Alfred Opolka

Collagen IX-deficiency seriously compromises growth cartilage development in mice.

For a large part, skeletal development, growth, and repair occur by endochondral ossification which comprises an orderly sequence of consecutive steps of proliferation and late differentiation of chondrocytes. After vascular invasion into hypertrophic cartilage, the tissue is remodelled into bone. At all stages, the process is under tight environmental control exerted by a combination of regulators, including nutritional supply and signalling through growth factors, hormones, and cell-matrix-interactions. Therefore, genetic elimination of collagen IX, a stabilizing component of the periphery of thin cartilage fibrils, is expected to compromise extracellular matrix properties and, hence, the chondrocyte environment required for normal cartilage development and homeostasis. Here, we have shown that growth plate cartilage morphology is markedly disturbed in mice lacking collagen IX. Abnormalities were most prominent in late proliferative, pre-hypertrophic, and hypertrophic zones whereas resting and early proliferative zones were less affected. In central epiphyseal regions of long bones, newborn animals show grossly abnormal areas with strongly reduced cell numbers, irregular distribution of glycosaminoglycans in the extracellular matrix, and a profoundly disturbed columnar arrangement of chondrocytes with an irregular beta1 integrin immunostaining. As a result, all long bones are shorter and broader in newborn Col9a1-/- mice. Remarkably, these abnormalities are attenuated in adult mice, but the number of cells per area still is too low due to reduced cell proliferation.

Substance P and norepinephrine modulate murine chondrocyte proliferation and apoptosis

OBJECTIVE Substance P (SP) and norepinephrine (NE) containing sensory and sympathetic nerve fibers innervate bone and fracture callus. They are involved in controlling vascularization and matrix differentiation during skeletal growth. Both types of nerve fibers are known to modulate growth and metabolic activity of osteoblasts and osteoclasts. The aim of this study was to understand the roles of SP and NE in chondrocyte metabolism and their impact on chondrocyte proliferation, apoptosis, and cell adhesion. METHODS Primary costal chondrocytes were isolated from newborn mice. Micromass and monolayer cell culture regimens were used to analyze the effects of SP and NE on matrix formation, as determined by quantitative polymerase chain reaction and immunohistochemistry. The effects of SP and NE on proliferation, adhesion, and apoptosis of chondrocytes were determined by enzyme-linked immunosorbent assay, bromodeoxyuridine, TUNEL, and morphometric analyses. RESULTS SP, neurokinin type 1 (NK-1) receptor, α-adrenergic receptor (α-AR), and β-AR were abundantly expressed in primary costal chondrocytes. Stimulation with SP or NE did not affect extracellular matrix formation with respect to types I, II, and IX collagen and aggrecan in micromass pellets. SP dose-dependently increased the rate of proliferation of chondrocytes via the NK-1 receptor, whereas NE decreased the apoptosis rate of chondrocytes by stimulating β-AR. Both neurotransmitters induced the formation of focal adhesion contacts. CONCLUSION Transmitters of sympathetic and sensory nerve fibers modulate the metabolic activity of chondrocytes. Endogenous SP, NK-1 receptor, and adrenoceptor expression in chondrocytes implicates as-yet-unknown, presumably trophic, functions of neurotransmitter for skeletal growth and might be of interest for use in cartilage regenerative medicine.

The melanocortin system in articular chondrocytes: Melanocortin receptors, pro‐opiomelanocortin, precursor proteases, and a regulatory effect of α‐melanocyte–stimulating hormone on proinflammatory cytokines and extracellular matrix components

OBJECTIVE The pro-opiomelanocortin (POMC)-derived neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) mediates its effects via melanocortin (MC) receptors. This study was carried out to investigate the expression patterns of the MC system and the effects of alpha-MSH in human articular chondrocytes. METHODS Articular chondrocytes established from human osteoarthritic joint cartilage were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting for the expression of MC receptors, POMC, and prohormone convertases (PCs). MC-1 receptor (MC-1R) expression in articular cartilage was further studied by immunohistochemistry. Ca(2+) and cAMP assays were used to monitor alpha-MSH signaling, while studies of alpha-MSH function were performed in cultures with chondrocyte micromass pellets stimulated with alpha-MSH. Expression of cytokines and extracellular matrix (ECM) components was determined by real-time RT-PCR, Western immunoblotting, and enzyme-linked immunosorbent assays. RESULTS MC-1R expression was detected in articular chondrocytes in vitro and in articular cartilage in situ. In addition, expression of transcripts for MC-2R, MC-5R, POMC, and PCs was detected in articular chondrocytes. Stimulation with alpha-MSH increased the levels of intracellular cAMP, but not Ca(2+), in chondrocytes. Both messenger RNA and protein expression of various proinflammatory cytokines, collagens, matrix metalloproteinases (MMPs), and SOX9 was modulated by alpha-MSH. CONCLUSION Human articular chondrocytes are target cells for alpha-MSH. The effects of alpha-MSH on expression of cytokines and MMPs suggest that this neuropeptide plays a role in inflammatory and degenerative processes in cartilage. It is conceivable that inflammatory reactions can be mitigated by the induction of endogenous MCs or administration of alpha-MSH to the affected joints. The induction pattern of regulatory and structural ECM components such as collagens as well as SOX9 and anabolic and catabolic cytokines points to a function of alpha-MSH as a trophic factor in skeletal development during endochondral ossification rather than as a factor in homeostasis of permanent cartilage.

