Cellular and Molecular Foundation for Fracture Healing in Children

Abstract Dealing with pediatric fracture patients requires a funded knowledge of complications and remodeling capability of the youth skeleton to find the accurate therapy decision and to avoid unnecessary invasive procedures. Due to the different mechanical environment, fractures in children occur at specific fracture-vulnerable areas. One of those is the growth plate, which on one hand gives rise to the unique ability of correcting angular deformities by specifically increasing the growth rate in definite regions, and on the other hand leads to complications like growth arrest or angular deformity. The pediatric diaphysis presents the exclusive greenstick fracture, only seen in the growing skeleton, which occurs because of the different composition of the pediatric bone. To understand these very specific features of the youth skeleton, the molecular and cellular basis should be taken into consideration. Therefore, this review will present the common characteristics of skeletal development and fracture healing. An insight into the mechanotransduction as part of the remodeling and self-correcting ability of pediatric bone is given to span the bridge between clinical treatment options and scientific background.     

Elective transplant pneumonectomy in a 38-year-old man.

Transplant pneumonectomy is a rarely performed procedure. It is occasionally carried out in the course of retransplantation. To our knowledge, resection of a transplanted lung without its replacement and with successful outcome in the adult has not been previously reported. We present a case of elective left transplant pneumonectomy in a 38-year-old man 6 years after left single-lung transplant. At 12 months after resection, the patient remains well, with good exercise tolerance.     

Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption.

Dissolution of the inorganic phase of bone by the osteoclasts mediated by V-ATPase and ClC-7 is a prerequisite for bone resorption. Inhibitors of osteoclastic V-ATPase or ClC-7 are novel approaches for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays, diphyllin was identified. We characterized diphyllin with respect to the pharmacological effects on osteoclasts. INTRODUCTION: Osteoclastic acidification of the resorption lacuna and bone resorption requires activity of both V-ATPase and the chloride channel ClC-7. Inhibition of these processes represents a novel approach for treatment of bone metabolic disorders. We identified diphyllin, a novel inhibitor of V-ATPase, and characterized this natural compound with respect to activity in human osteoclasts. MATERIALS AND METHODS: Diphyllin was tested in the acid influx assay and V-ATPase assay using bovine chromaffin granules. Human osteoclasts were generated from CD14+ monocytes cultured with macrophage-colony stimulating factor (M-CSF) and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was studied using acridine orange. The effect of diphyllin on bone resorption by osteoclasts was measured as release of C-terminal cross-linked telopeptide of type I collagen (CTX-I) and calcium into the supernatants and by scoring pit area. Osteoclast number, TRACP activity, and cell viability were measured. Furthermore, the effect of diphyllin on bone nodule formation was tested using the mouse osteoblast cell line MC3T3-E1. RESULTS: In the acid influx assay, diphyllin potently inhibited the acid influx (IC50 = 0.6 nM). We found that diphyllin inhibited V-ATPase with an IC50 value of 17 nM, compared with 4 nM for bafilomycin A1. Moreover, diphyllin dose-dependently inhibited lysosomal acidification in human osteoclasts. Furthermore, we found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50 = 14 nM), calcium release, and pit area, despite increasing TRACP activity, numbers of osteoclasts, and cell viability. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin A1 was toxic. CONCLUSIONS: We identified a natural compound that potently inhibits V-ATPase and thereby lysosomal acidification in osteoclasts, which leads to abrogation of bone resorption. Because recent studies indicate that inhibition of the osteoclastic acidification leads to inhibition of resorption without inhibiting formation, we speculate that diphyllin is a potential novel treatment for bone disorders involving excessive resorption.     

Are nonresorbing osteoclasts sources of bone anabolic activity?

