Gene Expression Of Matrix Proteins Research Articles

Regional Heterogeneity in Murine Lung Fibroblasts from Normal Mice or Mice Exposed Once to Cigarette Smoke

Chronic obstructive lung disease (COPD) is characterized by matrix deposition in the small airways but matrix loss from the parenchyma, phenomena which must depend on the ability of local fibroblasts to produce matrix after smoke exposure. To investigate this idea, we exposed C57Bl/6 mice once to cigarette smoke or to air (control) and prepared primary cultures of lung fibroblasts by microdissecting large airways (trachea, LAF), medium size airways (major bronchi, MAF) and parenchyma (PF). Control PF showed the lowest rate of wound closure and wound closure was depressed in all lines by a single in vivo smoke exposure. Gene expression of matrix proteins differed considerably among the sites; decorin, which may sequester TGFβ, was markedly higher in PF. PF showed higher intrinsic ratios of pSmad2/Smad2. Smoke caused much greater increases in secreted and matrix deposited collagens 1 and 3 in PF than in LAF or MAF. Expression of Thy-1, a gene that suppresses myofibroblast differentiation, was increased by smoke in PF. We conclude that there is considerable regional heterogeneity in murine lung fibroblasts in terms of matrix production, either basally or after in vivo smoke exposure; that PF have lower ability to repair wounds and higher intrinsic TGFβ signaling; and that a single exposure to smoke produces lasting changes in the pattern of matrix production and wound repair, changes that may be mediated in part by smoke-induced release of TGFβ. However, PF still retain the ability to repair by producing new matrix after a single in vivo smoke exposure.

Characterization of Human Endplate Biopsies: Preliminary Results on Cellular Composition, Metabolic Activity and Interactions with Disk Cells

Introduction Tissue alterations that result in an impaired endplate (EP) permeability are assumed to play a crucial role in the pathogenesis of intervertebral disk (IVD) degeneration. Therefore, disturbance of endplate permeability is one of the main problems that need to be solved for successful disk tissue engineering. However, the biological mechanisms underlying these alterations are poorly understood. To shed light on this aspect, a standardized analysis of EP structure and cellular composition is essential to understand the degeneration processes. Purpose of this study was the development of standardized protocols for a descriptive characterization of EP samples by histology, transmission electron microscopy (TEM), analysis of differentiation and metabolic activity of EP cells. In first preliminary experiments we also aimed to detect possible interactions of the EP with disk cells of the same donor patients in a bioassay with EP-conditioned media. The suitability of these methods for EP characterization and possible correlations with degenerative alterations is discussed. Materials and Methods Cartilaginous endplates obtained during spine surgery of total 27 human donors underwent the following procedures: (1) isolation of endplate cells, in vitro expansion and characterization of chondrogenic differentiation in 3D pellet cultures ( n = 6), (2) preparation of standardized EP-sections for histological and ultrastructural analyses ( n = 8), and (3) preparation of standardized endplate punches that were incubated for 24 hours in serum-free culture medium followed by determination of metabolic activity of endplate cells by MTT-test ( n = 13) and further analysis of conditioned media (CM) in a bioassay with NP cells of the same donor ( n = 2). Pellet cultures of EP cells were characterized for matrix protein expression by Alcian blue staining. Influences of CM on gene expression of matrix proteins, matrix degrading enzymes, and inflammation factors by NP cells were determined. Results Differentiation of endplate cells in pellet cultures varied strongly not only between the six individual donors, but also in different disk levels of the same patient. Part of the samples exhibited a very low proteoglycan production whereas others were strongly positive in the Alcian blue staining intensity suggesting high variations in the differentiation capacity of EP cells. Histological examination of the endplate samples allowed a characterization of both the endplates and an observation of the adjacent structures as the very thin endplate layer could not be clearly separated from adjacent tissue structures. High variations between tissue samples from different donors could be found. TEM images showed signs of degeneration both in the matrix and the EP cells whereas higher numbers of apoptotic cells and matrix degradation products could be found in stronger degenerated tissue samples. Standardized EP punches that were cultured for 24 hours in DMEM and incubated for 4 hours with MTT showed different staining intensities suggesting donor-specific variations in metabolic activity of EP cells. In first two experiments with CM of EP samples, a dose-dependent upregulation of inflammation-factors and matrix metalloproteinases (Interleukins IL-1β, IL-6, IL-8, MMP-3, and MMP13) by NP-cells of the same donors could be detected. The extent of this effect differed strongly between the two patients with different degeneration degree. Conclusion The present results suggest that a combination of different methods allows the characterization of EP tissue and possible interactions with the adjacent disk tissue. As the cartilaginous EP itself is very thin and with low cellularity, isolation of an almost homogeneous EP population is very difficult and the cell yield is very poor. Therefore, methods that directly analyze the cells in situ for example by laser capture micro-dissection might be more suitable to determine the gene expression profile of EP cells. Analysis of metabolic activity of standardized EP punches by MTT test is a promising approach provided that the samples are standardized with regard to diameter and height with no adjacent other tissue regions. TEM investigation is appropriate to investigate cellular details, but also with this method, the poor cellularity of EP tissue is a challenge. A very promising approach is the analysis of CM in a bioassay with NP cells of the same donor that allows the detection of possible interactions between both tissue areas. In ongoing investigations, we analyze higher numbers of EP samples with different degrees of degeneration to enable a correlation between the biological and clinical data of the patients. For this purpose, analyses of nondegenerated control samples are essential. In summary, all these methods together allow a holistic view on the mechanisms that contribute to the morphological and pathophysiological changes of the EP and potential interaction with the adjacent disk tissue. Acknowledgement Part of this research work was supported by the EU framework 7 project Geneodisc (FP7, 2007–2013) under grant agreement no. HEALTH-F2-2008-201626. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Differentiation of Nucleus Pulposus Cells Alters Their Responsivity to Mechanics and Glucose Reduction

