Aggrecan Gene Expression Research Articles

A comparison between nucleus pulposus-derived stem cell transplantation and nucleus pulposus cell transplantation for the treatment of intervertebral disc degeneration in a rabbit model

IntroductionIn recent years, nucleus pulposus cell (NPC) transplantation has been used to treat intervertebral disc degeneration (IDD); however, the degenerative nature of NPCs influences its effectiveness. Nucleus pulposus-derived stem cells (NPSCs), which are self-renewing, have high expansion potential and can adapt to the intervertebral disc (IVD) microenvironment and may have a better regenerative capacity, which is favourable for treating IDD. The aim of this study was to compare the effectiveness of transplantation with NPSCs and NPCs on the regeneration of the IVD in rabbit models. MethodsNPSCs and NPCs were isolated from human degenerate nucleus pulposus tissue by differential adhesion method, and stem cell surface markers were detected by flow cytometry. Degenerative discs in rabbits were randomly distributed into three groups: NPSCs, NPCs and vehicle control group; the normal discs served as the normal control group. Cells of the P3 generation were prepared for transplantation. About 20 μl of cell suspension (NPSCs or NPCs) or DMEM was injected into corresponding discs, while the normal discs were left untreated. After 8 weeks, disc height was evaluated using X-ray, water content was evaluated by MRI, and gene and protein expression levels of collagen II and aggrecan in the nucleus were determined by real-time PCR and ELISA. ResultsNPCs and NPSCs from the P3 generation were polygonal and spindle-shaped, respectively. Both NPSCs and NPCs strongly expressed surface markers CD73, CD90, and CD105 and weakly expressed CD34 and CD45. The relative rates of expression of CD73, CD90, and CD105 were higher in NPSCs than in NPCs. After 8 weeks, X-ray results showed no significant difference in disc height index among the groups (p > 0.05). MRI revealed that the intensity of the nucleus pulposus signal was increased in NPSCs (p < 0.05). The results from PCR and ELISA demonstrated that NPSCs promoted gene and protein expression of aggrecan instead of collagen II (p < 0.05). ConclusionCompared to NPCs, NPSCs harvested by differential adhesion method displayed a higher positive rate of stem cell surface markers and showed superior regenerative effectiveness for treating IDD in rabbit models. Therefore, NPSCs are potential candidates for cell therapy for the regeneration of the IVD.

Incorporation of chitosan nanoparticles into silk fibroin-based porous scaffolds: Chondrogenic differentiation of stem cells

ABSTRACTA silk fibroin-chondroitin sulfate-sodium alginate (SF-CHS-SA) porous scaffold containing chitosan nanoparticles (NPs) was investigated. The proliferation of adipose-derived stem cells (ASCs) was studied by SEM, fluorescent microscopy, alcian blue staining, dimethylmethylene blue assay, and real-time polymerization chain reaction. The results showed that incorporation of NPs into the scaffold improved compressive modulus (5.6 ± 0.15 MPa). The amount of glycosaminoglycan expression of the ASCs was reached to 8.9 ± 0.3 µg/mL. The gene expressions of aggrecan, collagen II, and SOX9 of the ASCs were significantly improved. This study revealed that the prepared scaffold can be used as a substrate for cartilage tissue engineering.

Evaluation of cell-laden polyelectrolyte hydrogels incorporating poly(l-Lysine) for applications in cartilage tissue engineering

