Can Rigid Femoral Nailing Be Used for Pediatric Femoral Shaft Fracture in Children 8 to 10 Years? Use of RIN in Patients With Femoral Shaft Fracture.

BACKGROUND: The treatment modalities for pediatric femoral shaft fractures are determined by their age, weight, and fracture pattern. Rigid intramedullary nailing (RIN) is usually recommended for patients >11 years of age, and elastic intramedullary nailing (EIN) has been used for patients under 10 years. However, little is known about the use of RIN in patients aged 8 to 10 years. We examined the differences in patients with femoral shaft fractures who were treated with EIN or RIN in terms of (1) fracture healing; (2) changes of anatomic parameters; and (3) related complications. METHODS: We retrospectively reviewed 54 patients between 8 and 10 years of age, with femoral shaft fractures, who were treated with either EIN or RIN between 2011 and 2020. Lateral trochanteric entry was used for RIN procedure. The mean follow-up period was 26.4 months (range, 6 to 113 mo). There were 17 patients in the EIN group and 37 patients in the RIN group. The mean age at the time of surgery was 1 year younger in the EIN group ( P <0.01). The mean weight of the patient was significantly heavier in the RIN group compared with the EIN group. RESULTS: Complete union of the fracture was achieved slightly faster in the RIN group at 3.4 months compared with 3.7 months in the EIN group ( P =0.04). There were no clinically significant changes of the anatomic parameters in either group, including neck shaft angle and articulotrochanteric distance. There was no evidence of avascular necrosis at the time of final follow-up for either group. There were no significant differences in postoperative complications between the groups. CONCLUSION: RIN using lateral trochanteric entry is a feasible surgical option for femoral shaft fractures in patients 8 to 10 years of age that are heavier than 40 kg or with unstable fracture patterns. LEVEL OF EVIDENCE: Level III, retrospective cohort study. See the Guidelines for Authors for a complete description of levels of evidence.

Intracapsular Pressures After Stable Slipped Capital Femoral Epiphysis.

PURPOSE: Stable slipped capital femoral epiphysis (SCFE) has been shown to have a lower rate of avascular necrosis than unstable SCFE. A recent study found increased intracapsular hip pressures in the setting of unstable SCFE, thus increasing the risk of osteonecrosis. The purpose of this study was to measure the intracapsular pressure in stable SCFE and compare it to the intracapsular pressure in normal hips and in unstable SCFE. METHODS: Thirteen hips with stable SCFE and 15 hips with unstable SCFE were identified. Using a side-bored needle, intracapsular hip pressures were measured at the time of surgery. Within these 2 study groups, 11 unaffected (normal) hips were also measured. Diastolic blood pressure and mean arterial pressure at the time of measurement were also recorded. RESULTS: The average intracapsular hip pressure in the stable SCFE group was 27.0 mm Hg, whereas the average pressure in the unstable SCFE group was 48.2 mm Hg and the average pressure in the normal group was 21.8 mm Hg. There was no significant difference between the normal and stable SCFE groups. There was a statistically significant difference between the stable SCFE and unstable SCFE groups (P<0.001). We found similar trends when comparing the intracapsular hip pressure as a percentage of the mean arterial pressure as well as the difference between diastolic blood pressure and hip pressure. CONCLUSIONS: As expected, the intracapsular pressure in the setting of stable SCFE approaches that of normal hips. This may explain why the risk of AVN in stable SCFE is significantly lower than that of unstable SCFE. It also supports the idea that capsulotomy is indicated for unstable slips to decrease the elevated hip pressure but not in stable SCFE.

Outcomes after salvage procedures for the painful dislocated hip in cerebral palsy.

BACKGROUND: The painful dislocated hip in the setting of cerebral palsy is a challenging problem. Many surgical procedures have been reported to treat this condition with varying success rates. The purpose of this study is to retrospectively evaluate and compare the outcomes of 3 different surgical procedures performed at our institution for pain relief in patients with spastic quadriplegic cerebral palsy and painful dislocated hips. METHODS: A retrospective chart review of the surgical procedures performed by 5 surgeons for spastic, painful dislocated hips from 1997 to 2010 was performed. The procedures identified were (1) proximal femoral resection arthroplasty (PFRA); (2) subtrochanteric valgus osteotomy (SVO) with femoral head resection; and (3) proximal femur prosthetic interposition arthroplasty (PFIA) using a humeral prosthesis. Outcomes based on pain and range of motion were determined to be excellent, good, fair, or poor by predetermined criteria. RESULTS: Forty-four index surgeries and 14 revision surgeries in 33 patients with an average follow-up of 49 months met the inclusion criteria. Of the index surgeries, 12 hips were treated with a PFRA, 21 with a SVO, and 11 with a PFIA. An excellent or good result was noted in 67% of PFRAs, 67% of SVOs, and 73% of PFIAs. No statistical significance between these procedures was achieved. The 14 revisions were performed because of a poor result from previous surgery, demonstrating a 24% reoperation rate overall. No patients classified as having a fair result underwent revision surgery. All patients receiving revision surgery were eventually classified as having an excellent or good result. CONCLUSIONS: Surgical treatment for the painful, dislocated hip in the setting of spastic quadriplegic cerebral palsy remains unsettled. There continue to be a large percentage of failures despite the variety of surgical techniques designed to treat this problem. These failures can be managed, however, and eventually resulted in a good outcome. We demonstrated a trend toward better outcomes with a PFIA, but further study should be conducted to prove statistical significance. LEVEL OF EVIDENCE: III.

