Projekte

Funding

DFG GR1301/22-1

Investigators

Internal/University of Regensburg
Dr. rer. nat. Nicole Sch?fer
Prof. Dr. rer. nat. Susanne Gr?ssel
Anja Pasoldt, UTA
Claudia G?ttl, PTA
Dr. med. Marianne Ehrnsperger, Dept. of Trauma Surgery
PD Dr. rer. nat. Jürgen Fritsch, Krankenhaushygiene und Infektiologie

External
Prof. Dr. med. Markus B?hm, Dept. of Dermatology und Venerology,
University Hospital of Münster
Prof. Dr. Brian Johnstone, Ph.D., Department of Orthopaedics and Rehabilitation,
Oregon Health & Science University, Portland, OR, United States

Role of α-Melanocyte-Stimulating-Hormone (MSH) in senescence/ageing of skin and cartilage

Compared to what is known about the proopiomelanocortin (POMC) system in skin much less is known for expression and effects of POMC peptides in cartilage. Our group demonstrated the presence of melanocortin receptors MC1R, MC2R and MC5R in human articular chondrocytes from patients with osteoarthritis (OA) (1). Differential zonal protein expression of the MC1R in these cells was confirmed on OA cartilage explants. Treatment of these chondrocytes with α-MSH was associated with functional cAMP coupling but not with a Ca2+ response. We and others (1, 2) also detected truncated forms of POMC mRNA (transcripts related to exon 2) in human articular chondrocytes obtained from end-stage OA patients. However, POMC transcripts related to exon 2–3 were not detectable (2), suggesting that articular chondrocytes cannot make functional POMC protein. In contrast to the unknown role of chondrocytes in the context of POMC peptide production, cartilage is a direct target for POMC-derived peptides. Kaneva et al. described the rapid response of articular chondrocytes to mechanical trauma – the speedy propagation of cartilage inflammation and chondrocyte death- and accentuate on the ability of α-MSH and [DTRP8]-γ-MSH to temper this response (3). Activation of MC1R prevents progression of trauma induced chondrocyte death and the consequential propagation of pro-inflammatory cytokines into non-impacted areas of cartilage, concomitantly promoting the release of reparative pro-resolving molecules. These data are in accordance with our study reporting that when challenged with OA, Mc1re/e mice (MC1R-signaling deficient mice) develop a more severe OA-pathology (4). We demonstrated that Mc1re/e mice have a cartilage phenotype prior to OA induction that increases in severity during OA-pathogenesis already in an early stage. Unchallenged Mc1re/e mice display smaller articular cartilage covered areas without OA-related surface erosions indicating that MC1R-signaling is critical for proper cartilage matrix formation and integrity. In addition, we suggest that absence of MC1R-signaling accelerates age-related structural cartilage alterations as demonstrated by loss of collagen II and increased number of MMP-13 positive chondrocytes. These observations are supported by a study where adenoviral vectors encoding the human POMC gene were injected intraarticular into the knee joints of rats after surgical induction of OA by ACLT (anterior cruciate ligament transection) (5). POMC gene transfer decreased the progression and severity of OA and reduced inflammation and angiogenesis in sub-synovial tissues. These effects may be triggered by reduced NF-κB signaling and IL-1? levels, which was demonstrated in chondrosarcoma cells(6).

Notably, in inflammatory arthritis, selective activation of MC1R via a highly potent synthetic small compound leads to anti-arthritic effects associated with induction of senescence in the synovial fibroblasts (SFs) and to cartilage protection (7). This approach may help restoring the disturbed homeostasis in the arthritic synovium, preventing the vicious cycle of reciprocal activation between SFs and other cells within the highly inflamed joint environment where SFs are over-activated and fail to return to a quiescent state. Of note, in OA-SFs MC1R activation via its natural ligand α-MSH suppressed pro-inflammatory cytokine secretion and increased adhesion to fibronectin as shown by us in a recent collaborative project (8).

