Karin Pichler

The effects of physical activity on apoptosis and lubricin expression in articular cartilage in rats with glucocorticoid-induced osteoporosis.

Glucocorticoids are considered the most powerful anti-inflammatory and immunomodulating drugs. However, a number of side-effects are well documented in different diseases, including articular cartilage, where increases or decreases in the synthesis of hormone-dependent extracellular matrix components are seen. The objective of this study has been to test the effects of procedures or drugs affecting bone metabolism on articular cartilage in rats with prednisolone-induced osteoporosis and to evaluate the outcomes of physical activity with treadmill and vibration platform training on articular cartilage. The animals were divided into 5 groups, and bone and cartilage evaluations were performed using whole-body scans and histomorphometric analysis. Lubricin and caspase-3 expression were evaluated by immunohistochemistry, Western blot analysis and biochemical analysis. These results confirm the beneficial effect of physical activity on the articular cartilage. The effects of drug therapy with glucocorticoids decrease the expression of lubricin and increase the expression of caspase-3 in the rats, while after physical activity the values return to normal compared to the control group. Our findings suggest that it might be possible that mechanical stimulation in the articular cartilage could induce the expression of lubricin, which is capable of inhibiting caspase-3 activity, preventing chondrocyte death. We can assume that the physiologic balance between lubricin and caspase-3 could maintain the integrity of cartilage. Therefore, in certain diseases such as osteoporosis, mechanical stimulation could be a possible therapeutic treatment. With our results we can propose the hypothesis that physical activity could also be used as a therapeutic treatment for cartilage disease such as osteoarthritis.

RANKL is downregulated in bone cells by physical activity (treadmill and vibration stimulation training) in rat with glucocorticoid-induced osteoporosis.

The aim of this study was to investigate bone tissue and plasma levels of RANKL and OPG in rats with prednisolone-induced osteoporosis and to evaluate the outcomes of physical activity on the skeletal system by treadmill and vibration platform training. Osteoporosis is a disease characterised by low bone mass and structural deterioration of bone tissue leading to bone fragility. Vibration exercise is a new and effective measure to prevent muscular atrophy and osteoporosis. The animals were divided into 5 groups. 1: control rats; 2: rats with osteoporosis receiving prednisolone; 3: rats receiving prednisolone and treadmill training; 4: rats receiving prednisolone and vibration stimulation training; 5: rats receiving prednisolone, treadmill and vibration stimulation training. For bone evaluations we used whole-body scans, histology and histomorphometric analysis. RANKL and OPG expression was evaluated by immunohistochemistry and biochemical analysis. After treatment, our data demonstrated that RANKL expression was significantly increased in groups 2 and 3 and decreased in groups 4 and 5. Conversely, OPG expression was significantly decreased in groups 2 and 3 and increased in groups 4 and 5. In conclusion, our findings suggest that mechanical stimulation inhibits the activity of RANKL. This finding provides new insights into the occurrence and progression of osteoporosis.

Aquaporin 1 (AQP1) expression in experimentally induced osteoarthritic knee menisci: An in vivo and in vitro study

Osteoarthritis (OA) of the knee is a major problem in our society. The development of new treatment options for OA is limited, because the pathophysiological mechanisms are not clearly understood, especially on the molecular level. Aquaporin 1 (AQP1) is a specific protein channels for water transport; it is expressed in articular chondrocytes, human synovitis, in chondrocytes of patients with rheumatoid arthritis or OA and in chondrocyte-like cells of human intervertebral disc. The aim of this study was to investigate the expression of AQP1, through immunohistochemistry, immunocytochemistry and Western blot, in experimentally induced OA knee menisci. AQP1 was studied in vivo in knee OA menisci from 36 rats that underwent medial or lateral meniscectomy, and in vitro on fibrochondrocytes derived from knee OA menisci rats. OA in rats was experimentally induced and tested by histomorphometric analysis. Histological results demonstrated structural alterations in OA menisci accompanied by a very strong AQP1 immunohistochemical and immunocytochemical staining. The Western blot analysis confirmed a strong expression of AQP1 in OA fibrochondrocytes cells. The results of the present research suggest that an activation of AQP1, induced by the OA process, may represent an endogenous mechanism, which can be used to control the tissue degeneration within OA articular joints.