Some osteopetrotic mutations lead to low resorption, increased numbers of osteoclasts, and increased bone formation, whereas other osteopetrotic mutations lead to low resorption, low numbers of osteoclasts, and decreased bone formation. Elaborating on these findings, we discuss the possibility that osteoclasts are the source of anabolic signals for osteoblasts. In normal healthy individuals, bone formation is coupled to bone resorption in a tight equilibrium. When this delicate balance is disturbed, the net result is pathological situations, such as osteopetrosis or osteoporosis. Human osteopetrosis, caused by mutations in proteins involved in the acidification of the resorption lacuna (ClC-7 or the a3-V-ATPase), is characterized by decreased resorption in face of normal or even increased bone formation. Mouse mutations leading to ablation of osteoclasts (e.g., loss of macrophage-colony stimulating factor [M-CSF] or c-fos) lead to secondary negative effects on bone formation, in contrast to mutations where bone resorption is abrogated with sustained osteoclast numbers, such as the c-src mice. These data indicate a central role for osteoclasts, and not necessarily their resorptive activity, in the control of bone formation. In this review, we consider the balance between bone resorption and bone formation, reviewing novel data that have shown that this principle is more complex than originally thought. We highlight the distinct possibility that osteoclast function can be divided into two more or less separate functions, namely bone resorption and stimulation of bone formation. Finally, we describe the likely possibility that bone resorption can be attenuated pharmacologically without the undesirable reduction in bone formation.     

Fidgetin-like 1 gene inhibited by basic fibroblast growth factor regulates the proliferation and differentiation of osteoblasts.

The FIGNL1 gene was proven to be a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). In this in vitro study, the AAA proteins inhibited osteoblast proliferation and stimulated osteoblast differentiation. We showed that FIGNL1 may play some regulatory role in osteoblastogenesis. INTRODUCTION: The fidgetin-like 1 (FIGNL1) gene encodes a new subfamily member of ATPases associated with diverse cellular activities (AAA proteins). Although the FIGNL1 protein localizes to both the nucleus and cytoplasm, the function of FIGNL1 remains unknown. In a previous study, we identified several genes that mediate the anabolic effects of basic fibroblast growth factor (bFGF) on bone by using microarray data. FIGNL1 was one of the genes that downregulated >2-fold in MC3T3-E1 cells after treatment with bFGF. Therefore, this study was aimed to identify and confirm the function of FIGNL1 on osteoblastogenesis. MATERIALS AND METHODS: We examined the effect of the FIGNL1 gene on proliferation, differentiation, and apoptosis in mouse osteoblast cells (MC3T3-E1 and mouse primary calvarial cells) using flow cytometry, RT-PCR, cell proliferation assay, and cell death assay. MC3T3-E1 cells and mouse calvarial cells were transfected with small interfering RNA (siRNA) directed against the FIGNL1 or nontargeting control siRNA and examined by cell proliferation and cell death assays. Also, FIGNL1 was fused to enhance green fluorescent protein (EGFP), and the EGFP-fused protein was transiently expressed in MC3T3-E1 cells. RESULTS: Reduced expression of FIGNL1 by bFGF and TGF-beta1 treatment was verified by RT-PCR analysis. Overexpression of FIGNL1 reduced the proliferation of MC3T3-E1 and calvarial cells, more than the mock transfected control cells did. In contrast, siFIGNL1 transfection significantly increased the proliferation of osteoblasts, whereas overexpression of FIGNL1 did not seem to alter apoptosis in osteoblasts. Meanwhile, overexpression of FIGNL1 enhanced the mRNA expression of alkaline phosphatase (ALP) and osteocalcin (OCN) in osteoblasts. In contrast, siFIGNL1 decreased the expression of ALP and OCN. A pEGFP-FIGNL1 transfected into MCT3-E1 cells had an initially ubiquitous distribution and rapidly translocated to the nucleus 1 h after bFGF treatment. CONCLUSIONS: From these results, we proposed that FIGNL1, a subfamily member of the AAA family of proteins, might play some regulatory role in osteoblast proliferation and differentiation. Further analyses of FIGNL1 will be needed to better delineate the mechanisms contributing to the inhibition of proliferation and stimulation of osteoblast differentiation.