Tino Felka

Hypoxia reduces the inhibitory effect of IL-1β on chondrogenic differentiation of FCS-free expanded MSC

OBJECTIVE Mesenchymal stromal cells (MSC) are a promising tool for tissue engineering of the intervertebral disc (ID). The IDs are characterized by hypoxia and, after degeneration, by an inflammatory environment as well. We therefore investigated the effects of inflammation induced with interleukin (IL)-1beta and of hypoxia (2% O(2)) on the chondrogenic differentiation of MSC. METHODS Bone-marrow-derived MSC (bmMSC) were cultured in a fetal-calf-serum-free medium and characterized according to the minimal criteria for multipotent MSC. Chondrogenic differentiation of MSC was induced following standard protocols, under hypoxic conditions, with or without IL-1beta supplementation. After 28 days of differentiation, micromasses were analyzed by histochemical staining and immunohistochemistry and by determining the mRNA level of chondrogenic marker genes utilizing quantitative RT-PCR. RESULTS Micromasses differentiated under IL-1beta supplementation are smaller and express less extracellular matrix (ECM) protein. Micromasses differentiated under hypoxia appear larger in size, display a denser ECM and express marker genes comparable to controls. The combination of hypoxia and IL-1beta supplementation improved chondrogenesis compared to IL-1beta supplementation alone. Micromasses differentiated under standard conditions served as controls. CONCLUSION Inflammatory processes inhibit the chondrogenic differentiation of MSC. This may lessen the regenerative potential of MSC in situ. Thus, for the cell therapy of IDs using MSC to be effective it will be necessary to manage the inflammatory conditions in situ. In contrast, hypoxic conditions exert beneficial effects on chondrogenesis and phenotype stability of transplanted MSC, and may improve the quality of the generated ECM.

Animal serum-free expansion and differentiation of human mesenchymal stromal cells

BACKGROUND AIMS Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products. METHODS MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation. CONCLUSIONS Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

Comparison of marker gene expression in chondrocytes from patients receiving autologous chondrocyte transplantation versus osteoarthritis patients

Currently, autologous chondrocyte transplantation (ACT) is used to treat traumatic cartilage damage or osteochondrosis dissecans, but not degenerative arthritis. Since substantial refinements in the isolation, expansion and transplantation of chondrocytes have been made in recent years, the treatment of early stage osteoarthritic lesions using ACT might now be feasible. In this study, we determined the gene expression patterns of osteoarthritic (OA) chondrocytes ex vivo after primary culture and subculture and compared these with healthy chondrocytes ex vivo and with articular chondrocytes expanded for treatment of patients by ACT. Gene expression profiles were determined using quantitative RT-PCR for type I, II and X collagen, aggrecan, IL-1β and activin-like kinase-1. Furthermore, we tested the capability of osteoarthritic chondrocytes to generate hyaline-like cartilage by implanting chondrocyte-seeded collagen scaffolds into immunodeficient (SCID) mice. OA chondrocytes ex vivo showed highly elevated levels of IL-1β mRNA, but type I and II collagen levels were comparable to those of healthy chondrocytes. After primary culture, IL-1β levels decreased to baseline levels, while the type II and type I collagen mRNA levels matched those found in chondrocytes used for ACT. OA chondrocytes generated type II collagen and proteoglycan-rich cartilage transplants in SCID mice. We conclude that after expansion under suitable conditions, the cartilage of OA patients contains cells that are not significantly different from those from healthy donors prepared for ACT. OA chondrocytes are also capable of producing a cartilage-like tissue in the in vivo SCID mouse model. Thus, such chondrocytes seem to fulfil the prerequisites for use in ACT treatment.