To address the lack of reliable long-term solutions for cartilage injuries, strategies in tissue engineering are beginning to leverage developmental processes to spur tissue regeneration. This study focuses on the use of poly(l-lysine) (PLL), previously shown to up-regulate mesenchymal condensation during developmental skeletogenesis in vitro, as an early chondrogenic stimulant of mesenchymal stem cells (MSCs). We characterized the effect of PLL incorporation on the swelling and degradation of oligo(poly(ethylene) glycol) fumarate) (OPF)-based hydrogels as functions of PLL molecular weight and dosage. Furthermore, we investigated the effect of PLL incorporation on the chondrogenic gene expression of hydrogel-encapsulated MSCs. The incorporation of PLL resulted in early enhancements of type II collagen and aggrecan gene expression and type II/type I collagen expression ratios when compared to blank controls. The presentation of PLL to MSCs encapsulated in OPF hydrogels also enhanced N-cadherin gene expression under certain culture conditions, suggesting that PLL may induce the expression of condensation markers in synthetic hydrogel systems. In summary, PLL can function as an inductive factor that primes the cellular microenvironment for early chondrogenic gene expression but may require additional biochemical factors for the generation of fully functional chondrocytes.

Estrogen Enhances Matrix Synthesis in Nucleus Pulposus Cell through the Estrogen Receptor β-p38 MAPK Pathway

Background/Aims: Matrix homeostasis within the disc nucleus pulposus (NP) tissue is important for disc function. Increasing evidence indicates that sex hormone can influence the severity of disc degeneration. This study was aimed to study the role of 17β-estradiol (E₂) in NP matrix synthesis and its underlying mechanism. Methods: Rat NP cells were cultured with (10^-5, 10^-7 and 10^-9 M) or without (control) E2 for48 hours. The estrogen receptor (ER)-β antagonist PHTPP and ERβ agonist ERB 041 were used to investigate the role mediated by ERβ. The p38 MAPK inhibitor SB203580 was used to investigate the role of p38 MAPK signaling pathway. Gene and protein expression of SOX9, aggrecan and collagen II, glycosaminoglycan (GAG) content, and immunostaining assay for aggrecan and collagen II were analyzed to evaluate matrix production in rat NP cells. Results: E2 enhanced NP matrix synthesis in a concentration-dependent manner regarding gene and proetin expression of SOX9, aggrecan and collagen II, protein deposition of aggrecan and collagen II, and GAG content. Moreover, activation of p38 MAPK signaling pathway was increased with elevating E2 concentration. Further analysis indicated that ERB 041 and PHTPP could respectively enhance and suppress effects of E2 on matrix synthesis in NP cells, as well as activation of p38 MAPK pathway. Additionally, inhibition of p38 MAPK signaling pathway significantly abolished the effects of E2 on matrix synthesis. Conclusion: E2 can enhance matrix synthesis of NP cells and the ERβ/p38 MAPK pathway is involved in this regulatory process.

The Extract of Fructus Psoraleae Promotes Viability and Cartilaginous Formation of Rat Chondrocytes In Vitro.

This study aimed to investigate the extract components of FP on rat chondrocyte function and cartilaginous formation in vitro. Petroleum ether extract (P-e) of FP extract components was selected to treat Sprague-Dawley rat chondrocytes. Cell viability was tested with different concentrations (0.1, 1, 10, and 100 μg/mL) of P-e treatment. Concentrations of 0.1 and 1 μg/mL P-e conditioned culture mediums were used for treating chondrocytes in experiments. Cell proliferation was measured via DNA incorporation assay. Type II collagen, aggrecan, and Sox-9 genes expression levels were measured with RT-PCR. Additionally, cartilaginous formation was analyzed with type II collagen immunofluorescence, H&E, and alcian blue staining. Concentrations of 0.1 and 1 μg/mL P-e showed low cytotoxicity and demonstrated stimulatory effects on chondrocyte proliferation in early stages. Following 6 days of P-e culture, aggrecan and Sox-9 gene expression levels of the 1 μg/mL P-e group were upregulated by 1.82- (p < 0.05) and 2.06-fold (p < 0.05), respectively, versus controls. Moreover, 1 μg/mL P-e significantly stimulated cell aggregation and type II collagen deposits after 1 week of treatment. Noteworthy, tight cartilaginous structures formed in the 10-day 1 μg/mL P-e conditioned culture. These findings suggest that P-e has the potential to treat cartilage degeneration induced by chondrocyte failure.

