Hyaluronic Acid Influence on Normal and Osteoarthritic Tissue-Engineered Cartilage

The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue- engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (−73%), although the extent was limited in the presence of HA (−39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level

Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

<p>Abstract</p> <p>Background</p> <p>The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors that trigger osteoblast differentiation in MSCs is therefore critical to promote the osteogenic potential of human MSCs. In this study, we used microarray analysis to identify signalling molecules that promote osteogenic differentiation in human bone marrow stroma derived MSCs.</p> <p>Results</p> <p>Microarray analysis and validation experiments showed that the expression of IGF2 and IGFBP2 was increased together with integrin alpha5 (ITGA5) during dexamethasone-induced osteoblast differentiation in human MSCs. This effect was functional since we found that IGF2 and IGFBP2 enhanced the expression of osteoblast phenotypic markers and <it>in vitro </it>osteogenic capacity of hMSCs. Interestingly, we showed that downregulation of endogenous ITGA5 using specific shRNA decreased IGF2 and IGFBP2 expression in hMSCs. Conversely, ITGA5 overexpression upregulated IGF2 and IGFBP2 expression in hMSCs, which indicates tight crosstalks between these molecules. Consistent with this concept, activation of endogenous ITGA5 using a specific antibody that primes the integrin, or a peptide that specifically activates ITGA5 increased IGF2 and IGFBP2 expression in hMSCs. Finally, we showed that pharmacological inhibition of FAK/ERK1/2-MAPKs or PI3K signalling pathways that are enhanced by ITGA5 activation, blunted IGF2 and IGFBP2 expression in hMSCs.</p> <p>Conclusion</p> <p>The results show that ITGA5 is a key mediator of IGF2 and IGFBP2 expression that promotes osteoblast differentiation in human MSCs, and reveal that crosstalks between ITGA5 and IGF2/IGFBP2 signalling are important mechanisms that trigger osteogenic differentiation in human bone marrow derived mesenchymal stromal cells.</p

Development of a High-Throughput Screening Assay Based on the 3-Dimensional Pannus Model for Rheumatoid Arthritis

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBi™-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time. (Journal of Biomolecular Screening 2007:956-965)BMBF, 0313604A, Verbundprojekt: Evaluierung eines interagierenden 3D Testsystems als Krankheitsmodell der rheumatoiden Arthritis (in vitro Pannus Modell) zur effektiven Prüfung von Wirkstoffen, Teilprojekt 1BMBF, 0313604B, Verbundprojekt: Entwicklung eines interagierenden 3D Testsystems als Krankheitsmodell der rheumatoiden Arthritis (in vitro Pannus Modell) zur effektiven Prüfung von Wirkstoffen, Teilprojekt

Quality assessment of surgical disc samples discriminates human annulus fibrosus and nucleus pulposus on tissue and molecular level

A discrimination of the highly specialised annulus fibrosus (AF) and nucleus pulposus (NP) cells in the mature human intervertebral disc (IVD) is thus far still not possible in a reliable way. The aim of this study was to identify molecular markers that distinguish AF and NP cells in human disc tissue using microarray analysis as a screening tool. AF and NP samples were obtained from 28 cervical discs. First, all samples underwent quality sorting using two novel scoring systems for small-sized disc tissue samples including macroscopic, haptic and histological evaluation. Subsequently, samples with clear disc characteristics of either AF or NP that were free from impurities of foreign tissue (IVD score) and with low signs of disc degeneration on cellular level (DD score) were selected for GeneChip analysis (HGU1332P). The 11 AF and 9 NP samples showed distinctly different genome-wide transcriptomes. The majority of differentially expressed genes (DEGs) could be specifically assigned to the AF, whereas no DEG was exclusively expressed in the NP. Nevertheless, we identified 11 novel marker genes that clearly distinguished AF and NP, as confirmed by quantitative gene expression analysis. The novel established scoring systems and molecular markers showed the identity of AF and NP in disc starting material and are thus of great importance in the quality assurance of cell-based therapeutics in regenerative treatment of disc degeneration

Antirheumatic drug response signatures in human chondrocytes: potential molecular targets to stimulate cartilage regeneration

Rheumatoid arthritis (RA) leads to progressive destruction of articular cartilage. This study aimed to disclose major mechanisms of antirheumatic drug action on human chondrocytes and to reveal marker and pharmacological target genes that are involved in cartilage dysfunction and regeneration

Human Cardiac-Derived Adherent Proliferating Cells Reduce Murine Acute Coxsackievirus B3-Induced Myocarditis