IntroductionNutritional supply of cells within the avascular intervertebral disk occurs mainly by diffusion from the blood vessels ending at the endplate of the vertebral body. With proceeding disk degeneration and calcification of the endplate the main route of transport of nutrients and metabolites is impaired. The consequential reduced nutrient supply is supposed to be a key contributor to disk degeneration. In short-term approaches using undifferentiated nucleus pulposus (NP) cells, gene expression of matrix proteins was decreased after glucose reduction but mechanosensitivity was not altered. Aim of this study was to investigate effects of long-term differentiation of NP cells regarding their responsivity to glucose reduction and mechanical stimulation.Materials and MethodsNP cells isolated from bovine caudal discs (n = 7) were cultured in alginate beads for 3 weeks for differentiation in high glucose (25 mM) chondrogenic medium supplemented with 10 ng/mL TGF-β1. After differentiation, part of the samples was exposed to reduced glucose concentration (5 mM) for 3 days. Afterwards glucose concentration was further reduced to either 0 mM, 0.5 mM, or 5 mM for 24 hours. Subsequently, half of all samples were mechanically loaded by hydrostatic pressure for 30 minutes at 0.1 Hz and 2.5 MPa or maintained at unloaded conditions. Gene expression of aggrecan, collagen type II and type I, MMP2, MMP3, MMP13, and aggrecanase was analyzed by real-time RT-PCR. Statistical significance was tested by Wilcoxon test (p < 0.05).ResultsTotal 3 weeks of differentiation in high-glucose (25 mM) chondrogenic medium caused a slight upregulation of aggrecan (2.7×), collagen type II (1.9×), and collagen type I (2.6×) expression in NP cells compared to day 0. While MMP2 (3.2×) expression increased significantly, a significant decrease of MMP3 (0.06×), MMP13 (0.006×), and aggrecanase (0.5×) expression was determined. Subsequent reduction of glucose concentration from high-glucose medium to medium containing 5 mM glucose decreased significantly expression of collagen type II (0.01×) and collagen type I (0.003×) to gene expression levels below that of day 0. Further reduction of glucose concentration revealed minor influences on gene expression.Effects of mechanical stimulation depended strongly on the medium in which NP cells were cultured. In NP cells, which were maintained in high-glucose chondrogenic medium without glucose reduction, mechanically induced effects were strongest. Within this group, mechanical loading increased significantly expression of c-FOS (3.3×) and collagen type I (4.6×), whereas expression of collagen type II (1.4×) increased only moderately. Furthermore, a decrease of gene expression of MMP2 (0.6×), MMP3 (0.02×), MMP13 (0.0005×) and aggrecanase (0.003×) after mechanical stimulation was observed. In cells exposed to glucose reduction, the mechanically induced changes in gene expression were minimal and no distinct correlation with glucose concentration could be pointed out.ConclusionAfter successful in vitro differentiation of NP cells, subsequent glucose deprivation revealed a strong downregulating impact on gene expression of matrix molecules and increased expression of matrix-degrading enzymes. Application of mechanical stimulation could not change these effects of glucose reduction. However, in high glucose chondrogenic medium supplemented with TGF-β1, hydrostatic pressure increased expression of matrix molecules and decreased expression of matrix-degrading enzymes. These findings indicate that mechanical sensitivity of NP cells depends on environmental conditions, as NP cells were less mechanosensitive under glucose deprivation. These interactions of glucose supply, presence of TGF-β1, and mechanical loading in the regulation of disk matrix turnover are investigated in further studies.Acknowledgement Funded by FP7 EU project GENODISK (HEALTH-F2-2008-201626).I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declared