The infrapatellar fat pad from diseased joints inhibits chondrogenesis of mesenchymal stem cells.

Cartilage repair by bone marrow derived mesenchymal stem cells (MSCs) can be influenced by inflammation in the knee. Next to synovium, the infrapatellar fat pad (IPFP) has been described as a source for inflammatory factors. Here, we investigated whether factors secreted by the IPFP affect chondrogenesis of MSCs and whether this is influenced by different joint pathologies or obesity. Furthermore, we examined the role of IPFP resident macrophages. First, we made conditioned medium from IPFP obtained from osteoarthritic joints, IPFP from traumatically injured joints during anterior cruciate ligament reconstruction, and subcutaneous adipose tissue. Additionally, we made conditioned medium of macrophages isolated from osteoarthritic IPFP and of polarised monocytes from peripheral blood. We evaluated the effect of different types of conditioned medium on MSC chondrogenesis. Conditioned medium from IPFP decreased collagen 2 and aggrecan gene expression as well as thionin and collagen type 2 staining. This anti-chondrogenic effect was the same for conditioned medium from IPFP of osteoarthritic and traumatically injured joints. Furthermore, IPFP from obese (Body Mass Index >30) donors did not inhibit chondrogenesis more than that of lean (Body Mass Index <25) donors. Finally, conditioned medium from macrophages isolated from IPFP decreased the expression of hyaline cartilage genes, as did peripheral blood monocytes stimulated with pro-inflammatory cytokines. The IPFP and the resident pro-inflammatory macrophages could therefore be targets for therapies to improve MSC-based cartilage repair.

Different responses of articular cartilage to strenuous running and joint immobilization

ABSTRACTObjective: To compare the pathological changes in cartilage derived from rats that developed osteoarthritis either by joint immobilization or by strenuous treadmill running in order to better understand their respective pathomechanism.Method: A total of 24 male Wistar rats were randomly assigned to three groups: sedentary control (CON), immobilization (IM), and strenuous running (SR). For rats in the IM group, unilateral knee joint was immobilized in flexion. Rats in the SR group underwent treadmill running with high intensity. Eight weeks later, all animals were sacrificed. Femoral condyles were collected to take histological observation for cartilage characteristic and immunohistochemistry for collagen type II. In addition, cartilage samples were obtained to assess gene expression of aggrecan, collagen type II, biglycan, and fibromodulin by quantitative RT-PCR.Results: Gross and histological observation showed osteoarthritic changes in groups SR and IM; however, more severe cartilage degradation was revealed in the latter. Proteoglycan and collagen II content decreased in groups SR and IM in comparison to group CON, with more loss in group IM. In group SR, mRNA levels in femoral cartilage were found to be unaltered for all the molecules measured. On the contrary, these molecules were significantly downregulated in group IM.Conclusion: Differences in gross observation, histological characteristics, and gene expression of proteoglycans and collagen II suggest that both knee immobilization and strenuous running would lead to degenerative change of cartilage, but at different stages of the degenerative process.

Growth Factors Cross-Linked to Collagen Microcarriers Promote Expansion and Chondrogenic Differentiation of Human Mesenchymal Stem Cells.