Objective: POMC-peptides, specifically melanocortin peptides, have osteo-/chondro-protective properties. They show immunomodulatory effects dampening inflammatory and presumably ageing-related processes but underlying molecular mechanisms as signaling pathways and intracellular effectors are mostly unknown yet. Possibly, application of POMC-derived peptides, i.e. α-MSH locally may serve as a potential adjuvant therapy for delaying the process of posttraumatic or even spontaneous OA and ageing processes in joint tissues (9).

Funding

DFG GR 1301/19-1/2 (TP4)
Research Consortium FOR 2407/2 Excarbon

Investigators

Dr. rer. nat. Patrick Pann
Prof. Dr. rer. nat. Susanne Gr?ssel
Paulina Osrodka, MTA
Mandy Vogel, MTA

Members of Excarbon

Substance P effects on bone metabolism

The extensive distribution of substance P (SP) in joint tissues and also bone has been described earlier, including its Neurokinin 1 receptor (NK1R) which is expressed in bone cells as osteoblasts, osteoclasts and osteocytes indicating a modulatory capacity of SP in bone remodeling (10). Recently, we reported alterations in osteoclast and osteoblast numbers during fracture callus maturation in SP-deficient (Tachykinin 1 – knock out) mice compared to wild type (WT) (11). In addition, we observed impaired bone microarchitecture in the fractured and more over in the contralateral non-fractured femora of SP-deficient mice indicating changes in bone metabolism and remodeling independent of trauma when SP is missing. These alterations in bone remodeling imply a strong and crucial neuro-osteogenic connection. Our recent data suggest that there indeed exist neuropeptide mediated, cell autonomous changes in bone cell metabolism (12). An important observation of this study is that endogenously produced SP is critical for metabolic activities of bone cells ultimately determining bone turnover rate. Cellular and nerve fibre derived SP could possibly differ in relevance during bone development and bone remodeling as impaired bone microarchitecture in SP-deficient mice let suggest developmental changes when cellular derived SP is missing while an osteoporotic bone phenotype in adult rats after NK1R blockade indicates nerve fiber mediated effects on bone remodelling (13). Contrary to the physiological situation, information on the effects of SP in bone in osteoarthritic conditions is sparse. Xiao et al. showed an increase in mean optical density for SP immunoreactivity in the cancellous bone of OA femoral heads compared to osteoporosis samples, which correlated positively with pain intensity analyzed by visual analog scale (VAS) but also with bone structural parameters analyzed by ?CT (14). Concluding from this, SP might be implicated in OA pain but also seems to preserve bone structure in OA pathophysiology. Zhen and co-workers elegantly demonstrated that anterior cruciate ligament transection lead to spatiotemporal uncoupling of bone remodeling with an increase of osteoblast and osteoclast activity in an OA mouse model (15). How local release of SP in bone tissue might be involved needs to be further elucidated but acting as enhancer on both cell types, SP could contribute to these observations and SP targeted therapies could potentially target OA bone phenotypes too. ?