Differentiation of Nucleus Pulposus Cells Alters Their Responsivity to Mechanics and Glucose Reduction

Introduction Nutritional supply of cells within the avascular intervertebral disk occurs mainly by diffusion from the blood vessels ending at the endplate of the vertebral body. With proceeding disk degeneration and calcification of the endplate the main route of transport of nutrients and metabolites is impaired. The consequential reduced nutrient supply is supposed to be a key contributor to disk degeneration. In short-term approaches using undifferentiated nucleus pulposus (NP) cells, gene expression of matrix proteins was decreased after glucose reduction but mechanosensitivity was not altered. Aim of this study was to investigate effects of long-term differentiation of NP cells regarding their responsivity to glucose reduction and mechanical stimulation. Materials and Methods NP cells isolated from bovine caudal discs ( n = 7) were cultured in alginate beads for 3 weeks for differentiation in high glucose (25 mM) chondrogenic medium supplemented with 10 ng/mL TGF-β1. After differentiation, part of the samples was exposed to reduced glucose concentration (5 mM) for 3 days. Afterwards glucose concentration was further reduced to either 0 mM, 0.5 mM, or 5 mM for 24 hours. Subsequently, half of all samples were mechanically loaded by hydrostatic pressure for 30 minutes at 0.1 Hz and 2.5 MPa or maintained at unloaded conditions. Gene expression of aggrecan, collagen type II and type I, MMP2, MMP3, MMP13, and aggrecanase was analyzed by real-time RT-PCR. Statistical significance was tested by Wilcoxon test ( p &lt; 0.05). Results Total 3 weeks of differentiation in high-glucose (25 mM) chondrogenic medium caused a slight upregulation of aggrecan (2.7×), collagen type II (1.9×), and collagen type I (2.6×) expression in NP cells compared to day 0. While MMP2 (3.2×) expression increased significantly, a significant decrease of MMP3 (0.06×), MMP13 (0.006×), and aggrecanase (0.5×) expression was determined. Subsequent reduction of glucose concentration from high-glucose medium to medium containing 5 mM glucose decreased significantly expression of collagen type II (0.01×) and collagen type I (0.003×) to gene expression levels below that of day 0. Further reduction of glucose concentration revealed minor influences on gene expression. Effects of mechanical stimulation depended strongly on the medium in which NP cells were cultured. In NP cells, which were maintained in high-glucose chondrogenic medium without glucose reduction, mechanically induced effects were strongest. Within this group, mechanical loading increased significantly expression of c-FOS (3.3×) and collagen type I (4.6×), whereas expression of collagen type II (1.4×) increased only moderately. Furthermore, a decrease of gene expression of MMP2 (0.6×), MMP3 (0.02×), MMP13 (0.0005×) and aggrecanase (0.003×) after mechanical stimulation was observed. In cells exposed to glucose reduction, the mechanically induced changes in gene expression were minimal and no distinct correlation with glucose concentration could be pointed out. Conclusion After successful in vitro differentiation of NP cells, subsequent glucose deprivation revealed a strong downregulating impact on gene expression of matrix molecules and increased expression of matrix-degrading enzymes. Application of mechanical stimulation could not change these effects of glucose reduction. However, in high glucose chondrogenic medium supplemented with TGF-β1, hydrostatic pressure increased expression of matrix molecules and decreased expression of matrix-degrading enzymes. These findings indicate that mechanical sensitivity of NP cells depends on environmental conditions, as NP cells were less mechanosensitive under glucose deprivation. These interactions of glucose supply, presence of TGF-β1, and mechanical loading in the regulation of disk matrix turnover are investigated in further studies. Acknowledgement Funded by FP7 EU project GENODISK (HEALTH-F2-2008-201626). I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Tendon structure changes after maximal exercise in the Thoroughbred horse: Use of ultrasound tissue characterisation to detect in vivo tendon response

Investigations into the response of the superficial digital flexor tendon (SDFT) of the Thoroughbred horse to mechanical stimuli have been limited to in vitro cell culture studies focused primarily on gene expression of critical matrix proteins. It is uncertain how well in vitro outcomes translate to the tendon of the horse during exercise. The current study examined changes in tendon structure in response to maximal exercise using ultrasound tissue characterisation (UTC) to scan the SDFT prior to and after competitive racing. UTC uses contiguous transverse ultrasound images to assess the dynamics of the echopattern, which has a close relationship with changes in the 3-D ultra-structure of the tendon. Using UTC, it was possible to detect subtle changes in the dynamics of the echopattern, with a reduction in pixels that represent aligned and integer collagen tendon bundles on days 1 and 2 post-race when compared to pre-race (P<0.05). The echopattern of these tendons returned to baseline on day 3. This change in echopattern was not seen in control horses. It was concluded that short-term changes in the SDFT following maximal exercise could be detected using UTC.