Tissue engineering is a field in progressive expansion and requires constant updates in methods and devices. One of the central fields is the development of biocompatible, biodegradable, and injectable scaffolds, such as collagen microcarriers. To enhance cell attachment and produce a cost-effective cell culture solution with local stimulation of cells, basic fibroblast growth factor (bFGF) or transforming growth factor-β1 (TGF-β1) was covalently immobilized on microcarriers either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) or riboflavin/UV (RB/UV) light-mediated cross-linking. Collagen microcarriers cross-linked with bFGF or TGF-β1 were used for expansion and chondrogenic differentiation of human mesenchymal stem cells (MSCs). Evaluation methods included cell viability test, chondrogenic marker expression (aggrecan and collagen type I and type II), histological detection of proteoglycans, and immunohistochemical analysis. Cross-linking strengthened the collagen structure of the microcarriers and reduced collagenase-mediated degradation. MSCs effectively proliferated on microcarriers cross-linked with bFGF, especially by EDC/NHS cross-linking. Chondrogenic differentiation of MSCs was induced by TGF-β1 cross-linked on microcarriers, promoting gene expression and protein accumulation of aggrecan and collagen type I and type II, as well as proteoglycans. Cross-linking by RB/UV enhanced chondrogenesis more than any other group. In addition, cross-linking reduced scaffold shrinkage exerted by MSCs during chondrogenesis, a desirable feature for microcarriers if used as tissue defect filler. In conclusion, cross-linking of bFGF or TGF-β1 to collagen microcarriers supported in vitro proliferation and chondrogenesis, respectively. If translated in vivo and in clinical practice, such approach might lead a step closer to development of a cost-effective and locally acting device for cell-based therapy.

The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells.

Annulus fibrosus (AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc (IVD) degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells (AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.

In vitro effects of glutamate and N-methyl-D-aspartate receptor (NMDAR) antagonism on human tendon derived cells.

It is known that extracellular glutamate concentrations are increased in tendinopathy but the effects of glutamate upon human tendon derived cells are unknown. The primary purpose was to investigate the effect of glutamate exposure on human tendon-derived cells in terms of viability, protein, and gene expression. The second purpose was to assess whether NMDAR antagonism would affect the response of tendon-derived cells to glutamate exposure. Human tendon-derived cells were obtained from supraspinatus tendon tissue obtained during rotator cuff repair (tendon tear derived cells) and from healthy hamstring tendon tissue (control cells). The in vitro impact of glutamate exposure and NMDAR antagonism (MK-801) was measured using the Alamar blue cell viability assay, immunocytochemistry, and quantitative real-time PCR. Glutamate reduced cell viability at 24 h in tendon tear derived cells but not in control cells at concentrations of 7.5 mM and above. Cell viability was significantly reduced after 72 h of 1.875 mM glutamate in both cell groups; this deleterious effect was attenuated by NMDAR antagonism with 10 µM MK-801. Both 24 and 72 h of 1.875 mM glutamate exposure reduced Type 1 alpha 1 collagen (COL1A1) and Type 3 alpha 1 collagen (COL3A1) gene expression, but increased Aggrecan gene expression. We propose that these effects of glutamate on tendon derived cells including reduced cell viability and altered matrix gene expression contribute to the pathogenesis of tendinopathy.

Expression of matrix factors in the process of neovascularization of intervertebral disc

&lt;sec&gt;&lt;title&gt;OBJECTIVE:&lt;/title&gt;&lt;p&gt; To investigate the effects of proteins products of endothelial cells (ECs) on the annulus fibrosus (AF) cell metabolism in an in vitro culture.&lt;/p&gt;&lt;/sec&gt;&lt;sec&gt;&lt;title&gt;METHODS:&lt;/title&gt;&lt;p&gt;Human AF cells were expanded in monolayer cultures and treated with proteins from the medium of cell line HMEC-1 (Human Microvascular Endothelial Cells) (125µg/ml). After 72h of treatment RNA was isolated from AF cells for analysis of gene expression and the culture medium was collected for protein expression analysis.&lt;/p&gt;&lt;/sec&gt;&lt;sec&gt;&lt;title&gt;RESULTS:&lt;/title&gt;&lt;p&gt; The qRT-PCR analysis demonstrated increased gene expression of matrix metalloproteinases (MMPs) in AF cells treated with protein products of endothelial cells compared with cells from control group of AF cells: MMP-1 243.10 times (p&lt;0.05), MMP-2 1.37 time (p&lt;0.05), MMP-3 39.83 times (p&lt;0.05) and MMP-13 5.70 times (p&lt;0.05). In contrast, tissue inhibitors of metalloproteinases (TIMPs) were suppressed; TIMP-2 (0.55 time) (p&lt;0.05) and TIMP-3 (0.60 time) (p&lt;0.05) in the exposed groups. The expression of aggrecan gene (0.83 time) (p&lt;0.05), an important extracellular matrix component, was also reduced. MMP-1 and MMP-3 detection was performed, confirming the results of PCR by Western Blot technique.&lt;/p&gt;&lt;/sec&gt;&lt;sec&gt;&lt;title&gt;CONCLUSIONS:&lt;/title&gt;&lt;p&gt; In this study, we observed that the proteins produced by ECs induced the MMPs expression and suppressed the TIMPs as well as the aggrecan in primary cells of the human intervertebral disc, targeting the development of potential treatments for intervertebral disc degeneration and associated discogenic pain.&lt;/p&gt;&lt;/sec&gt;