αCGRP effects on bone metabolism

Expression of the main calcitonin gene-related peptide (CGRP) receptor complex composed of CRLR and RAMP1 (its co-receptor) has been demonstrated on different bone cells (16, 17). Addition of CGRP to bone marrow stromal cells, subjected to osteogenic differentiation, enhanced proliferation and expression of osteoblastic genes like Runx2, ALP, bglap2 (osteocalcin) and col1a1 (18). Increasing the OPG/Rankl ratio and thus favoring osteoblast differentiation would shift the skeletal balance towards bone formation, and emphasizes the anabolic character of CGRP.? The indirect inhibitory effect of CGRP on bone resorption was additionally confirmed in the MC3T3-E1 pre-osteoblastic cell line where addition of αCGRP downregulated Rankl and upregulated OPG independently from mechanical stimulation (19). Supporting the preserving effect of CGRP on bone matrix, different studies reported an inhibitory influence of CGRP on osteoclastogenesis. CGRP inhibited IL-1 induced osteoclastic bone resorption in a co-culture setup with osteoblasts on ivory slices either directly or indirectly (20). In fetal rat osteoblasts, CGRP was able to inhibit the LPS and IL-1-induced production of TNF and weakly induced IL-6 in these osteoblasts (21). Recently, we demonstrated that both sensory neuropeptides, SP and αCGRP, and their receptors are involved in murine macrophage mechano-transduction affecting neuropeptide impact on adhesion and ROS activity (22). Loading induced NK1R and CRLR/Ramp1 gene expression and, also increased their protein expression in RAW264.7 macrophages. Whether these effects are beneficial or deleterious in vivo remains elusive, but could indicate the higher mobility and the activity of macrophages in musculoskeletal tissues subjected to loading. As for SP, not much is known about effects and involvement of αCGRP in OA progression. With respect to OA associated subchondral bone alterations, it was shown that OA caused an upregulation of αCGRP in subchondral bone afferents over time which displayed a strong correlation with the subchondral bone damage score (23). An elegant study by Zhu et al. showed that an early increase of osteoclasts in an ACLT murine OA model was strongly related to an induction of CGRP positive nerves in the subchondral bone. They found strong evidence that osteoclast derived netrin-1 promoted sensory nerve innervation which very likely is involved in mediating chronic OA pain.

Objective: Based on our hypotheses the overall goal of this project in the 2nd funding period was to further specify the role of sensory neuropeptides on molecular and cellular basis in OA pathophysiology.

Funding

Bayerisch-Tschechische Hochschulagentur (BTHA-JC-2022-36)

Investigators

Internal/University of Regensburg
Prof. Dr. rer. nat. Susanne Gr?ssel
Jian Mei, cand. med.
Dr. med. Marianne Ehrnsperger
Anja Pasoldt, UTA
Dr. rer. nat. Nicole Sch?fer

External
Prof. Dr. rer. nat. Denitsa Docheva, Muskuloskelettales Zentrum Würzburg, Universit?t Würzburg
Prof. Dr. rer. nat. Eva Matalova, Department of Physiology, University of Veterinary Sciences Brno, Brno, Czech Republic; Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Brno, Czech Republic.
Prof. Dr. Brian Johnstone, Ph.D., Department of Orthopaedics and Rehabilitation, Oregon Health & Science University, Portland, OR, United States

Caspases and osteoarthritis

Caspases belong to cysteine proteases and are well known from intracellular processes associated with apoptosis (programmed cell death) and inflammation. Gradually, caspases were even assigned as a possible switch between life and death (25) and as key player in PANoptosis (26), a hot topic covering an array of medical applications including OA (27). Recently, non-apoptotic functions of caspases such as in cell proliferation, differentiation and migration become emphasized (28, 29). A research hotspot considering caspases is their accumulating impact on extracellular communication, particularly in the biogenesis of extracellular vesicles (30). This inside out insight is challenging also for OA (31)? where caspases have been considered in therapeutic strategies (32) .
Among these, apical caspases as key caspases, which are responsible for activation of the corresponding pathways, were emphasized. These include caspase-8 for the receptor mediated apoptotic pathway, caspase-9 for the intrinsic one and caspase-1 as the key inflammatory caspase being involved also in OA pathogenesis (33).
Caspases in their classical roles were widely investigated also in chondrocytes (34, 35) and bone and cartilage associated pathologies including OA (36) or rheumatoid arthritis (37). Besides senescence, chondrocyte apoptosis is known to contribute to articular cartilage damage and is correlated to a number of cartilage disorders. In particular, after hyperthermia, chondrocytes experience caspase-6 activation even if the executive caspase-3, activated during classical apoptosis, appears down-regulated (38). However, not much is known about the non-apoptotic functions of the pro-apoptotic caspases and how these affect OA pathology, which will be topic of this project. There are recent publications indicating particular OA-related genes are affected by caspase inhibition in healthy chondrocytes (39), non-apoptotic roles of OA-associated caspase-1 (40) and specification of genes affected by individual caspase inhibition, such as Mmp9 in the case of caspase-9 inhibition (41). These key findings were further evaluated with proteomic analysis of non-apoptotic functions of caspase-9 in chondrocytes (42).