Mechanical Destabilization Induced by Controlled Annular Incision of the Intervertebral Disc Dysregulates Metalloproteinase Expression and Induces Disc Degeneration

An investigation of mechanical destabilization of the lumbar ovine intervertebral disc (IVD) inducing IVD degeneration (IVDD) as determined by multiparameter outcome measures (magnetic resonance imaging [MRI], IVD composition, biomechanical testing, gene profiling, immunohistochemistry, and immunoblotting). To assess the effect of IVD mechanical destabilization on matrix protein and metalloproteinase gene expression to investigate the pathophysiological mechanisms of lumbar IVDD. Several earlier studies have used annular transection to induce IVDD in sheep, but none have optimized or validated the most appropriate lesion size. The annulus fibrosus (AF) incision inducing maximal change in IVD biomechanics was applied to L1-L2, L3-L4, and L5-L6 discs in vivo to compare with a sham procedure at 3 months post operation. IVDs were evaluated by MRI, biomechanics, histopathology, proteoglycan and collagen content, gene expression, and aggrecan proteolysis by Western blotting. Significant changes were observed in lesion (6 × 20 mm(2)) compared with sham IVDs at 3 months post operation: reduced disc height on MRI; increased neutral zone in biomechanical testing; depleted proteoglycan and collagen content in the nucleus pulposus (NP) and lesion half of the AF but not in the contralateral AF; increased messenger RNA for collagen I and II, aggrecan, versican, perlecan, matrix metalloproteinase (MMP)-1 & 13, and ADAMTS-5, in the lesion-site AF and NP but not in the contralateral AF. ADAMTS-4 messenger RNA was increased in the lesion-site AF but decreased in the NP. Despite an upregulation in MMPs, there was no change in MMP- or ADAMTS-generated aggrecan neoepitopes in any region of the IVD in lesion or sham discs. Lumbar IVDD was reproducibly induced with a 6 × 20 mm(2) annular lesion, with focal dysregulation of MMP gene expression, cell cloning in the inner AF, loss of NP aggrecan, and disc height. Loss of aggrecan from the NP was not attributable to increased proteolysis in the interglobular domain by MMPs or ADAMTS.

The role of matrix proteins in the control of nacreous layer deposition during pearl formation

To study the function of pearl oyster matrix proteins in nacreous layer biomineralization in vivo, we examined the deposition on pearl nuclei and the expression of matrix protein genes in the pearl sac during the early stage of pearl formation. We found that the process of pearl formation involves two consecutive stages: (i) irregular calcium carbonate (CaCO(3)) deposition on the bare nucleus and (ii) CaCO(3) deposition that becomes more and more regular until the mature nacreous layer has formed on the nucleus. The low-expression level of matrix proteins in the pearl sac during periods of irregular CaCO(3) deposition suggests that deposition may not be controlled by the organic matrix during this stage of the process. However, significant expression of matrix proteins in the pearl sac was detected by day 30-35 after implantation. On day 30, a thin layer of CaCO(3), which we believe was amorphous CaCO(3), covered large aragonites. By day 35, the nacreous layer had formed. The whole process is similar to that observed in shells, and the temporal expression of matrix protein genes indicated that their bioactivities were crucial for pearl development. Matrix proteins controlled the crystal phase, shape, size, nucleation and aggregation of CaCO(3) crystals.

Interactions of environmental conditions and mechanical loads have influence on matrix turnover by nucleus pulposus cells

Disc degeneration is associated with several changes in the physicochemical environment of intervertebral disc cells. Nucleus pulposus (NP) cells in the center of degenerated discs are exposed to decreased glucose supply, osmolarity, pH, and oxygen levels. To understand the complexity of these interactions on a cellular level, we designed standardized experiments in which we compared responses to these environmental factors under normal levels with those seen under two different degrees of disc degeneration. We hypothesized that these changes in environmental stimuli influence gene expression of matrix proteins and matrix degrading enzymes and alter their responses to cyclic hydrostatic pressure (HP). Our results suggest that a simulation of degenerative conditions influences the degradation of disc matrix through impairing matrix formation and accelerating matrix resorption via up- or down-regulation of the respective target genes. The greatest effects were seen for decreases in glucose concentration and pH. Low oxygen had little influence. HP had little direct effect but appeared to counteract matrix degradation by reducing or inverting some of the adverse effects of other stimuli. For ongoing in vitro studies, interactions between mechanical stimuli and factors in the physicochemical environment should not be ignored as these could markedly influence results.

