Anti-inflammatory effects of cell-based therapy with tyrosine hydroxylase-positive catecholaminergic cells in experimental arthritis

Objectives: Studies in rheumatoid arthritis (RA), osteoarthritis (OA) and mice with arthritis demonstrated tyrosine hydroxylase-positive (TH+) cells in arthritic synovium and parallel loss of sympathetic nerve fibres. The exact function of TH+ cells and mode of TH induction are not known. Methods: Synovial cells of RA/OA were isolated and cultured under normoxic/hypoxic conditions with/without stimulating enzyme cofactors of TH and inhibitors of TH. We studied TH expression and release of cytokines/catecholamines. In vivo function was tested by cell therapy with TH+ neuronal precursor cells (TH+ neuronal cells) in DBA/1 mice with collagen type II-induced arthritis (CIA). Results: Compared with normoxic conditions, hypoxia increased TH protein expression and catecholamine synthesis and decreased release of tumour necrosis factor (TNF) in OA/RA synovial cells. This inhibitory effect on TNF was reversed by TH inhibition with α-methyl-para-tyrosine (αMPT), which was particularly evident under hypoxic conditions. Incubation with specific TH cofactors (tetrahydrobiopterin and Fe2+) increased hypoxia-induced inhibition of TNF, which was also reversed by αMPT. To address a possible clinical role of TH+ cells, murine TH+ neuronal cells were generated from mesenchymal stem cells. TH+ neuronal cells exhibited a typical catecholaminergic phenotype. Adoptive transfer of TH+ neuronal cells markedly reduced CIA in mice, and 6-hydroxydopamine, which depletes TH+ cells, reversed this effect. Conclusions: The anti-inflammatory effect of TH+ neuronal cells on experimental arthritis has been presented for the first time. In RA/OA, TH+ synovial cells have TH-dependent anti-inflammatory capacities, which are augmented under hypoxia. Using generated TH+ neuronal cells might open new avenues for cell-based therapy

Extracellular Vesicles in Musculoskeletal Pathologies and Regeneration

The incidence of musculoskeletal diseases is steadily increasing with aging of the population. In the past years, extracellular vesicles (EVs) have gained attention in musculoskeletal research. EVs have been associated with various musculoskeletal pathologies as well as suggested as treatment option. EVs play a pivotal role in communication between cells and their environment. Thereby, the EV cargo is highly dependent on their cellular origin. In this review, we summarize putative mechanisms by which EVs can contribute to musculoskeletal tissue homeostasis, regeneration and disease, in particular matrix remodeling and mineralization, pro-angiogenic effects and immunomodulatory activities. Mesenchymal stromal cells (MSCs) present the most frequently used cell source for EV generation for musculoskeletal applications, and herein we discuss how the MSC phenotype can influence the cargo and thus the regenerative potential of EVs. Induced pluripotent stem cell-derived mesenchymal progenitor cells (iMPs) may overcome current limitations of MSCs, and iMP-derived EVs are discussed as an alternative strategy. In the last part of the article, we focus on therapeutic applications of EVs and discuss both practical considerations for EV production and the current state of EV-based therapies

β2-Adrenoceptor Deficiency Results in Increased Calcified Cartilage Thickness and Subchondral Bone Remodeling in Murine Experimental Osteoarthritis

Purpose: Recent studies demonstrated a contribution of adrenoceptors (ARs) to osteoarthritis (OA) pathogenesis. Several AR subtypes are expressed in joint tissues and the β2-AR subtype seems to play a major role during OA progression. However, the importance of β2-AR has not yet been investigated in knee OA. Therefore, we examined the development of knee OA in β2-AR-deficient (Adrb2-/- ) mice after surgical OA induction. Methods: OA was induced by destabilization of the medial meniscus (DMM) in male wildtype (WT) and Adrb2-/- mice. Cartilage degeneration and synovial inflammation were evaluated by histological scoring. Subchondral bone remodeling was analyzed using micro-CT. Osteoblast (alkaline phosphatase - ALP) and osteoclast (cathepsin K - CatK) activity were analyzed by immunostainings. To evaluate β2-AR deficiency-associated effects, body weight, sympathetic tone (splenic norepinephrine (NE) via HPLC) and serum leptin levels (ELISA) were determined. Expression of the second major AR, the α2-AR, was analyzed in joint tissues by immunostaining. Results: WT and Adrb2-/- DMM mice developed comparable changes in cartilage degeneration and synovial inflammation. Adrb2-/- DMM mice displayed elevated calcified cartilage and subchondral bone plate thickness as well as increased epiphyseal BV/TV compared to WTs, while there were no significant differences in Sham animals. In the subchondral bone of Adrb2-/- mice, osteoblasts activity increased and osteoclast activity deceased. Adrb2-/- mice had significantly higher body weight and fat mass compared to WT mice. Serum leptin levels increased in Adrb2-/- DMM compared to WT DMM without any difference between the respective Shams. There was no difference in the development of meniscal ossicles and osteophytes or in the subarticular trabecular microstructure between Adrb2-/- and WT DMM as well as Adrb2-/- and WT Sham mice. Number of α2-AR-positive cells was lower in Adrb2-/- than in WT mice in all analyzed tissues and decreased in both Adrb2-/- and WT over time. Conclusion: We propose that the increased bone mass in Adrb2-/- DMM mice was not only due to β2-AR deficiency but to a synergistic effect of OA and elevated leptin concentrations. Taken together, β2-AR plays a major role in OA-related subchondral bone remodeling and is thus an attractive target for the exploration of novel therapeutic avenues