BACKGROUND: Under conventional heart failure therapy, inflammatory cardiomyopathy typically has a progressive course, indicating a need for alternative therapeutic strategies to improve long-term outcomes. We recently isolated and identified novel cardiac-derived cells from human cardiac biopsies: cardiac-derived adherent proliferating cells (CAPs). They have similarities with mesenchymal stromal cells, which are known for their anti-apoptotic and immunomodulatory properties. We explored whether CAPs application could be a novel strategy to improve acute Coxsackievirus B3 (CVB3)-induced myocarditis. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the safety of our approach, we first analyzed the expression of the coxsackie- and adenovirus receptor (CAR) and the co-receptor CD55 on CAPs, which are both required for effective CVB3 infectivity. We could demonstrate that CAPs only minimally express both receptors, which translates to minimal CVB3 copy numbers, and without viral particle release after CVB3 infection. Co-culture of CAPs with CVB3-infected HL-1 cardiomyocytes resulted in a reduction of CVB3-induced HL-1 apoptosis and viral progeny release. In addition, CAPs reduced CD4 and CD8 T cell proliferation. All CAPs-mediated protective effects were nitric oxide- and interleukin-10-dependent and required interferon-γ. In an acute murine model of CVB3-induced myocarditis, application of CAPs led to a decrease of cardiac apoptosis, cardiac CVB3 viral load and improved left ventricular contractility parameters. This was associated with a decline in cardiac mononuclear cell activity, an increase in T regulatory cells and T cell apoptosis, and an increase in left ventricular interleukin-10 and interferon-γ mRNA expression. CONCLUSIONS: We conclude that CAPs are a unique type of cardiac-derived cells and promising tools to improve acute CVB3-induced myocarditis

Differentiation- and migration potential of mesenchymal stem- and progenitor cells for in situ tissue engineering