Post-Traumatic Caspase-3 Expression in the Adjacent Areas of Growth Plate Injury Site: A Morphological Study

The epiphyseal plate is a hyaline cartilage plate that sits between the diaphysis and the epiphysis. The objective of this study was to determine the impact of an injury in the growth plate chondrocytes through the study of histological morphology, immunohistochemistry, histomorphometry and Western Blot analyses of the caspase-3 and cleaved PARP-1, and levels of the inflammatory cytokines, Interleukin-6 (IL-6) and Tumor Necrosis Factor alpha (TNF-α), in order to acquire more information about post-injury reactions of physeal cell turnover. In our results, morphological analysis showed that in experimental bones, neo-formed bone trabeculae—resulting from bone formation repair—invaded the growth plate and reached the metaphyseal bone tissue (bone bridge), and this could result in some growth arrest. We demonstrated, by ELISA, increased expression levels of the inflammatory cytokines IL-6 and TNF-α. Immunohistochemistry, histomorphometry and Western Blot analyses of the caspase-3 and cleaved PARP-1 showed that the physeal apoptosis rate of the experimental bones was significantly higher than that of the control ones. In conclusion, we could assume that the inflammation process causes stress to chondrocytes that will die as a biological defense mechanism, and will also increase the survival of new chondrocytes for maintaining cell homeostasis. Nevertheless, the exact stimulus leading to the increased apoptosis rate, observed after injury, needs additional research to understand the possible contribution of chondrocyte apoptosis to growth disturbance.

Ameliorative Effects of PACAP against Cartilage Degeneration. Morphological, Immunohistochemical and Biochemical Evidence from in Vivo and in Vitro Models of Rat Osteoarthritis

Osteoarthritis (OA); the most common form of degenerative joint disease, is associated with variations in pro-inflammatory growth factor levels, inflammation and hypocellularity resulting from chondrocyte apoptosis. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide endowed with a range of trophic effects in several cell types; including chondrocytes. However; its role in OA has not been studied. To address this issue, we investigated whether PACAP expression is affected in OA cartilage obtained from experimentally-induced OA rat models, and then studied the effects of PACAP in isolated chondrocytes exposed to IL-1β in vitro to mimic the inflammatory milieu of OA cartilage. OA induction was established by histomorphometric and histochemical analyses. Changes in PACAP distribution in cartilage, or its concentration in synovial fluid (SF), were assessed by immunohistochemistry and ELISA. Results showed that PACAP abundance in cartilage tissue and SF was high in healthy controls. OA induction decreased PACAP levels both in affected cartilage and SF. In vitro, PACAP prevented IL-1β-induced chondrocyte apoptosis, as determined by MTT assay; Hoechst staining and western blots of apoptotic-related proteins. These changes were also accompanied by decreased i-NOS and COX-2 levels, suggesting an anti-inflammatory effect. Altogether, these findings support a potential role for PACAP as a chondroprotective agent for the treatment of OA.

Cellular reactions to biodegradable magnesium alloys on human growth plate chondrocytes and osteoblasts

PurposeIn recent decades operative fracture treatment using elastic stable intramedullary nails (ESINs) has mainly taken precedence over conservative alternatives in children. The development of biodegradable materials that could be used for ESINs would be a further step towards treatment improvement. Due to its mechanical and elastic properties, magnesium seems to be an ideal material for biodegradable implant application. The aim of this study was therefore to investigate the cellular reaction to biodegradable magnesium implants in vitro.MethodsPrimary human growth plate chondrocytes and MG63 osteoblasts were used for this study. Viability and metabolic activity in response to the eluate of a rapidly and a slower degrading magnesium alloy were investigated. Furthermore, changes in gene expression were assessed and live cell imaging was performed.ResultsA superior performance of the slower degrading WZ21 alloy’s eluate was detected regarding cell viability and metabolic activity, cell proliferation and morphology. However, the ZX50 alloy’s eluate induced a favourable up-regulation of osteogenic markers in MG63 osteoblasts.ConclusionsThis study showed that magnesium alloys for use in biodegradable implant application are well tolerated in both osteoblasts and growth plate chondrocytes respectively.

Towards a better understanding of bone bridge formation in the growth plate - an immunohistochemical approach.

