Viable osteoblastic potential of cortical reamings from intramedullary nailing.

To analyze the effect of intramedullary reaming on fracture healing, we investigated whether or not cortical reamings contain viable bone cells. There are several tissue components contained in medullary reamings including blood, bone marrow and cortical bone. This study is focused on the cortical reamings, which are produced during reaming of the medullary cavity. They may stimulate fracture healing but it is still unclear if they contain vital bone cells. We tested the hypothesis that these cortical reamings are a source of viable bone cells and compared cell cultures with cultivated cells from iliac crest biopsies. Responses of protein content and ALP activity to vitD stimulation in the cells were considered as properties of viability. Ten in tact living sheep femora were fully reamed and the cortical reamings were cultivated in a standard manner and compared with cultivated cells from ipsi-lateral iliac crest biopsies from the same animals. Cells started to grow from the reamings as well as the iliac crest within 2-5 days, and covered the entire culture flask within 9-13 days. Protein content and ALP activity in cells from both reamings and iliac crest were significantly responsive to vitD stimulation. Cortical reamings from intramedullary nailing have osteoblastic potential and contain living bone cells similar to bone cells from the iliac crest. These findings may further explain the superior healing of fractures, treated with reamed nailing.     

Pulsating fluid flow modulates gene expression of proteins involved in Wnt signaling pathways in osteocytes

Strain‐derived flow of interstitial fluid activates signal transduction pathways in osteocytes that regulate bone mechanical adaptation. Wnts are involved in this process, but whether mechanical loading modulates Wnt signaling in osteocytes is unclear. We assessed whether mechanical stimulation by pulsating fluid flow (PFF) leads to functional Wnt production, and whether nitric oxide (NO) is important for activation of the canonical Wnt signaling pathway in MLO‐Y4 osteocytes. MC3T3‐E1 osteoblasts were studied as a positive control for the MLO‐Y4 osteocyte response to mechanical loading. MLO‐Y4 osteocytes and MC3T3‐E1 osteoblasts were submitted to 1‐h PFF (0.7 ± 0.3 Pa, 5 Hz), and postincubated (PI) without PFF for 0.5–3 h. Gene expression of proteins related to the Wnt canonical and noncanonical pathways were studied using real‐time polymerase chain reaction (PCR). In MLO‐Y4 osteocytes, PFF upregulated gene expression of Wnt3a, c‐jun, connexin 43, and CD44 at 1–3‐h PI. In MC3T3‐E1 osteoblasts, PFF downregulated gene expression of Wnt5a and c‐jun at 0.5–3‐h PI. In MLO‐Y4 osteocytes, gene expression of PFF‐induced Wnt target genes was suppressed by the Wnt antagonist sFRP4, suggesting that loading activates the Wnt canonical pathway through functional Wnt production. The NO inhibitor L‐NAME suppressed the effect of PFF on gene expression of Wnt target genes, suggesting that NO might play a role in PFF‐induced Wnt production. The response to PFF differed in MC3T3‐E1 osteoblasts. Because Wnt signaling is important for bone mass regulation, osteocytes might orchestrate loading‐induced bone remodeling through, among others, Wnts. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1280–1287, 2009     

Multidrug resistance proteins in rhabdomyosarcomas: comparison between children and adults

BACKGROUND: Pediatric rhabdomyosarcomas (RMS) have a more advantageous prognosis after multimodality treatment compared with adult RMS, which might be related to a decreased sensitivity to chemotherapy in adults. Resistance to chemotherapy might be conveyed by the multidrug resistance (MDR)-associated proteins P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP). It was therefore suggested that these proteins were expressed differently in pediatric and adult patients. METHODS: The expression of P-gp, MRP1, and LRP was assessed immunohistochemically in 45 specimens of untreated RMS: 29 were obtained from children younger than 16 years old and 16 were obtained from adults. All children had an embryonal or botryoid RMS. Among the adults, there were 10 embryonal, 3 alveolar, and 3 pleomorphic RMS. Samples were scored as negative or positive according to the percentage of immunoreactive tumor cells: 0.5 (1-5%), 1 (5-25%), 2 (26-50%), 3 (51-75%), or 4 (> 75%). RESULTS: Expression of LRP was more pronounced in embryonal and pleomorphic RMS in adults compared with RMS in children. In addition, LRP expression correlated with age at diagnosis. Alveolar RMS had remarkably low LRP expression. Expression of P-gp and MRP1 did not differ significantly between children and adults. CONCLUSIONS: In this series of embryonal and pleomorphic RMS, an increased LRP expression was observed in adults, which may explain their worse response to chemotherapy reported in other studies. In alveolar RMS, a low LRP expression was observed, suggesting that other mechanisms are responsible for the resistant phenotype in most of these tumors.     

Induction of the cholesterol biosynthesis pathway in differentiated Caco-2 cells by the potato glycoalkaloid alpha-chaconine.

Glycoalkaloids are naturally occurring toxins in potatoes, which at high levels may induce toxic effects in humans, mainly on the gastrointestinal tract by cell membrane disruption. In order to better understand the molecular mechanisms underlying glycoalkaloid toxicity, we examined the effects of alpha-chaconine on gene expression in the Caco-2 intestinal epithelial cell line using DNA microarrays. Caco-2 cells were exposed for 6h to 10 microM alpha-chaconine in three independent experiments (randomized block design). The most prominent finding from our gene expression and pathway analyses was the upregulation of expression of several genes involved in cholesterol biosynthesis. This to some extent is in line with the literature-described mechanism of cell membrane disruption by glycoalkaloids. In addition, various growth factor signaling pathways were found to be significantly upregulated. This study is useful in understanding the mechanism(s) of alpha-chaconine toxicity, which may be extended to other potato glycoalkaloids more generally.     

Increased calcification of growth plate cartilage as a result of compressive force in vitro

The influence of intermittent compressive force (ICF) and of continuous compressive force (CCF) on calcification of growth plate cartilage was investigated, using organ cultures of fetal mouse cartilaginous long bone rudiments. Sixteen-day-old metatarsal rudiments, still consisting of uncalcified cartilage, were isolated and cultured for 5 days. Initial calcification of hypertrophic cartilage occurred under control conditions (atmospheric pressure), and under the influence of ICF or CCF by intermittently or continuously compressing the gas phase above the culture medium. Calcification was monitored by means of 45Ca and 32P incorporation into calcium-phosphate mineral and by morphometric methods. Both ICF and CCF increased cartilage calcification, but ICF was about twice as effective as CCF. Killed rudiments did not calcify during the culture period, nor did ICF or CCF increase the incorporation of label. The effects of ICF and CCF on calcification could not be mimicked by increasing the PO2 and PCO2 levels in the gas phase. The length of the central zone of calcified cartilage was significantly increased by ICF and CCF. We conclude that hypertrophic chondrocytes respond directly to ICF and CCF by an increased deposition of calcium-phosphate mineral in the matrix. Discontinuous mechanical stimulation evokes a higher cellular response than does continuous stimulation.     

A man with pain and weakness in his legs

A 75-year-old man came to the clinic because of pain and weakness in his legs. He had fallen a year earlier. The pain and weakness were caused by a bilateral rupture of the tendon of the M. rectus femoris.