Verschiedene Studien weisen darauf hin, dass im Falle akuter Erkrankungen wie dem Herzinfarkt oder dem Schlaganfall, mesenchymale Stammzellen (MSC) zu den betroffenen Geweben migrieren und an deren Regeneration beteiligt sind. Solche Beobachtungen sind die Grundlage für die Entwicklung des in situ Tissue Engineering (TE). Hier werden zellfreie Biomaterialien mit chemotaktisch aktiven Faktoren wie Chemokinen sowie Differenzierungs-faktoren kombiniert und in einen Gewebedefekt implantiert. Die Faktoren ermöglichen eine Rekrutierung geeigneter Zellen und deren Entwicklung zum Ersatzgewebe. In dieser Dissertation wurden Fragestellungen untersucht, die in vitro vor der Durchführung präklinischer in situ Knorpel TE Studien beantwortet werden müssen. Aus dem Knochen-mark isolierte und expandierte humane MSC präsentierten das in der Literatur beschriebene Profil an Oberflächenmarkern (SH2+, SH3+, CD34-). In hochdichten Zellaggregaten konnte histologisch, immunhistochemisch und mittels PCR erstmals eine chondrogene Induktion dieser Zellen durch BMP2 nachgewiesen werden. Eine BMP2 induzierte Weiterentwicklung zu hypertrophem Knorpel oder eine Differenzierung in die osteo- bzw. adipogene Richtung wurde nicht beobachtet. Zusätzlich konnte auf der mRNA- und der Proteinebene erstmals das komplette Chemokinrezeptor Expressionsprofil humaner MSC bestimmt werden und im 96-Well Chemotaxis Assay ein dosisabhängiger migratorischer Effekt der Chemokine IL8 und SDF1α gemessen werden. Insgesamt wurde das Proof of Principal für die Eignung von Chemokinen als Chemoattraktanten humaner MSC erbracht und gezeigt, dass BMP2, IL8 und SDF1α interessante Kandidaten für präklinische in situ Knorpel TE Studien sind. Hinsichtlich der Etablierung eines Großtiermodells für das in situ TE wurden aus porcinem und equinem Knochenmark MSC isoliert und charakterisiert. Porcine und humane MSC verhielten sich sehr ähnlich, während equine MSC nicht in die adipogene Entwicklungslinie gelenkt werden konnten. Dennoch wurden die equinen MSC dazu verwendet, um erstmals einen chondrogenen Effekt der gelenkrelevanten Faktoren Hyaluronsäure und autologe Synovialflüssigkeit auf MSC zu demonstrieren. Zusammengefasst sind beide Faktoren interessante Kandidaten für eine präklinische Testung. Das Schwein und auch das Pferd repräsentieren hierfür geeignete Großtiermodelle. In einem weiteren Teil der Arbeit konnte erstmals gezeigt werden, dass die Periostzellen des humanen Mastoids ein adipogenes Potential haben, sie basierend auf FACS und genomweiten Mikroarray Analysen zahlreiche MSC Marker präsentieren, sie ein MSC-ähnliches Chemokinrezeptor Profil aufweisen und dass sie nach Stimulation mit SDF1α im Chemotaxis Assay migrieren. Die Periostzellen zeigen somit einen MSC-ähnlichen Charakter und sind für das in situ TE von Interesse. Periostzellen wurden oftmals als multipotente MSC bezeichnet, ohne das bisher ein mit den MSC vergleichbares Potential aufgezeigt wurde. Insgesamt sind die Ergebnisse zum Differenzierungspotential der für klinische Anwendungen zugelassenen Faktoren BMP2, Hyaluronsäure und Synovialflüssigkeit für die in situ Knorpelregeneration viel versprechend. Die Wirkung dieser Faktoren und auch die der Chemokine sollte in vivo bestätigt werden. Hierfür stehen mit dem Pferd und mit dem Schwein zwei Großtiermodelle zur Verfügung.Different studies indicate that in case of acute diseases, such as myocardial infarction or stroke, mesenchymal stem cells (MSC) migrate to the affected tissue and are involved in the regeneration of this tissue. Such observations are the basis for the development of in situ tissue engineering (TE) applications. Here, cell free biomaterials are combined with chemotactic active factors, such as chemokines, as well as differentiation factors and are implanted into a tissue defect. The factors allow the recruitment of appropriate cells and their subsequent development into a repair tissue. In this dissertation questions were investigated that have to be answered in vitro before performing preclinical in situ cartilage TE studies. Bone marrow-derived and culture expanded human MSC presented a cell surface marker profile known from the literature (SH2+, SH3+, CD34-). In high density cell aggregates, based on histological and immunohistochemical staining and on PCR, a chondrogenic effect of BMP2 on these cells could be demonstrated for the first time. A BMP2 induced development to hypertropic cartilage or differentiation into the osteo- or adipogenic lineage was not observed. Moreover, the entire chemokine receptor expression profile of human MSC was determined on the mRNA and protein level. Using a 96-well chemotaxis assay a dose-dependent migratory effect of the chemokines IL8 and SDF1α on human MSC was measured. All in all, the proof of principal for the suitability of chemokines as chemoattractants of human MSC was established and it was shown, that BMP2, IL8 und SDF1α are interesting candidates for preclinical in situ cartilage TE studies. Regarding the establishment of a large animal model for in situ TE, MSC were isolated from porcine and equine bone marrow and were characterized. Porcine and human MSC showed a similar behavior, whereas equine MSC could not be directed into the adipogenic lineage. Nevertheless, equine MSC were used to demonstrate a chondrogenic effect of the joint relevant factors hyaluronic acid and autologous synovial fluid on MSC for the first time. Summarized, both factors are interesting candidates for preclinical studies. Pigs and also horses represent an appropriate large animal model for such testings. In a further part of this work, it was demonstrated that human mastoid-derived periosteal cells differentiate into the adipogenic lineage. Based on FACS and genome-wide microarray analysis they presented several MSC marker. Furthermore, they showed a chemokine receptor profile similar to MSC and also migrate stimulated by SDF1α in a chemotaxis assay. Therefore, periosteal cells have an MSC-like character and are of interest for in situ TE. So far, periosteal cells have often been named multipotent MSC without showing their similar potential. In conclusion, the results regarding the differentiation potential of the clinical approved factors BMP2, hyaluronic acid and autologous synovial fluid are very promising for in situ cartilage regeneration. The effect of these factors and also of chemokines should be analyzed in more detail in vivo. For this, the pig and the horse present appropriate large animal models

Tissue engineering in the rheumatic diseases

Diseases such as degenerative or rheumatoid arthritis are accompanied by joint destruction. Clinically applied tissue engineering technologies like autologous chondrocyte implantation, matrix-assisted chondrocyte implantation, or in situ recruitment of bone marrow mesenchymal stem cells target the treatment of traumatic defects or of early osteoarthritis. Inflammatory conditions in the joint hamper the application of tissue engineering during chronic joint diseases. Here, most likely, cartilage formation is impaired and engineered neocartilage will be degraded. Based on the observations that mesenchymal stem cells (a) develop into joint tissues and (b) in vitro and in vivo show immunosuppressive and anti-inflammatory qualities indicating a transplant-protecting activity, these cells are prominent candidates for future tissue engineering approaches for the treatment of rheumatic diseases. Tissue engineering also provides highly organized three-dimensional in vitro culture models of human cells and their extracellular matrix for arthritis research