Analysis of Bone Matrix Composition and Trabecular Microarchitecture of the Femoral Metaphysis in Patients with Osteonecrosis of the Femoral Head

Osteonecrosis of the femoral head (ONFH) usually affects young individuals. In advanced stages of ONFH, total hip replacement is the golden standard. However, survivorship after total hip replacement has been reported to be poorer in patients with ONFH compared to patients with primary osteoarthritis (OA). In radiological and histological studies, an impaired bone quality was found not only for the femoral head, but also for the intertrochanteric and metaphyseal region. We hypothesize that alterations of bone quality in the femoral metaphysis might contribute to early stem loosening. The objective of this study was to assess the gene expression levels of factors regulating bone formation and remodeling of the intertrochanteric regions and the proximal femoral canal in patients with ONFH and those with primary OA. The cellular and macromolecular composition of the bone matrix was assessed by osteocalcin immunohistochemistry, and the three‐dimensional organization of trabecular bone was characterized by µCT analysis. Gene expression of BMP‐2 is twofold higher in the proximal femur in the region of the greater trochanter of patients with ONFH compared to those with OA. The number of osteoblasts in the greater trochanter of patients with ONFH (253/mm2) is increased compared to patients with OA (156/mm2). Trabecular properties in ONFH bone are altered for bone volume (OA: 32 mm3, ONFH: 51 mm3) and structure model index (OA: 2.2, ONFH: 1.6) in the proximal femoral canal, but not in the trochanteric regions. These alterations in bone metabolism and architecture might contribute to the higher rates of stem loosening after total hip replacement in patients with ONFH, however, further experimental and clinical studies are needed to support our findings.

Modulation of cartilage differentiation by melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP)

Melanoma inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from malignant melanoma cells and from chondrocytes. Recently, we revealed that MIA/CD-RAP can modulate bone morphogenetic protein (BMP)2-induced osteogenic differentiation into a chondrogenic direction. In the current study we aimed to find the molecular details of this MIA/CD-RAP function. Direct influence of MIA on BMP2 by protein-protein-interaction or modulating SMAD signaling was ruled out experimentally. Instead, we revealed inhibition of ERK signaling by MIA/CD-RAP. This inhibition is regulated via binding of MIA/CD-RAP to integrin α5 and abolishing its activity. Active ERK signaling is known to block chondrogenic differentiation and we revealed induction of aggrecan expression in chondrocytes by treatment with MIA/CD-RAP or PD098059, an ERK inhibitor. In in vivo models we could support the role of MIA/CD-RAP in influencing osteogenic differentiation negatively. Further, MIA/CD-RAP-deficient mice revealed an enhanced calcified cartilage layer of the articular cartilage of the knee joint and disordered arrangement of chondrocytes. Taken together, our data indicate that MIA/CD-RAP stabilizes cartilage differentiation and inhibits differentiation into bone potentially by regulating signaling processes during differentiation.

Influence of Factors Regulating Bone Formation and Remodeling on Bone Quality in Osteonecrosis of the Femoral Head

Osteonecrosis of the femoral head (ONFH) usually affects young individuals and has a major impact on lifestyle. Notably, the pathogenetic mechanisms of osteonecrosis are unresolved and no effective treatment exists. The objective of this study was to assess the gene expression levels of factors regulating bone formation and remodeling (bone morphogenetic protein [BMP]-2, BMP-7, Runx2, osteocalcin, osteoprotegerin [OPG]) in patients with ONFH and to compare them to those of patients with primary osteoarthritis (OA). The cellular and macromolecular composition of the bone matrix was assessed by osteocalcin immunohistochemistry, and the three-dimensional organization of trabecular bone was characterized by micro-computed tomographic analysis. Our results demonstrate that gene expression of BMP-2, BMP-7, and Runx2 is elevated in patients with ONFH. We observed increased extracellular osteocalcin deposition, presumably caused by a higher number of osteoblasts in concordance with increased activity of Runx2. Constant gene expression level of OPG implies an unchanged osteoclast differentiation rate in ONFH bone. We found no significant change in bone volume, connectivity, and structural model index; further, no significant differences were detected for trabecular properties in ONFH bone. In conclusion, we have shown increased gene expression of factors regulating bone formation and remodeling in the femoral head and/or neck of patients with ONFH. Further, we observed an increase in osteocalcin immunoreactivity and osteoblast/osteocyte cell number, while no significant changes in trabecular microarchitecture were detected. This study increases our understanding of the pathophysiology and repair process following ONFH and might help in the development of new treatment strategies in the future.