GPR30 Deficiency Causes Increased Bone Mass, Mineralization, and Growth Plate Proliferative Activity in Male Mice

Estrogen regulation of the male skeleton was first clearly demonstrated in patients with aromatase deficiency or a mutation in the ERα gene. Estrogen action on the skeleton is thought to occur mainly through the action of the nuclear receptors ERα and ERβ. Recently, in vitro studies have shown that the G protein–coupled receptor GPR30 is a functional estrogen receptor (ER). GPR30-deficient mouse models have been generated to study the in vivo function of this protein; however, its in vivo role in the male skeleton remains underexplored. We have characterized size, body composition, and bone mass in adult male Gpr30 knockout (KO) mice and their wild-type (WT) littermates. Gpr30 KO mice weighed more and had greater nasal-anal length (p < .001). Both lean mass and percent body fat were increased in the KO mice. Femur length was greater in Gpr30 KO mice, as was whole-body, spine, and femoral areal bone mineral density (p < .01). Gpr30 KO mice showed increased trabecular bone volume (p < .01) and cortical thickness (p < .001). Mineralized surface was increased in Gpr30 KO mice (p < .05). Bromodeoxyuridine (BrdU) labeling showed greater proliferation in the growth plate of Gpr30 KO mice (p < .05). Under osteogenic culture conditions, Gpr30 KO femoral bone marrow cells produced fewer alkaline phosphatase–positive colonies in early differentiating osteoblast cultures but showed increased mineralized nodule deposition in mature osteoblast cultures. Serum insulin-like growth factor 1 (IGF-1) levels were not different. These data suggest that in male mice, GPR30 action contributes to regulation of bone mass, size, and microarchitecture by a mechanism that does not require changes in circulating IGF-1. © 2011 American Society for Bone and Mineral Research

Der Einfluss von Steroidhormonen auf die chondrogene Differenzierung humaner mesenchymaler Stammzellen

Gelenkknorpelläsionen können aufgrund der fehlenden Möglichkeit zur Selbstregeneration nicht behoben werden und führen zur Osteoarthrose. Eine der größten Herausforderungen ist deshalb, die Entstehung einer sekundären Osteoarthrose zu verzögern bzw. zu verhindern. Durch Verwendung von autologen mesenchymalen Stammzellen bietet Tissue Engineering eine vielversprechende Option für die Regeneration von fokalen artikulären Knorpeldefekten. Aufgrund fehlender Kenntnisse über den Einfluss von Wachstumsfaktoren oder Hormonen des Körpers auf die chondrogene Differenzierung ist die klinische Anwendung noch nicht möglich. Sexualhormone sind im Gelenk in hohen Konzentrationen vorzufinden und beeinflussen verschiedene Prozesse im Knorpelgewebe. Das Ziel der vorliegenden Arbeit ist, die Wirkung von Sexualhormonen auf die Chondrogenese humaner mesenchymaler Stammzellen zu untersuchen. Die spezifischen Androgen- und Estrogenrezeptoren sowie der neu entdeckte Estrogenrezeptor GPR30 konnten in den chondrogen differenzierenden mesenchymalen Stammzellen nachgewiesen werden. Die Behandlung mit Sexualhormonen in der Proliferationsphase hatte weder einen Einfluss auf die Zellzahl oder Zellvitalität noch auf die Qualität der darauffolgenden chondrogenen Differenzierung. Die Zugabe von Steroidhormonen in der Differenzierungsphase zeigte, dass Dexamethason für die Chondrogenese erforderlich ist und dass die Sexualhormone diese chondrogenesefördernde Rolle nicht ersetzen können. Durch die Applikation von Sexualhormonen in Kombination mit Dexamethason konnte die Chondrogenesequalität nicht verbessert werden. Im Gegenteil, Estradiol verursachte eine dosisabhängige Reduktion von chondrogenen Markern und beschleunigte gleichzeitig die Hypertrophie. Der Einsatz von spezifischen Rezeptor-Agonisten und -Antagonisten zeigte, dass die estrogenabhängige Reduktion der Chondrogenese nicht von den klassischen intrazellulären Estrogenrezeptoren (ER alpha/beta), sondern vom membranassoziierten Rezeptor GPR30 vermittelt wird. Die Ergebnisse der vorliegenden Arbeit liefern wichtige Erkenntnisse über den möglichen Einfluss von Sexualhormonen auf die Qualität, Einheilung und Reifung von chondrogenen Implantaten und dienen als Basis für weitere Untersuchungen in Richtung klinischer Anwendung