Biomimetic Nucleus Pulposus Replacement for the Treatment of Degenerative Disc Disease

Introduction Nucleus pulposus (NP) replacement offers an alternative minimally invasive treatment for traditional spinal fusion or total disc replacement for degenerative disc disease (DDD). Recently, a novel polyurethane scaffold (PUS) with swelling capability in situ was developed to restore the mechanical functions of the intervertebral disc (IVD). In addition, a fibrinogen–hyaluronic acid (FBG–HA) conjugate-based hydrogel was manufactured to mimic the native NP extracellular matrix. The aim of this study is to evaluate the PUS and the FBG–HA hydrogel, with/without transglutaminase crosslinked bone morphogenetic protein (TG-BMP) 2/7 heterodimer in an organ culture system under dynamic load. Materials and Methods Discoid/ravioli-shaped polyurethane scaffolds were manufactured with a PU hydrogel-based core with swelling capacity in between two electrospun nanofiber envelope sheets. In addition, FBG–HA conjugate solution was synthesized with 235 kDa HA at FBG/HA w/w ratio of 17:1. FBG–HA hydrogels were prepared by mixing ⅔ volume of FBG–HA conjugate solution with ⅓ volume of thrombin solution (5.2 U/mL). Bovine IVDs with endplates were nucleotomized by incision through the endplate and refilled with either (1) PUS, (2) PUS surrounded by 50 to 80 µL FBG–HA or (3) PUS surrounded by 50 to 80µL FBG–HA containing 5,000 ng/mL TG–BMP 2/7. The endplate defect was closed with an endogenous endplate stopper and sealed with polymethyl methacrylate. Empty discs served as negative controls. To assess the mechanical compatibility of the different implants, dynamic compressive stiffness modulus (DCSM) was measured for each disc at different time points: intact, after nucleotomy, after refilling with biomaterial and free swelling recovery, after 3-hour dynamic load at 0 to 0.1 MPa, 0.1 Hz within a bioreactor system, and after free swelling recovery overnight. Moreover, biomaterials were evaluated in an organ culture system under dynamic load for 14 days at 0 to 0.1 MPa, 0.1 Hz for 3 hours/d. Disc height was recorded after load and recovery at day 1, 7, and 14. After 14 days, disc tissue was harvested and gene expression analyzed using real-time PCR. Glycosaminoglycan- (GAG) and collagen-content of the disc tissue was assessed and proteoglycan synthesis was analyzed by Sulfur-35 (35S) incorporation measurement. Histology was performed using Safranin O/Fast Green staining. One-way ANOVA was used to determine the statistical significance. Results After dynamic load, all three implant groups maintained their disc height, while it dropped by 7% in the empty controls ( p &lt; 0.001 vs. implant groups). The PUS and addition of FBG–HA hydrogel caused immediate restoration of DCSM (PUS: 73 ± 21%; p &lt; 0.05 vs. empty control: 25 ± 5%). While addition of FBG–HA hydrogel surrounding the PUS delayed the stiffness restoring effect, stiffness also increased to 69 ± 16% after dynamic load and free swelling recovery ( p &lt; 0.05 vs. empty control: 27 ± 5%). GAG-/collagen-content was maintained in all three implant groups. Aggrecan and collagen-2 gene expression were upregulated in NP cells by TG-BMP2/7 and FBG–HA hydrogel, respectively, although not significantly. Conclusion Results indicate that PUS is able to restore the mechanical functions of nucleotomized discs. Addition of FBG–HA hydrogel does not affect the swelling capacity of PUS in situ. FBG–HA hydrogel and TG-BMP2/7 may further support the biological repair of NP tissue. Acknowledgment Funded by the European Commission under the FP7–NMP Project NPMimetic.