Human Nucleus Pulposus Cells React to IL-6

STUDY DESIGN.: Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1β or TNF-α. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. OBJECTIVE.: To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-α actions in these cells. SUMMARY OF BACKGROUND DATA.: Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. METHODS.: NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1β or TNF-α. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. RESULTS.: IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-α stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-α. MMP-13 induction by TNF-α was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-α fourfold. CONCLUSION.: The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-α, thus, helping preserve disc composition, structure, and function.

Quantitative expression of shell matrix protein genes and their correlations with shell traits in the pearl oyster Pinctada fucata

Shell matrix proteins play an important role in the regulation of shell growth and formation in the pearl oyster, Pinctada fucata. This study compared the expression levels of seven shell matrix protein genes ( aspein, prismalin-14, n16, n19, nacrein, msi7, and efcbp) in pearl oysters of various sizes [large (L), medium (M), and small (S)] and analyzed the correlations between expression level and shell size. Except for efcbp and msi7, significant correlations were detected between the expression levels of matrix protein genes and shell size: n19 expression displayed a negative correlation ( P < 0.01) with increasing shell size, whereas expression levels of aspein, prismalin-14, n16, and nacrein were greater in oysters with larger shell sizes. Furthermore, stronger correlations were found with shell height for genes related to prismatic layer of shell than shell weight; and with shell weight for genes related to nacreous layer of shell, which makes sense because prismatic proteins are considered to contribute to the extension of the shell length; and nacreous proteins are responsible for the shell thickness. These results contribute to a better understanding of the shell growth mechanisms of the pearl oyster.

The involvement of interleukin-1 and interleukin-4 in the response of human annulus fibrosus cells to cyclic tensile strain: an altered mechanotransduction pathway with degeneration

IntroductionRecent evidence suggests that intervertebral disc (IVD) cells derived from degenerative tissue are unable to respond to physiologically relevant mechanical stimuli in the 'normal' anabolic manner, but instead respond by increasing matrix catabolism. Understanding the nature of the biological processes which allow disc cells to sense and respond to mechanical stimuli (a process termed 'mechanotransduction') is important to ascertain whether these signalling pathways differ with disease. The aim here was to investigate the involvement of interleukin (IL)-1 and IL-4 in the response of annulus fibrosus (AF) cells derived from nondegenerative and degenerative tissue to cyclic tensile strain to determine whether cytokine involvement differed with IVD degeneration.MethodsAF cells were isolated from nondegenerative and degenerative human IVDs, expanded in monolayers and cyclically strained in the presence or absence of the cytokine inhibitors IL-1 receptor antagonist (IL-1Ra) or IL-4 receptor antibody (IL-4RAb) with 10% strain at 1.0 Hz for 20 minutes using a Flexcell strain device. Total RNA was extracted from the cells at time points of baseline control and 1 or 24 hours poststimulation. Quantitative real-time polymerase chain reaction was used to analyse the gene expression of matrix proteins (aggrecan and type I collagen) and enzymes (matrix metalloproteinase 3 (MMP3) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 4 (ADAMTS4)).ResultsExpression of catabolic genes (MMP3 and ADAMTS4) decreased in AF cells derived from nondegenerative tissue in response to 1.0-Hz stimulation, and this decrease in gene expression was inhibited or increased following pretreatment of cells with IL-1Ra or IL-4RAb respectively. Treatment of AF cells derived from degenerative tissue with an identical stimulus (1.0-Hz) resulted in reduced anabolic gene expression (aggrecan and type I collagen), with IL-1Ra or IL-4RAb pretreatment having no effect.ConclusionsBoth IL-1 and IL-4 are involved in the response of AF cells derived from nondegenerative tissue to 1.0-Hz cyclic tensile strain. Interestingly, the altered response observed at 1.0-Hz in AF cells from degenerative tissue appears to be independent of either cytokine, suggesting an alternative mechanotransduction pathway in operation.

