Thilo John

Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

Resveratrol Inhibits IL‐1β–Induced Stimulation of Caspase‐3 and Cleavage of PARP in Human Articular Chondrocytes in Vitro

Abstract:  Resveratrol is a polyphenolic phytoalexin that is present in various fruits, in the skin of red grapes and peanuts. Recent studies have shown that resveratrol exhibits potent antioxidant properties and is able to exert anti‐inflammatory and anti‐catabolic properties in several cell types. The pro‐inflammatory cytokine interleukin‐1β (IL‐1β) plays a pivotal role in the pathogenesis of osteoarthritis (OA) in humans and animals. In this article we investigated whether resveratrol is able to block the effects of IL‐1β, specifically the activation of caspase‐3 and subsequent cleavage of poly (ADP‐ribose) polymerase (PARP) in human articular chondrocytes. Cultures of human chondrocytes were prestimulated with 10 ng/mL IL‐1β for 1, 12, and 24 h before being co‐treated with IL‐1β and 100 μM resveratrol or 50 μM of the caspase inhibitor Z‐DEVD‐FMK for 1, 12, and 24 h, respectively in vitro. Resveratrol significantly reduced the IL‐1β‐induced inhibition of expression of cartilage‐specific collagen type II and signal transduction receptor β1‐integrin in a time‐dependent manner. Incubation of chondrocytes with IL‐1β resulted in the activation of caspase‐3 and PARP cleavage. These effects were abolished through co‐treatment with resveratrol. Furthermore, co‐treatment of IL‐1β‐stimulated cells with the caspase inhibitor Z‐DEVD‐FMK blocked activation of caspase‐3 and PARP cleavage, suggesting that this process is a caspase‐dependent pathway. In summary, our results confirm that resveratrol is an effective inhibitor of chondrocyte apoptosis in vitro. These findings suggest that this dietary polyphenolic compound may have future applications in the nutraceutical‐based therapy of human and animal OA.

Effect of Pro-Inflammatory and Immunoregulatory Cytokines on Human Tenocytes

Tendon injury induces a local inflammatory response, characterized by the induction of pro‐inflammatory cytokines. The aim of the present study was to analyze the effects of TNFα, IL‐6 and IL‐10 on key parameters of tendon homeostasis. Cultured primary human tenocytes were treated with the recombinant cytokines IL‐6, IL‐10, TNFα, or combinations of TNFα with IL‐6 and IL‐10 (10 ng/mL, 6, 24 h). Expression of type I collagen, elastin, MMP‐1, TNFα, IL‐1β, IL‐6, IL‐10, and suppressors of cytokine signaling (SOCS1, 3) was analyzed with the use of RTD‐PCR, immunocytochemistry, and Western blot analysis. In response to TNFα, tenocytes reduced their type I collagen deposition but increased their elastin gene expression and highly upregulated their expression for MMP‐1, pro‐inflammatory (TNFα, IL‐1β) and immunoregulatory (IL‐6, IL‐10) cytokines. TNFα stimulation augmented SOCS1, whereas SOCS3 expression in tenocytes was also induced by IL‐6. The treatment of tenocytes with IL‐6 and IL‐10 had no effect on cytokine expression. Neither IL‐6 nor IL‐10 modulated the observed effects of TNFα significantly. These results indicate that TNFα strongly activates the tenocytes to amplify their own TNFα expression and, subsequently, that of other regulatory cytokines and matrix degrading enzymes. However, the impact of IL‐6 and IL‐10 on tenocytes remains unclear.

Effects of Curcumin (Diferuloylmethane) on Nuclear Factor κB Signaling in Interleukin‐1β‐Stimulated Chondrocytes

Abstract: Curcumin (diferuloylmethane) is a nontoxic dietary pigment in tumeric and curry and a potent inhibitor of the common transcription factor Nuclear Factor κB (NF‐κB) in several cell types. It is well established that some of the catabolic effects of the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α in osteoarthritis are regulated by the activation of NF‐κB. Therefore, the aim of this study was to determine whether curcumin modifies the catabolic response of chondrocytes to IL‐1β. Human articular chondrocytes were prestimulated with 10 ng/mL IL‐1β for 0, 4, 8, 12, or 24 h and then cotreated with 50 μM curcumin for 0, 12, 24, 36, or 48 h. Synthesis of the cartilage‐specific collagen type II and matrix‐degrading enzyme matrix metalloproteinase‐3 (MMP‐3) was investigated in chondrocytes by Western blot analysis. Activation and nuclear translocation of NF‐κB were observed by immunofluorescence microscopy. IL‐1β induced a decrease in collagen type II and upregulation of MMP‐3 in a time‐dependent manner. Upregulation of MMP‐3 was inhibited by curcumin in a time‐dependent manner. In addition, IL‐1β‐induced a decrease in type II collagen, which was relieved by curcumin treatment. In response to IL‐1β, NF‐κB translocated to the nucleus, but translocation was inhibited by curcumin, as revealed by immunofluorescence microscopy. Taken together, these results confirmed an IL‐1β‐mediated upregulation of proinflammatory MMP‐3 in chondrocytes via an NF‐κB activation mechanism. Curcumin protected chondrocytes from the catabolic effects of IL‐1β, such as MMP‐3 upregulation, and interestingly also relieved cytokine‐induced suppression of matrix protein synthesis. Therefore, curcumin antagonizes crucial catabolic effects of IL‐1β signaling that are known to contribute to the pathogenesis of osteoarthritis.

Extracellular matrix expression of human tenocytes in three-dimensional air-liquid and PLGA cultures compared with tendon tissue: implications for tendon tissue engineering.

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue.