Focal experimental injury leads to widespread gene expression and histologic changes in equine flexor tendons.

It is not known how extensively a localised flexor tendon injury affects the entire tendon. This study examined the extent of and relationship between histopathologic and gene expression changes in equine superficial digital flexor tendon after a surgical injury. One forelimb tendon was hemi-transected in six horses, and in three other horses, one tendon underwent a sham operation. After euthanasia at six weeks, transected and control (sham and non-operated contralateral) tendons were regionally sampled (medial and lateral halves each divided into six 3cm regions) for histologic (scoring and immunohistochemistry) and gene expression (real time PCR) analysis of extracellular matrix changes. The histopathology score was significantly higher in transected tendons compared to control tendons in all regions except for the most distal (P ≤ 0.03) with no differences between overstressed (medial) and stress-deprived (lateral) tendon halves. Proteoglycan scores were increased by transection in all but the most proximal region (P < 0.02), with increased immunostaining for aggrecan, biglycan and versican. After correcting for location within the tendon, gene expression for aggrecan, versican, biglycan, lumican, collagen types I, II and III, MMP14 and TIMP1 was increased in transected tendons compared with control tendons (P < 0.02) and decreased for ADAMTS4, MMP3 and TIMP3 (P < 0.001). Aggrecan, biglycan, fibromodulin, and collagen types I and III expression positively correlated with all histopathology scores (P < 0.001), whereas lumican, ADAMTS4 and MMP14 expression positively correlated only with collagen fiber malalignment (P < 0.001). In summary, histologic and associated gene expression changes were significant and widespread six weeks after injury to the equine SDFT, suggesting rapid and active development of tendinopathy throughout the entire length of the tendon. These extensive changes distant to the focal injury may contribute to poor functional outcomes and re-injury in clinical cases. Our data suggest that successful treatments of focal injuries will need to address pathology in the entire tendon, and that better methods to monitor the development and resolution of tendinopathy are required.

Activin receptor-like kinase ALK5 and ALK1 are both required for TGFβ-initiated chondrogenic differentiation of mesenchymal stem cells

Activin receptor-like kinase ALK5 and ALK1 are both required for TGFβ-initiated chondrogenic differentiation of mesenchymal stem cells

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Terminology of CCN1-6 should not be applicable for their fragments and be limited to only full length CCN1-6

Effects of nuclear factor kappa B signaling pathway in human intervertebral disc degeneration.

IL-1β (interleukin-1β) can activate human nucleus pulposus cells with or without nuclear factor kappa B (NF-κB) inhibition. We undertook a descriptive and mechanistic investigation of catabolic effects of NF-κB signaling pathway in intervertebral disc degenerative changes. To clarify the mediatory role of NF-κB signaling pathway in human intervertebral disc degeneration (IDD). IDD is a major cause of lower back pain, but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic activation of NF-κB in many degenerative diseases, but its role in IDD has not been adequately explored. Human nucleus pulposus cells in monolayer culture were exposed to IL-1β, which increases matrix-degrading enzyme activity in the nucleus pulposus, with or without NF-κB inhibition by BAY11-7082; ribonucleic acid was isolated for real-time polymerase chain reaction analysis of gene expression, Western blot analysis was performed to detect the changes of protein expression. NF-κB specific inhibitor BAY11-7082 significantly inhibited IL-1β-induced NF-κB activation. IL-1β-dependent gene upregulation of matrix metalloproteinase (MMP)-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5 was significantly reduced by NF-κB inhibition. The decreased gene expression of aggrecan and type II collagen, induced by IL-1β was also reversed by BAY11-7082. NF-κB inhibition reversed the IL-1β-induced changes of protein expression of MMP-3, MMP-9, MMP-13, ADAMTS-4, ADAMTS-5, aggrecan, and type II collagen. These findings demonstrate that the NF-κB signaling pathway is a key mediator of IDD and represents a therapeutic target for mitigating disc degenerative diseases. N/A.