Changes in matrix protein gene expression associated with mineralization in the differentiating chick limb-bud micromass culture system

Chick limb-bud mesenchymal stem cells plated in high density culture in the presence of 4 mM inorganic phosphate and vitamin C differentiate and form a mineralizable matrix, resembling that of the chick growth plate. To further elucidate the mechanism that allows these cultures to form physiologic hydroxyapatite deposits, and how the process can be manipulated to gain insight into mineralization mechanisms, we compared gene expression in mineralizing (with 4 mM inorganic phosphate) and non-mineralizing cultures (containing only 1 mM inorganic phosphate) at the start of mineralization (day 11) and after mineralization reached a plateau (day 17) using a chick specific microarray. Based on replicate microarray experiments and K-cluster analysis, several genes associated with the mineralization process were identified, and their expression patterns confirmed throughout the culture period by quantitative RT-PCR. The functions of bone morphogenetic protein 1, BMP1, dentin matrix protein 1, DMP1, the sodium phosphate co-transporter, NaPi IIb, matrix metalloprotease 13. MMP-13, and alkaline phosphatase, along with matrix protein genes (type X collagen, bone sialoprotein, and osteopontin) usually associated with initiation of mineralization are discussed.

Contribution of Circulating Fibrocytes to Atherosclerotic Plaque Stability

Background: Atherosclerotic plaque rupture in part results from inadequate compensation for lost collagens in the fibrous cap. Fibrocytes are circulating mesenchymal progenitors, which may serve as a source of new population of fibroblasts during wound healing, tumour formation and fibrosis. We hypothesised that reduced circulatingfibrocytesmaybe associatedwith plaque instability. Methods: Patients with acute myocardial infarction (MI) (n= 22), stable angina (n= 18) or healthy controls (n= 22) were recruited. The number of circulating fibrocytes (CD45+/CD34+/collagen I+) were measured by flow cytometry. PBMCs were isolated from blood and cultured for two weeks in six well plates and fibrocytes were then measured by morphology and flow cytometry (CD45+/collagen I+). Gene expression of matrix proteins in cells remaining after culturewasmeasured by real-time PCR. Results: Patients with acute MI had a lower absolute outcomes. Our aim was to compare coronary artery sizes between SA and CA. Method: A retrospective review of a database of CT coronary angiograms of 1200 patients was undertaken. A group, self-identified as SA, was analysed as were a gender, age, body surface are and coronary dominance matched cohort of CA. Luminal areas of left main, proximal left anterior descending, proximal left circumflex and proximal right coronary arteries were measured in all patients. All studies were analysed by a cardiologist and a radiologist whowere blinded to the patient demographics and ethnicity. Results: Eighty-eight subjects were included in both groups. The luminal area measurements (mean+SD) revealed

The response of human anulus fibrosus cells to cyclic tensile strain is frequency‐dependent and altered with disc degeneration

Mechanical loads are important for homeostasis of the intervertebral disc (IVD) cell matrix, with physiologic and nonphysiologic loads leading to matrix anabolism and catabolism, respectively. Previous investigations into the effects of load on disc cells have predominantly used animal models, with the limited number of human studies focusing primarily on nucleus pulposus cells. The aim of this study was to examine the effect of cyclic tensile strain (CTS) on human anulus fibrosus (AF) cells to ascertain whether the response was frequency-dependent and to compare AF cells derived from nondegenerated and degenerated tissue samples. AF cells were isolated from nondegenerated and degenerated human IVDs, expanded in monolayer, and cyclically strained for 20 minutes, applying 10% strain at a frequency of 1.0 Hz or 0.33 Hz with the use of a Flexcell strain device. Total RNA was extracted from the cells at baseline (control) and at 1, 3, and 24 hours following application of CTS. Real-time quantitative polymerase chain reaction was used to analyze gene expression of matrix proteins (aggrecan, type I collagen, and type II collagen) and enzymes (matrix metalloproteinases [MMPs] 3, 9, 13, and ADAMTS-4). The expression of catabolic genes (MMP-3 and ADAMTS-4) in AF cells derived from nondegenerated tissue decreased in response to 1.0 Hz of CTS, whereas changing the frequency to 0.33 Hz resulted in a shift toward matrix catabolism. Application of 1.0 Hz of CTS reduced anabolic gene expression (aggrecan and type I collagen) in AF cells derived from degenerated tissue, with 0.33 Hz of CTS resulting in increased catabolic gene expression. The response of human AF cells to CTS is frequency-dependent and is altered by degeneration.