Caspase inhibitors and osteoarthritis

Studies in humans and in animal models demonstrate a key role for chronic, low-grade inflammation in the pathogenesis of OA and OA associated pain; and synovitis is increasingly recognized as a characteristic feature of the OA joint even in the early stages and a prognostic factor for OA progression (43). Since caspases play an important role in modulating inflammation, caspase inhibitors have been widely utilized to study diseases involving inflammation in animal models and clinical trials (32). Caspase inhibitors have been investigated for potential treatment of inflammatory arthritis, mostly? rheumatoid arthritis (RA). In collagenase treated female Balb/c mice and male STR/1N mice, which spontaneously develop OA, oral application of Pralnacasan, a reversible caspase-1 inhibitor, reduced joint damage (44). In a rabbit model of anterior cruciate ligament transection (ACLT)-induced OA, Z-VAD-FMK (a broad spectrum caspase inhibitor) reduced the level of active caspase-3 in chondrocytes and inhibited the cleavage of PARP, resulting in the alleviation of cartilage lesions and confirmed the role of cell death in OA pathogenesis (45). However, inhibiting caspases affect various cellular functions, signalling pathways and tissue homeostasis besides the effects on inflammation and cell death. The recent challenges associated with therapeutic application of caspase inhibitors have been reviewed in Dhani et al., 2021 (32) and have been included in the project strategy. The project focuses on correlation of caspase activity with critical cellular events but also on investigation of the impact of caspase inhibition.

Objective: Taken together, a complex understanding of the spectrum of intracellular functions of caspases in the range between cell death and survival might lead towards new therapeutic opportunities for a plethora of diseases including OA. Therefore, the project aims to provide data to specific functions of caspases inside and outside of chondrocytes considering both, physiological as well as pathophysiological (OA-like) conditions.

Funding

Braun/Aeskulap/TETEC

Investigators

Internal/University of Regensburg
Alissa Behn, MSc
Dr. rer. nat. Nicole Sch?fer
Prof. Dr. rer. nat. Susanne Gr?ssel
Dr. med. Magadalena Zborilova
Dr. med. Marianne Ehrnsperger
Prof. Dr. med. Dr. h.c. Joachim Grifka
Claudia G?ttl, PTA

External
Dr. rer. nat. Karin Benz, TETEC AG
Prof. Dr. med. habil. Dr.-Ing. Thomas M. Grupp, Dr. med. Saskia Brendle,
Research and Development, Aesculap AG, Tuttlingen, Germany
Department of Orthopaedic and Trauma Surgery, Musculoskeletal University Center Munich (MUM), LMU Munich, Munich, Germany

Current OA treatments primarily focus on symptomatic relief through nonsteroidal anti-inflammatory drugs, acetaminophen, opioids, and intra-articular injections. However, these treatments often have limited efficacy and safety concerns. Disease-modifying OA drugs (DMOADs) aim to slow or reverse joint damage but face unresolved challenges due to disease heterogeneity (46-48).
Recent advances include the development of DMOADs targeting extracellular matrix homeostasis and chondrocyte metabolism. For example, the recombinant fibroblast growth factor 18, Sprifermin, promoted dose-dependent cartilage thickness but no significant changes in pain scores (Hochberg et al., 2019). An ADAMTS-5 inhibitor, S201086/GLPG 1972, and the WNT-β-catenin pathway inhibitor Lorecivivint (SM04690) have demonstrated potential in reducing cartilage loss and are currently under clinical investigation (48-50).
Orthobiologics, such as autologous nanofat, introduced by Tonnard et al., in 2013, refers to? techanically emulsified lipoaspirates used in regenerative medicine, dermatology, and orthopedics. It has emerged as a promising new therapeutic option for alleviating pain in OA due to its regenerative potential and ability to modulate the inflammatory environment within joints. Its application in OA could offer a novel approach to managing the disorder, addressing not only pain relief but also potentially slowing disease progression (51). Unlike enzymatically prepared adipose-derived stromal vascular fraction, nanofat contains mesenchymal stem cells (MSCs), stromal cells, ECM macromolecules, and numerous paracrine factors (52, 53). Nanofat has shown comparable success to cellular stromal vascular fractions in reducing joint pain and improving mobility (54). This study aims to elucidate the molecular composition of a critical nanofat component, the fluid phase of the SVF (sSVF, containing both–intact adipocytes and secretomes of chopped adipocytes), on metabolism of chondrocytes and synoviocytes isolated from OA affected knee joints.