Probing the role of scaffold dimensionality and media composition on matrix production and phenotype of fibroblasts.

Porous sponges, hydrogels, and micro/nanofibrous matrix are most commonly used three dimensional (3D) biomaterials in tissue engineering; however, reciprocal interaction between internal dimensionality of biomaterials and fibroblasts remains largely unexplored. Such studies would have potential to generate valuable insights about wound healing, tissue morphogenesis and homeostasis. To the best of our knowledge this is the first study to evaluate functionality of porous collagen matrix and collagen gels for in vitro culture of fibroblasts while investigating the role of culture media composition in modulating morphology, phenotype, extracellular matrix (ECM)-related gene expression and protein synthesis by fibroblasts. Encapsulation of fibroblasts in collagen gel was found to be more effective for ECM production compared to scaffold-based culture, as evidenced by enhanced collagen type I, elastin, lysyl oxidase, aggrecan gene expression. High glucose media induced spindle like morphology of typical in vivo fibroblasts and enhanced collagen production compared to other media. This variation in biosynthesis in different glucose concentrations was possibly due to endogenous activation of TGF-β or by an increase in ATP consuming anabolic pathways in high glucose concentration.

Strategies for faster detachment of corneal cell sheet using micropatterned thermoresponsive matrices.

The development of transplantable cell sheets of functional keratocytes embedded within an aligned collagen type I matrix is a viable approach for constructing a bioequivalent of corneal stroma. Thermoresponsive materials based on poly(N-isopropylacrylamide) (PolyNIPA) have been utilized to recover carrier-free corneal cell sheets by inducing temperature changes. In this study, we employed direct-write assembly (DWA) to develop microperiodic parallel patterns of silk-PolyNIPA and gelatin-PolyNIPA. Semi-interpenetrating networks of PolyNIPA hybrids (with silk/gelatin) exhibited temperature-responsive nature and thereby have potential use in cell sheet engineering. Silk-PolyNIPA and gelatin-PolyNIPA hybrids demonstrated a hydrophobic surface at 37 °C (i.e. above their lower critical solution temperature) with a contact angle of 59°± 0.3° and 55°± 3°, respectively, whereas the surface roughness of silk-PolyNIPA was double that of gelatin-PolyNIPA. The reduction of temperature to 20 °C resulted in a decrease in the value of surface roughness and water contact angle for both hybrids. All four parallel patterned substrates guided corneal cell alignment along the direction of the patterns. Collagen type-I and aggrecan gene expression was higher when the cells were grown over the gelatin-PolyNIPA matrix after 3 weeks of culture when compared to silk-PolyNIPA. In addition, a significantly higher metabolic activity as well as enhanced vinculin expression of keratocytes on the gelatin-PolyNIPA matrix indicated the improved cytocompability compared to the silk, gelatin and silk-PolyNIPA matrices. Interestingly, the detachment of keratocytes cell sheet was achieved from the silk-PolyNIPA and gelatin-PolyNIPA planar films only within 10 min and 30 min, respectively, but the patterns could not yield intact sheet recovery. Hence, we conclude that while gelatin-PolyNIPA hybrids with parallel patterns fabricated using DWA will benefit from the application of cellular alignment, some optimization in the pattern parameters may be required for rapid sheet recovery from such substrates. Understanding the keratocytes responses to such hybrid biomaterials suggests viable options to develop a corneal stromal bioequivalent.