The impact of luteal phase support on gene expression of extracellular matrix protein and adhesion molecules in the human endometrium during the window of implantation following controlled ovarian stimulation with a GnRH antagonist protocol

To evaluate the impact of two different luteal phase support protocols on gene expression of extracellular matrix (ECM) proteins and adhesion molecules in the human endometrium. Eighty-four ECM protein and adhesion molecule genes were analyzed using array-based reverse-transcription polymerase chain reaction. Academic hospital. Nine oocyte donors. Endometrial biopsies were obtained on the day of oocyte retrieval (group I) and 3-5 days later (group II) after randomization into 3 groups. Group IIa had no luteal phase support, group IIb had luteal support with micronized progesterone, and group IIc had luteal support with progesterone plus 17β-estradiol. Gene expression profiles in relation to different types of luteal phase support protocols. Compared with the day of retrieval (group I), 24 genes were significantly up-regulated (4 in group IIa, 14 in group IIb, 22 in group IIc) and 14 genes were down-regulated (5 in group IIa, 2 in group IIb, 10 in group IIc) on day 3-5 (P<.05). In the luteal support group, up- regulation occurred predominantly in genes encoding for matrix metallopeptidases (MMP10, MMP3, MMP9), integrins (ITGA3, ITGA5, ITGB3, ITGB4), and laminin (LAMB3). In contrast, the most significant suppression was documented in genes encoding for catenin-D2, collagen-11A1, and the laminins (LAMA1 and LAMA3). Significant changes between groups IIb and IIc were also observed in 9 genes. Luteal phase support following controlled ovarian stimulation has a profound impact on the ECM pathway targeted genes.

Cryopreserved intervertebral disc with injected bone marrow–derived stromal cells: a feasibility study using organ culture

Background Context A recent clinical study demonstrated that cryopreserved allogeneic intervertebral disc transplantation relieved pain and preserved motion, thus opening up a new treatment option for degenerative disc disease. However, these transplanted discs continued to degenerate, possibly due to a lack of viable cells. Bone marrow–derived stromal cell (BMSC) implantation has been shown to delay disc degeneration. Purpose This study examined the viability over time of endogenous and injected BMSCs in cryopreserved disc under simulated-physiological loading conditions. Study Design/ Setting An in vitro study of BMSCs injected into cryopreserved bovine caudal discs. Methods Bovine caudal discs were harvested and cryopreserved at −196°C. After thawing, PKH-26–labeled BMSCs embedded in peptide hydrogel carrier were injected into the nucleus pulposus. Two BMSC injection quantities, that is, 1×10 5 and 2.5×10 5 were examined. Discs with injected cells were maintained in a bioreactor for 7 days under simulated-physiological loading. Cell viability (staining), gene expression (reverse transcription-polymerase chain reaction) profile, and proteoglycan content (histologically) were evaluated. Results Forty percent of endogenous cell viability was maintained after freeze thawing. Over the 7-day culture, this did not change further. However, there was upregulation of Col1a2 and Mmp-13 and downregulation of Col2a1gene expression. Sixty percent of BMSCs survived the initial injection procedure, and only 20% remained alive after 7 days of culture. Bone marrow–derived stromal cell implantation did not alter the viability of the endogenous cells, but discs injected with 1×105 BMSCs showed significantly higher ACAN expression than sham discs. Conclusions Although only 40% of cells survived cryopreservation, these endogeneous cells continued to survive over 7 days if maintained under simulated-physiological loading conditions. Although only a small portion of injected BMSCs survived, they did have some effect on the matrix protein gene expression profile. Their influence on native cells requires long-term evaluation.

A Three-Dimensional Traction/Torsion Bioreactor System for Tissue Engineering

Purpose The aim of this study was to design, develop and validate a simple, compact bioreactor system for tissue engineering. The resulting bioreactor was designed to achieve ease-of-use and low costs for automated cell-culturing procedures onto three-dimensional scaffolds under controlled torsion/traction regimes. Methods Highly porous poly-caprolactone-based scaffolds were used as substrates colonized by fibroblast cells (3T3 cell line). Constructs were placed within the cylindrical culture chamber, clumped at the ends and exposed to controlled sequences of torsional stimuli (forward/back-forward sequential cycles of 100° from neutral position at a rate of 600°/min) through a stepper-motor; working settings were defined via PC by an easy user-interface. Cell adhesion, morphology, cytoskeletal fiber orientation and gene expression of extracellular matrix proteins (collagen type I, tenascin C, collagen type III) were evaluated after three days of torsional stimulation in the bioreactor system. Results and Conclusions The 3D bioreactor system was validated in terms of sterility, experimental reproducibility and flexibility. Cells adhered well onto the polymeric scaffolds. Collagen type I, tenascin C and collagen type III gene expression were significantly up-regulated when cells were cultured under torsion in the bioreactor for three days. In conclusion, we have developed a simple, efficient and versatile 3D cell-culture system to engineer ligament grafts. This system can be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system for possible clinical use in the field of regenerative medicine.