Objective: We compared nanofat prepared using the Lipocube? Nano and Adinizer? filter systems to each other and to unfiltered lipoaspirates. Soluble paracrine factors were analyzed with Luminex Multiplex-ELISA, and cellular responses were evaluated in OA-chondrocytes and OA-synoviocytes in vitro. This study aimed to deepen our understanding regarding the influence of nanofat on OA pain, progression and treatment outcomes by systematically identifying and analyzing its specific components and their effects.

Funding

DFG proposal in preparation

Investigators

Internal:
Dr. rer. nat. Nicole Sch?fer
PD Dr. med. Fabian Westhauser
Prof. Dr. rer. nat. Susanne Gr?ssel
Prof. Dr. med. Tobias Renkawitz
Claudia G?ttl, PTA

External:
Prof. Dr.-Ing. Aldo R. Boccaccini, Dept. of Biomaterials, FAU, Erlangen-Nürnberg
Dr. rer. nat. Yvonne Reinders, Leibniz-Institut für Analytische Wissenschaften –ISAS – e.V., Dortmund

Bioactive Glass–Immune Interfaces: Modulating Complement, Senescence, and Regeneration in Joint Tissues

Bioactive glasses (BGs) are silica-based, osteoconductive materials originally exemplified by Hench’s 45S5 Bioglass. 45S5-BG has been extensively studied for its osteoconductive and osteoinductive properties in bone tissue engineering applications. 45S5-BG, known for its bone regenerative properties, has recently shown potential in modulating inflammatory processes in joint tissues. Current research reveals that 45S5-BG influences synovial membrane cells from osteoarthritis (OA) patients by affecting matrix metalloproteinase release and cytokine production. OA is the most common joint disease worldwide, characterized by progressive cartilage degradation, subchondral bone remodeling, synovial inflammation, and changes in periarticular tissues. The complement system plays a significant role in OA pathogenesis through dysregulated activation contributing to chronic inflammation. Recent studies indicate that the release of anaphylatoxins, promotes OA progression by contributing to cartilage calcification, and that deposition of the membrane attack complex (MAC) further advances OA by inducing chondrosenescence through MAC-mediated cytokine release. Key regulators of the complement system like Factor H (FH) and Factor H-Related protein 5 (FHR-5) can alleviate joint inflammation and inhibit osteoclast differentiation. Granzyme K (GZMK), a serine protease expressed by immune cells, can independently activate the complement cascade, creating a direct link to joint inflammation. In OA joints, chronic inflammation can induce oxidative stress in resident cells, accelerating senescence. Conversely, the accumulation of senescent cells exacerbates inflammation, creating a feedback loop.
Therefore, combining the regenerative properties of bioactive glass with the inhibition of complement activation may attenuate inflammation and cellular senescence associated with OA, offering promising therapeutic potential.

The main objective of this project investigates how different BGs impact joint cell inflammation and senescence in OA, with particular focus on filling subchondral bone defects. By elucidating mechanisms through which bioactive glass interacts with joint tissues and modulates inflammatory processes in the subchondral region, the research seeks to develop novel therapeutic strategies for OA. The project will examine how BG scaffolds in subchondral bone defects influence the local inflammatory environment and cellular senescence, potentially mitigating disease progression. The research will utilize advanced modeling techniques and explore siRNA as tools for Granzyme K inhibition and FH, FHR-5 release in the context of bioactive glass applications for treating subchondral lesions in OA.