keratin 18 ( KRT 18), aggrecan ( AGG), collagen type 1 ( COL1A2), collagen type 2 ( COL2A1) was analyzed through semi-quantitative reverse transcription-polymerase chain reaction ( RT-PCR).Results: Notochordal cells were maintained in culture on PPE:N for up to 14 days with no loss in cell viability. Except for VIM, gene expression varied depending on the culture periods and [ N] concentration of the substratum. Generally, PPE: N surfaces altered gene expression significantly when cells were cultured for 3 or 7 days.Conclusion: The present study has shown that notochordal cells from dogs can attach to and grow on PPE: N surfaces. Analysis of the expression of different genes in these cells cultured on different N-functionalized surfaces indicates that cellular behaviour is gene-specific and time-dependent. Further studies are required to better understand the roles of specific surface functionalities on receptor sites, and their effects on cellular phenotypes

The Potential of N-Rich Plasma-Polymerized Ethylene (PPE:N) Films for Regulating the Phenotype of the Nucleus Pulposus

We recently developed a nitrogen-rich plasma-polymerized biomaterial, designated “PPE:N” (N-doped plasma-polymerized ethylene) that is capable of suppressing cellular hypertrophy while promoting type I collagen and aggrecan expression in mesenchymal stem cells from osteoarthritis patients. We then hypothesized that these surfaces would form an ideal substrate on which the nucleus pulposus (NP) phenotype would be maintained. Recent evidence using microarrays showed that in young rats, the relative mRNA levels of glypican-3 (GPC3) and pleiotrophin binding factor (PTN) were significantly higher in nucleus pulposus (NP) compared to annulus fibrosus (AF) and articular cartilage. Furthermore, vimentin (VIM) mRNA levels were higher in NP versus articular cartilage. In contrast, the levels of expression of cartilage oligomeric matrix protein (COMP) and matrix gla protein precursor (MGP) were lower in NP compared to articular cartilage. The objective of this study was to compare the expression profiles of these genes in NP cells from fetal bovine lumbar discs when cultured on either commercial polystyrene (PS) tissue culture dishes or on PPE:N with time. We found that the expression of these genes varies with the concentration of N ([N]). More specifically, the expression of several genes of NP was sensitive to [N], with a decrease of GPC3, VIM, PTN, and MGP in function of decreasing [N]. The expression of aggrecan, collagen type I, and collagen type II was also studied: no significant differences were observed in the cells on different surfaces with different culture time. The results support the concept that PPE:N may be a suitable scaffold for the culture of NP cells. Further studies are however necessary to better understand their effects on cellular phenotypes

Novel Nitrogen Rich Polymers and Chitosan for Tissue Engineering of Intervertebral Discs

ABSTRACT: Degenerative disc disease has been implicated as a major component of spine pathology. However, though biological repair of the degenerate disc would be the ideal treatment, there is a lack of a universally accepted scaffold for tissue engineering of intervertebral discs (IVD) and little is known of how to differentiate mesenchymal stem cells (MSCs) to a disc-like phenotype. We show that 2.5% Protasan® UP G213 cross-linked to 5% genipin might be a promising scaffold for disc tissue engineering. Furthermore, we have developed extremely N-rich plasma polymer layers, which we call "PPE:N" (N-doped plasma-polymerized ethylene, containing up to 36% [N]). We show that PPE:N almost completely suppresses the expression not only of type X collagen, but also of osteogenic marker genes such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). In contrast, neither aggrecan nor types 1 collagen expression were significantly affected. These results indicate that PPE:N coatings may be suitable surfaces for inducing MSCs to a chondrocyte or disc-like phenotype for tissue engineering of cartilage or IVDs, in which hypertrophy and osteogenesis are suppressed