Abstract The growth plate at the end of long bones is the cartilaginous organ responsible for longitudinal bone growth in children. Trauma to the growth plate, i.e. fractures, can severely impair longitudinal bone growth, leading to growth disorders due to destruction of the epiphyseal circulation and formation of a bone bridge. From the clinical experience it is known that in some patients this bone bridge eventually disappears during the growth process. However, the molecular mechanisms involved in bone bridge formation and dissolution have not been clarified yet. The aim of this study was to investigate the spatial and temporal protein level of molecules potentially involved in these processes, i.e. RANKL, OPG, DKK-1, Coll 10, BMP-2 and IL-6, in an experimental rat model using an immunohistochemical approach. The results from our study suggest that bone bridge formation might be an early event starting immediately after growth plate injury and involving several pro-osteoblastic molecules, i.e. IL-6, BMP-2 as well as OPG and Coll X. In the late studied time points 3- and 9-month post-injury expression of anti-osteoblastic proteins, i.e. DKK1 and RANKL, was increased. This indicates that bone bridge dissolution might be a late event and potentially linked to Wnt signaling inhibition and RANK/RANKL signaling activation.

Behaviour of human physeal chondro-progenitorcells in early growth plate injury response in vitro.

PurposeThe aim of this study was to investigate the proliferation and differentiation behaviour of a defined cell population gained from the human growth plate, namely, chondro-progenitorcells (CPCs), in the initial inflammatory phase of growth plate injury response in vitro.MethodsGrowth plate cells were sorted via FACS and differentiated along adipogenic and osteogenic lineage to confirm their progenitor features. To mimic the inflammatory phase of injury response at the growth plate they were treated with IL-1β and exposed to cyclic mechanical loading. A BrdU assay was used to investigate CPC proliferation. CPC differentiation behaviour was analysed by RT-PCR.ResultsCPCs (CD45-, CD34-, CD73+, CD90+, and CD105+) showed a successful differentiation along adipogenic and osteogenic lineage. Under conditions simulating the inflammatory phase of injury response at the growth plate in vitro CPCs differentiated towards hypertrophy while chondrogenesis and ossification were inhibited. Proliferation was not significantly altered.ConclusionThis study showed that CPCs can be isolated from the human growth plate and expanded in vitro. In the first phase of injury response at the growth plate these cells differentiate towards hypertrophy. As longitudinal growth is obtained by chondrocyte proliferation and volume increase during hypertrophy this maturation might be the first step towards post-traumatic growth disorders such as unwanted premature ossification of the growth plate.

BMP‐6 and BMPR‐1a are up‐regulated in the growth plate of the fractured tibia

Bone overgrowth is a known phenomenon occurring after fracture of growing long bones with possible long‐term physical consequences for affected children. Here, the physeal expression of bone morphogenetic proteins (BMPs) was investigated in a fracture‐animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of bone formation, thus stimulating bone overgrowth. Sprague–Dawley rats (male, 4 weeks old), were subjected to a unilateral mid‐diaphyseal tibial fracture. Kinetic expression of physeal BMP‐2, ‐4, ‐6, ‐7, and BMP receptor‐1a (BMPR‐1a) was analyzed in a monthly period by quantitative real time‐polymerase chain reaction and immunohistochemistry. On Days 1, 3, 10, and 14 post‐fracture, no changes in physeal BMPs gene‐expression were detected. Twenty‐nine days post‐fracture, when the fracture was consolidated, physeal expression of BMP‐6 and BMPR‐1a was significantly upregulated in the growth plate of the fractured and contra‐lateral intact bone compared to control (p < 0.005). This study demonstrates a late role of BMP‐6 and BMPR‐1a in fracture‐induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory “cross‐talk” mechanism between the lower limbs whose function could be to limit leg‐length‐discrepancies following the breakage of growing bones.

Bisphosphonates in multicentric osteolysis, nodulosis and arthropathy (MONA) spectrum disorder – an alternative therapeutic approach

Multicentric osteolysis, nodulosis and arthropathy (MONA) spectrum disorder is a rare inherited progressive skeletal disorder caused by mutations in the matrix metalloproteinase 2 (MMP2) gene. Treatment options are limited. Herein we present successful bisphosphonate therapy in three affected patients. Patients were treated with bisphosphonates (either pamidronate or zoledronate) for different time periods. The following outcome variables were assessed: skeletal pain, range of motion, bone densitometry, internal medical problems as well as neurocognitive function. Skeletal pain was dramatically reduced in all patients soon after initiation of therapy and bone mineral density increased. Range of motion did not significantly improve. One patient is still able to walk with aids at the age of 14 years. Neurocognitive development was normal in all patients. Bisphosphonate therapy was effective especially in controlling skeletal pain in MONA spectrum disorder. Early initiation of treatment seems to be particularly important in order to achieve the best possible outcome.