Healing parameters in a rabbit partial tendon defect following tenocyte/biomaterial implantation

Although rabbits are commonly used as tendon repair model, interpretative tools are divergent and comprehensive scoring systems are lacking. Hence, the aim was to develop a multifaceted scoring system to characterize healing in a partial Achilles tendon defect model. A 3 mm diameter defect was created in the midsubstance of the medial M. gastrocnemius tendon, which remained untreated or was filled with a polyglycolic-acid (PGA) scaffold + fibrin and either left cell-free or seeded with Achilles tenocytes. After 6 and 12 weeks, tendon repair was assessed macroscopically and histologically using self-constructed scores. Macroscopical scoring revealed superior results in the tenocyte seeded PGA + fibrin group compared with the controls at both time points. Histology of all operated tendons after 6 weeks proved extracellular matrix (ECM) disorganization, hypercellularity and occurrence of irregular running elastic fibres with no significance between the groups. Some inflammation was associated with PGA implantation and increased sulphated proteoglycan deposition predominantly with the empty defects. After 12 weeks defect areas became hard to recognize and differences between groups, except for the increased sulphated proteoglycans content in the empty defects, were almost nullified. We describe a partial Achilles tendon defect model and versatile scoring tools applicable for characterizing biomaterial-supported tendon healing.

Interleukin-10 and Articular Cartilage: Experimental Therapeutical Approaches in Cartilage Disorders

Interleukin (IL)-10 is a well known anti-inflammatory and immunoregulatory cytokine, mainly released by, and acting on cells of the immune system such as monocytes, macrophages, T cells, NK cells, and B cells. IL-10 is also produced by a few connective tissue cell types including chondrocytes and is involved in processes such as connective tissue extracellular matrix remodelling, although its exact function in articular cartilage remains unclear. This review article summarizes after a short insight into functions of IL-10 in the immune system most of the published literature on the role of IL-10 in articular cartilage homeostasis and disorders. A critical analysis of the present literature was undertaken leading to a survey of the significance of IL-10 in rheumatoid arthritis (RA), osteoarthritis (OA) and blood induced cartilage damage with particular focus on the direct impact of IL-10 on chondrocyte biology. IL-10 is up-regulated in RA and OA and therapeutic effects of IL-10 in experimental arthritis using several gene therapeutic approaches were reported, mainly through an immune cell mediated immunosuppression mechanism. Recently, a direct anti-inflammatory, -catabolic and -apoptotic potential of IL-10 in cartilage was described, suggesting a chondroprotective effect of IL-10, not only in RA and OA, but also in non-RA and non-systemic cartilage disorders. Chondroprotection by IL-10 may be a promising tool in arthritis therapy, as IL-10 plays a role in joint and cartilage immunoregulation and homeostasis. However, a crucial problem remains to be the optimisation of local and continuous therapeutic effective levels of IL-10 administration in the joint.

IL-10 overexpression differentially affects cartilage matrix gene expression in response to TNF-α in human articular chondrocytes in vitro

Cartilage-specific extracellular matrix synthesis is the prerequisite for chondrocyte survival and cartilage function, but is affected by the pro-inflammatory cytokine TNF-alpha in arthritis. The aim of the present study was to characterize whether the immunoregulatory cytokine IL-10 might modulate cartilage matrix and cytokine expression in response to TNF-alpha. Primary human articular chondrocytes were treated with either recombinant IL-10, TNF-alpha or a combination of both (at 10ng/mL each) or transduced with an adenoviral vector overexpressing human IL-10 and subsequently stimulated with 10ng/ml TNF-alpha for 6 or 24h. The effects of IL-10 on the cartilage-specific matrix proteins collagen type II, aggrecan, matrix-metalloproteinases (MMP)-3, -13 and pro-inflammatory cytokines were evaluated by real-time RT-PCR and immunohistochemistry. Transduced chondrocytes overexpressed high levels of IL-10 which significantly up-regulated collagen type II expression. TNF-alpha suppressed collagen type II and aggrecan, but increased MMP and cytokine expression in chondrocytes compared to the non-stimulated controls. The TNF-alpha mediated down-regulation of aggrecan expression was significantly antagonized by IL-10 overexpression, whereas the suppression of collagen type II was barely affected. The MMP-13 and IL-1beta expression by TNF-alpha was slightly reduced by IL-10. These results suggest that IL-10 overexpression modulates some catabolic features of TNF-alpha in chondrocytes.

Differing in vitro biology of equine, ovine, porcine and human articular chondrocytes derived from the knee joint: an immunomorphological study

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.

Current Concepts in the Treatment of Proximal Humeral Fractures

Preoperative classification of proximal humeral fractures in addition to thorough knowledge of the specific anatomy and vascular blood supply is more important for successful treatment than the choice of implant. If reduction and fixation is necessary, aggressive reduction maneuvers can compromise humeral head perfusion with subsequent humeral head necrosis regardless of the implant used. Modern implants such as intramedullary proximal humeral nails and anatomically designed proximal humeral angular stable plates offer high primary stability even in osteoporotic bone with preservation of periosteal blood supply to the humeral head. These implants allow early functional exercises and showed good to excellent results in the majority of patients with an acceptable complication rate. Increasing experience with these relatively new implants and further technical development might improve clinical results and reduce complications. Minimally invasive, percutaneous techniques also demonstrate favorable results comparable to those mentioned above, although mean patient age tends to be younger in these studies and complications requiring reoperation tend to be more pronounced in elderly patients due to poor bone quality. Alternatively, nonoperative treatment of displaced two- and three-part fractures in elderly patients with severe morbidity and high perioperative risks should be considered. In elderly patients with selected displaced four-part fractures or fracture dislocations and head-split fractures, hemiarthroplasty offers high subjective patient satisfaction despite moderate function with most of the patients being pain free.