Enhanced cartilage regeneration in MIA/CD-RAP deficient mice

Melanoma inhibitory activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP) is a small soluble protein secreted from chondrocytes. It was identified as the prototype of a family of extracellular proteins adopting an SH3 domain-like fold. In order to study the consequences of MIA/CD-RAP deficiency in detail we used mice with a targeted gene disruption of MIA/CD-RAP (MIA−/−) and analyzed cartilage organisation and differentiation in in vivo and in vitro models. Cartilage formation and regeneration was determined in models for osteoarthritis and fracture healing in vivo, in addition to in vitro studies using mesenchymal stem cells of MIA−/− mice. Interestingly, our data suggest enhanced chondrocytic regeneration in the MIA−/− mice, modulated by enhanced proliferation and delayed differentiation. Expression analysis of cartilage tissue derived from MIA−/− mice revealed strong downregulation of nuclear RNA-binding protein 54-kDa (p54nrb), a recently described modulator of Sox9 activity. In this study, we present p54nrb as a mediator of MIA/CD-RAP to promote chondrogenesis. Taken together, our data indicate that MIA/CD-RAP is required for differentiation in cartilage potentially by regulating signaling processes during differentiation.

Coculture between periosteal explants and articular chondrocytes induces expression of TGF-β1 and collagen I

OBJECTIVE Repair of focal articular cartilage lesions is usually performed by employing cell-based therapeutic strategies such as autologous chondrocyte implantation (ACI). The aim of this study was to determine whether periosteum exerts pro-chondrogenic effects on the transplanted cells beyond its biomechanical role in ACI. METHODS Micromass pellets of human articular chondrocytes were cocultured for up to 28 days with human periosteal explants either with physical contact or separated by a membrane allowing paracrine interactions only. Quantitative reverse transcription (RT)-PCR, ELISA, immunohistochemistry and collagen isolation were used to analyse the expression and secretion of TGF-beta1, collagens I and II and chondrogenic differentiation markers such as MIA (CD-RAP) and aggrecan. RESULTS TGF-beta1 gene expression was induced significantly in paracrine cocultures in periosteum, whereas it was repressed in physical contact cocultures. However, a higher TGF-beta1 secretion rate was observed in physical contact cocultures compared with periosteal monocultures. The expression of COL2A1, melanoma inhibitory activity (cartilage-derived retinoic acid-sensitive protein) [MIA (CD-RAP)] and aggrecan was mainly unaffected by culture conditions, whereas COL1A1 gene expression was increased in periosteal paracrine cocultures. Collagen I staining was induced in micromass pellets from paracrine cocultures, whereas it was repressed in chondrocytes from physical contact cocultures. CONCLUSIONS We found evidence for a bidirectional regulating system with paracrine signalling pathways between periosteum and articular chondrocytes. Stimulation of TGF-beta1 and COL1A1 gene expression in periosteal paracrine cocultures and the increased release of TGF-beta1 protein in physical contact conditions indicate an anabolic, and not merely chondrogenic micro-environment in this in vitro model for periosteal-based ACI.

Collagen XVI induces formation of focal contacts on intestinal myofibroblasts isolated from the normal and inflamed intestinal tract.

In Crohns disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF) provided by intact tissue is disturbed due to inflammation and thus, cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original ECM leads ISEMF to exit this cycle. In contrast, during fibrosis, ISEMF persist. It is known that ISEMF produce and deposit collagen types I, III, IV and V; however synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed the distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell-matrix interactions in different phases of the inflammatory process and their effect on cell spreading, proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall where it co-localizes with fibrillin-1 and integrin alpha1. ISEMF reveal increasing gene and protein expression of collagen XVI concurrent to increasing inflammation. ISEMF reveal more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This involves recruitment of alpha1beta1 integrin, which shows increased cell surface expression on ISEMF in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and, even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell-matrix interaction preferentially through recruitment of alpha1ss1 integrin into the tips of the focal adhesion contacts.

Interaction of Periosteal Explants with Articular Chondrocytes Alters Expression Profile of Matrix Metalloproteinases

Periosteal tissue is a source of growth factors and of osteochondral progenitor cells which makes it suitable for implantation in chondral defects as known in autologous chondrocyte implantation. The aim of this study was to determine the interaction between periosteal tissue and articular chondrocytes with respect to catabolic effectors such as matrix metalloproteinases (MMPs) and IL‐6. Human articular chondrocytes were cultured for up to 28 days as micromass pellets in coculture either with physical contact to periosteal explants or allowing paracrine interactions only. Expression, secretion, and activation of MMPs and IL‐6 were analyzed in chondrocytes, periosteum, and culture supernatants. Both coculture conditions influence gene expression levels of MMPs and IL‐6 in a time‐, culture‐, and tissue‐dependent manner. Coculturing of periosteum with chondrocytes promotes gene expression and secretion of IL‐6. In periosteum, physical contact inhibits MMP‐2 and MMP‐13 gene expression while paracrine coculture induces expression of IL‐6, MMP‐2, ‐7, and ‐13. Pro‐MMP‐2, ‐7, and ‐13 were detected in supernatants of all culture regimens whereas pro‐MMP‐9 was secreted from periosteum only. As a balanced amount of MMP activity is likely required to achieve sufficient integration of the regenerate tissue with the surrounding healthy cartilage, an exceeding expression of proteinases might result in degradation, hypertrophy or rejection of the graft.