Human mesenchymal stromal cells express CD14 cross‐reactive epitopes

Mesenchymal stromal cells (MSCs) do not express a unique definite epitope or marker gene. As such, minimal criteria were recently established for defining multipotent MSC. These criteria include expression of CD73, CD90, CD105, and a lack of hematopoietic marker expression. However, we detected binding of a CD14 antibody on bone marrow‐ and placenta‐derived MSC and investigated the staining of CD14 antibodies on these MSC in more detail. The MSC were isolated from human bone marrow and placenta tissue, expanded, characterized by quantitative RT‐PCR, flow cytometry, and immunocytochemistry and differentiated to generate osteoblasts, chondrocytes, and adipocytes. The CD14‐cross‐reactive MSCs were enriched by cell sorting. Human peripheral blood mononuclear cells, fibroblasts, and hematopoietic cell lines served as controls. Utilizing four different clones of CD14 monoclonal antibodies, we found that three CD14 reagents stained the MSC. Two CD14 antibodies (HCD14 and M5E2) clearly marked the CD90+ MSC population with distinct intensities, clone 134 620 generated a shift in flow cytometry histograms, but clone MΦP9 did not stain MSC. Transcripts encoding CD14 or the CD14 protein were not detected in MSC. We confirm that bone marrow‐ and placenta‐derived MSC do not express CD14 and that the CD14 antibody MΦP9 discriminates between monocytes and MSC more efficiently than the other antibodies employed here. This investigation does not contradict previous work but provides a more accurate characterization of MSC.

Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells

Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes in vitro and in vivo. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1–5 fmol/µL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P<0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications.

Stress-vs-time signals allow the prediction of structurally catastrophic events during fracturing of immature cartilage and predetermine the biomechanical, biochemical, and structural impairment

OBJECTIVE Trauma-associated cartilage fractures occur in children and adolescents with clinically significant incidence. Several studies investigated biomechanical injury by compressive forces but the injury-related stress has not been investigated extensively. In this study, we hypothesized that the biomechanical stress occurring during compressive injury predetermines the biomechanical, biochemical, and structural consequences. We specifically investigated whether the stress-vs-time signal correlated with the injurious damage and may allow prediction of cartilage matrix fracturing. METHODS Superficial and deeper zones disks (SZDs, DZDs; immature bovine cartilage) were biomechanically characterized, injured (50% compression, 100%/s strain-rate), and re-characterized. Correlations of the quantified functional, biochemical and histological damage with biomechanical parameters were zonally investigated. RESULTS Injured SZDs exhibited decreased dynamic stiffness (by 93.04±1.72%), unresolvable equilibrium moduli, structural damage (2.0±0.5 on a 5-point-damage-scale), and 1.78-fold increased sGAG loss. DZDs remained intact. Measured stress-vs-time-curves during injury displayed 4 distinct shapes, which correlated with histological damage (p<0.001), loss of dynamic stiffness and sGAG (p<0.05). Damage prediction in a blinded experiment using stress-vs-time grades was 100%-correct and sensitive to differentiate single/complex matrix disruptions. Correlations of the dissipated energy and maximum stress rise with the extent of biomechanical and biochemical damage reached significance when SZDs and DZDs were analyzed as zonal composites but not separately. CONCLUSIONS The biomechanical stress that occurs during compressive injury predetermines the biomechanical, biochemical, and structural consequences and, thus, the structural and functional damage during cartilage fracturing. A novel biomechanical method based on the interpretation of compressive yielding allows the accurate prediction of the extent of structural damage.

Modeling chondrocyte patterns by elliptical cluster processes.

Superficial zone chondrocytes (CHs) of human joints are spatially organized in distinct horizontal patterns. Among other factors, the type of spatial CH organization within a given articular surface depends on whether the cartilage has been derived from an intact joint or the joint is affected by osteoarthritis (OA). Furthermore, specific variations of the type of spatial organization are associated with particular states of OA. This association may prove relevant for early disease recognition based on a quantitative structural characterization of CH patterns. Therefore, we present a point process model describing the distinct morphology of CH patterns within the articular surface of intact human cartilage. This reference model for intact CH organization can be seen as a first step towards a model-based statistical diagnostic tool. Model parameters are fitted to fluorescence microscopy data by a novel statistical methodology utilizing tools from cluster and principal component analysis. This way, the complex morphology of surface CH patters is represented by a relatively small number of model parameters. We validate the point process model by comparing biologically relevant structural characteristics between the fitted model and data derived from photomicrographs of the human articular surface using techniques from spatial statistics.