Regulation of bone development and extracellular matrix protein genes by RUNX2

RUNX2 is a multifunctional transcription factor that controls skeletal development by regulating the differentiation of chondrocytes and osteoblasts and the expression of many extracellular matrix protein genes during chondrocyte and osteoblast differentiation. This transcription factor plays a major role at the late stage of chondrocyte differentiation: it is required for chondrocyte maturation and regulates Col10a1 expression in hypertrophic chondrocytes and the expression of Spp1, Ibsp, and Mmp13 in terminal hypertrophic chondrocytes. It is essential for the commitment of pluripotent mesenchymal cells to the osteoblast lineage. During osteoblast differentiation, RUNX2 upregulates the expression of bone matrix protein genes including Col1a1, Spp1, Ibsp, Bglap, and Fn1 in vitro and activates many promoters including those of Col1a1, Col1a2, Spp1, Bglap, and Mmp13. However, overexpression of Runx2 inhibits osteoblast maturation and reduces Col1a1 and Bglap expression. The inhibition of RUNX2 in mature osteoblasts does not reduce the expression of Col1a1 and Bglap in mice. Thus, RUNX2 directs pluripotent mesenchymal cells to the osteoblast lineage, triggers the expression of major bone matrix protein genes, and keeps the osteoblasts in an immature stage, but does not play a major role in the maintenance of the expression of Col1a1 or Bglap in mature osteoblasts. During bone development, RUNX2 induces osteoblast differentiation and increases the number of immature osteoblasts, which form immature bone, whereas Runx2 expression has to be downregulated for differentiation into mature osteoblasts, which form mature bone. During dentinogenesis, Runx2 expression is downregulated, and RUNX2 inhibits the terminal differentiation of odontoblasts.

Influence of Three-Dimensional Hyaluronic Acid Microenvironments on Mesenchymal Stem Cell Chondrogenesis

Mesenchymal stem cells (MSCs) are multipotent progenitor cells whose plasticity and self-renewal capacity have generated significant interest for applications in tissue engineering. The objective of this study was to investigate MSC chondrogenesis in photo-cross-linked hyaluronic acid (HA) hydrogels. Because HA is a native component of cartilage, and MSCs may interact with HA via cell surface receptors, these hydrogels could influence stem cell differentiation. In vitro and in vivo cultures of MSC-laden HA hydrogels permitted chondrogenesis, measured by the early gene expression and production of cartilage-specific matrix proteins. For in vivo culture, MSCs were encapsulated with and without transforming growth factor beta-3 (TGF-beta3) or pre-cultured for 2 weeks in chondrogenic medium before implantation. Up-regulation of type II collagen, aggrecan, and sox 9 was observed for all groups over MSCs at the time of encapsulation, and the addition of TGF-beta3 further enhanced the expression of these genes. To assess the influence of scaffold chemistry on chondrogenesis, HA hydrogels were compared with relatively inert poly(ethylene glycol) (PEG) hydrogels and showed enhanced expression of cartilage-specific markers. Differences between HA and PEG hydrogels in vivo were most noticeable for MSCs and polymer alone, indicating that hydrogel chemistry influences the commitment of MSCs to undergo chondrogenesis (e.g., approximately 43-fold up-regulation of type II collagen of MSCs in HA over PEG hydrogels). Although this study investigated only early markers of tissue regeneration, these results emphasize the importance of material cues in MSC differentiation microenvironments, potentially through interactions between scaffold materials and cell surface receptors.

Use of staurosporine, an actin-modifying agent, to enhance fibrochondrocyte matrix gene expression and synthesis

Modulation of the actin cytoskeleton in chondrocytes has been used to prevent or reverse dedifferentiation and to enhance protein synthesis. We have hypothesized that an actin-modifying agent, staurosporine, could be used with fibrochondrocytes to increase the gene expression and synthesis of critical fibrocartilage proteins. A range of concentrations (0.1-100 nM) was applied to fibrochondrocytes in monolayer and evaluated after 24 h and after 4 days. High-dose staurosporine treatment (10-100 nM) increased cartilage oligomeric matrix protein 60- to 500-fold and aggrecan gene expression two-fold. This effective range of staurosporine was then applied to scaffoldless tissue-engineered fibrochondrocyte constructs for 4 weeks. Whereas glycosaminoglycan synthesis was not affected, collagen content doubled, from 27.6 +/- 8.8 microg in the untreated constructs to 55.2 +/- 12.2 microg per construct with 100 nM treatment. When analyzed for specific collagens, the 10-nM group showed a significant increase in collagen type I content, whereas collagen type II was unaffected. A concomitant dose-dependent reduction was noted in construct contraction, reflecting the actin-disrupting action of staurosporine. Thus, staurosporine increases the gene expression for important matrix proteins and can be used to enhance matrix production and reduce contraction in tissue-engineered fibrocartilage constructs.