The effect of estrogen on the expression of cartilage-specific genes in the chondrogenesis process of adipose-derived stem cells.

Background:During adolescence, sex hormones play an important role in regulating proliferation, differentiation, maturation, and the scheduled death of chondrocytes. Although some studies have reported the regulatory role of estrogen in the development and progression of cartilage, some of the mechanisms still remain unclear, including the role of estrogen in the expression of cartilage-specific genes in chondrogenesis process, which we cover in this study.Materials and Methods:In the present study, we used adipose-derived stem cells (ADSCs) to differentiate into cartilage. Differentiated cartilage cells were used in the control (without estrogen E2 in the culture medium) and experimental (with estrogen in the culture medium) groups to evaluate the expression of type II collagen and aggrecan as chondrogenic genes markers, with -real-time polymerase chain reaction technique.Results:Our results indicated that estrogen leads to inhibition of type II collagen gene expression and reduction of aggrecan gene expression.Conclusion:Therefore, estrogen probably has negative effects on chondrogenesis process of ADSCs.

Bone marrow mesenchymal stem cells slow intervertebral disc degeneration through the NF-κB pathway

Background contextPrevious studies have demonstrated the use of bone marrow mesenchymal stem cells (BMSCs) in tissue-engineering treatments to slow or reverse diseased intervertebral discs. Several approaches have successfully used the coculturing of stem cells with disc-native nucleus pulposus cells (NPCs) with the evidence of transformed BMSCs into NP-like cells, increased activity and matrix production by NPCs, or elements of both. The influence of the cytokine transforming growth factor-beta (TGF-β) in the differentiation of BMSCs into NP-like cells and its upregulation in coculture to increase matrix production are well established. However, the role of the inflammatory signaling molecule nuclear factor kappa B (NF-κB) in intervertebral disc degeneration is far less clear, although there is some existing evidence suggesting its role in the pathogenesis and progression of disc disease. A limited number of studies in other pathologies have alluded to the antagonistic relationship between both proteins. To date, there is no such investigation of their dynamic role in coculture of BMSCs and NPCs. PurposeThe purpose of this study was to investigate the relationship of the regenerative effects of BMSCs cocultured with NPCs. The authors hypothesized that as levels of TGF-β increase in the coculture, the levels of NF-κB will concomitantly decrease. This would in turn be reflected by an increase in the expression of messenger RNA markers of the nucleus pulposus matrix that includes aggrecan, Type II collagen (CII), and SOX-9 and an increase in the cellular proliferation. Study design/settingThis study is based on a coculture with the contact of rabbit NPCs and BMSCs. MethodsBone marrow mesenchymal stem cells were cocultured with NPCs at a ratio of 1:1 and compared with BMSC and NPC controls cultured alone. Cell proliferation was evaluated by Cell Counting Kit-8 from 3 to 9 days. Gene expressions of aggrecan, CII, and SOX-9 was assayed by reverse transcription-polymerase chain reaction from 5 to 14 days. Detection of TGF-βl and NF-κB was determined by enzyme-linked immunosorbent assay, and immunohistochemical staining was carried out to evaluate CII synthesis. ResultsAfter 3 days, cellular proliferation of the cocultured group exceeded that of controls. After 11 days, the expression of SOX-9 in the cocultured group had also exceeded controls. Furthermore, after 14 days, expressions of aggrecan and CII significantly exceeded controls. Immunohistochemical stains of CII in the NPC control group were positive at each point in time and demonstrated strongest expression at 14 days. Coculturing BMSCs with NPCs, therefore, seems to have resulted in the promotion of aggrecan, CII, and SOX-9 gene expressions. Finally, after 11 days, TGF-βl content of the cocultured group significantly exceeded control levels, whereas NF-κB content had significantly lowered. ConclusionsCoculture of BMSCs may be able to delay NPC matrix degeneration potentially through the concomitant upregulation of TGF-β and the downregulation of NF-κB pathway.