Nitric Oxide Activates Signaling by c-Raf, MEK, p-JNK, p38 MAPK and p53 in Human Mesenchymal Stromal Cells inhibits their Osteogenic Differentiation by Blocking Expression of Runx2

Introduction: Mesenchymal stromal cells (MSC) are a promising therapy for wound healing and regeneration of inflamed tissues. They are used clinically for different symptoms and diseases and are being investigated in clinical trials world wide at an increasing rate. However, depending on the application protocol and site of treatment, MSC may face an inflammatory environment. Objective: Nitric oxide (NO) is one of the soluble factors produced in acute and chronic inflammation and influences growth, apoptosis, proliferation and differentiation of cells. NO therefore my have an influence on MSC injected into inflamed sites. Thus we investigated the effects of NO radicals on human MSC. Methods: Human MSC were expanded and characterized. Expression of the mesenchymal linage markers was determined by flow cytometry and their tri-lineage differentiation was explored in vitro. MSC were incubated with the NO-donor sodium nitroprusside (SNP) at different concentrations (5 μM-5 mM) and over different periods of time (15 min–24 hrs), and analyzed for their respiratory activity, gene expression responses, cell signalling pathways, and differentiation potential. Results: Human MSC expressed the mesenchymal marker proteins CD73, CD90, CD105, CD146, but failed to express the hematopoietic markers CD11b, CD14, CD34, and CD45. Activation of the MSC in vitro by nitric oxide activated c-Raf-, p-38-MAPK, and p-JNK- mediated signalling in a dose dependent manner, and also significantly regulated genes involved in cellular proliferation (cyclin D1, GAS1), apoptotis (p53), and induced an intense nuclear factor E2-related factor (NRF2)-associated stress response. Moreover, NO inhibited the entry of MSC in the osteogenic differentiation pathway and NO-treated MSC expressed less of the transcription factor Runx2. In contrast, the expression of the adipogenic marker gene PPARγ2 remained unchanged. Conclusion: We conclude that NO modulates the metabolism of MSC and compromises their osteogenic differentiation potential, which may have detrimental consequences for bone remodelling or bone regeneration.

The TGF-β1-Induced Expression of Matrix Metalloproteinases in Mesenchymal Stromal Cells is Influenced by Type of Substrate

Transforming growth factor (TGF)-?1 activates the expression of matrix metalloproteinases (MMPs) in fibroblasts. Attachment of these cells to laminin-111 further raises the TGF-?1-induced expression of MMP-3 and MMP-10. Mesenchymal stromal cells (MSC) attach to a variety of extracellular matrix proteins during development and wound healing. We therefore investigated the TGF-?1-regulated expression of MMPs in MSC upon attachment to laminin-111 and type I collagen. The expression of MMPs was determined by quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The TGF-?1 signalling pathways were investigated by immunoblot and by pharmacological blocking of Smad2, MEK/ERK and p38MAPK activities. Overall, TGF-?1 significantly activated the expression of mRNA encoding MMP-3 (p=0.05), MMP-13 (p=0.05) and TIMP-1 (p=0.01) in MSC. Induction of MMP-10 was not significant. In contrast to our observation on fibroblasts, the attachment of MSC to laminin-111 did not affect the TGF-?1-induced expression of MMP-3 and MMP-10. Attachment to type I collagen reduced the TGF-?1-induced secretion of MMP-3 and MMP-10 compared to cells grown on laminin-111 or tissue culture plastic dishes. The expression of MMP-3 was induced by TGF-?1 via Smad2, ERK1/2 and p38MAPK. The expression of MMP-10 was regulated by Smad2 and ERK/1/2, whereas the expression of MMP-13 was shown to be p38 MAPkinase dependent. We conclude that the regulation of MMP-3, MMP-10, and MMP-13 by TGF-?1 proceeds via distinct signalling routes. In contrast to the regulatory pathways in fibroblasts, we could not prove a co-signalling of TGF-?1- and integrin-dependent pathways for the regulation of MMP-3 and MMP-10 in MSC upon attachment to laminin-111. Therefore, MSC respond differently to TGF-?1 and extracellular matrix molecules compared to fibroblasts.