Septoclast deficiency accompanies postnatal growth plate chondrodysplasia in the toothless (tl) osteopetrotic, colony-stimulating factor-1 (CSF-1)-deficient rat and is partially responsive to CSF-1 injections.

The septoclast is a specialized, cathepsin B-rich, perivascular cell type that accompanies invading capillaries on the metaphyseal side of the growth plate during endochondral bone growth. The putative role of septoclasts is to break down the terminal transverse septum of growth plate cartilage and permit capillaries to bud into the lower hypertrophic zone. This process fails in osteoclast-deficient, osteopetrotic animal models, resulting in a progressive growth plate dysplasia. The toothless rat is severely osteopetrotic because of a frameshift mutation in the colony-stimulating factor-1 (CSF-1) gene (Csf1(tl)). Whereas CSF-1 injections quickly restore endosteal osteoclast populations, they do not improve the chondrodysplasia. We therefore investigated septoclast populations in Csf1(tl)/Csf1(tl) rats and wild-type littermates, with and without CSF-1 treatment, at 2 weeks, before the dysplasia is pronounced, and at 4 weeks, by which time it is severe. Tibial sections were immunolabeled for cathepsin B and septoclasts were counted. Csf1(tl)/Csf1(tl) mutants had significant reductions in septoclasts at both times, although they were more pronounced at 4 weeks. CSF-1 injections increased counts in wild-type and mutant animals at both times, restoring mutants to normal levels at 2 weeks. In all of the mutants, septoclasts seemed misoriented and had abnormal ultrastructure. We conclude that CSF-1 promotes angiogenesis at the chondroosseous junction, but that, in Csf1(tl)/Csf1(tl) rats, septoclasts are unable to direct their degradative activity appropriately, implying a capillary guidance role for locally supplied CSF-1.

Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation.

Microarray and real-time RT-PCR were used to examine expression changes in primary bone marrow cells and RAW 264.7 cells in response to RANKL. In silico sequence analysis was performed on a novel gene which we designate OC-STAMP. Specific siRNA and antibodies were used to inhibit OC-STAMP RNA and protein, respectively, and tartrate-resistant acid phosphatase (TRAP)+ multinucleated osteoclasts were counted. Antibodies were used to probe bone tissues and western blots of RAW cell extracts +/- RANKL. cDNA overexpression constructs were transfected into RAW cells and the effect on RANKL-induced differentiation was studied. OC-STAMP was very strongly up-regulated during osteoclast differentiation. Northern blots and sequence analysis revealed two transcripts of 2 and 3.7 kb differing only in 3'UTR length, consistent with predictions from genome sequence. The mRNA encodes a 498 amino acid, multipass transmembrane protein that is highly conserved in mammals. It has little overall homology to other proteins. The carboxy-terminal 193 amino acids, however, are significantly similar to the DC-STAMP family consensus sequence. DC-STAMP is a transmembrane protein required for osteoclast precursor fusion. Knockdown of OC-STAMP mRNA by siRNA and protein inhibition by antibodies significantly suppressed the formation of TRAP+, multinucleated cells in differentiating osteoclast cultures, with many TRAP+ mononuclear cells present. Conversely, overexpression of OC-STAMP increased osteoclastic differentiation of RAW 264.7 cells. We conclude that OC-STAMP is a previously unknown, RANKL-induced, multipass transmembrane protein that promotes the formation of multinucleated osteoclasts.

Increased intracapsular pressures after unstable slipped capital femoral epiphysis.

BACKGROUND: Osteonecrosis of the femoral head is the most dreaded complication associated with an unstable slipped capital femoral epiphysis (SCFE). We hypothesize that the hip joint pressure will be increased in unstable slips, confirming that emergent treatment and decompression are warranted. METHODS: Thirteen unstable SCFE hips were evaluated. Hip pressure monitoring was performed. Postcapsulotomy measurements were also performed in all of the patients. Five of these under gentle manipulation. Six patients underwent measurement of the hip pressure on the unaffected side. RESULTS: The mean pressure on the affected hip was 48 mm Hg. The mean pressure on the unaffected side was 23 mm Hg. There was a significant increase in intraarticular hip pressure after attempted manipulation (mean, 75 mm Hg). DISCUSSION: Hip pressures are increased in unstable SCFE to levels higher for those of a compartment syndrome probably causing a tamponade effect. There is a need to perform a capsulotomy if manipulation is performed.

Selective Targeting of Bromodomains of the Bromodomain-PHD Fingers Family Impairs Osteoclast Differentiation.

Histone acetyltransferases of the MYST family are recruited to chromatin by BRPF scaffolding proteins. We explored functional consequences and the therapeutic potential of inhibitors targeting acetyl-lysine dependent protein interaction domains (bromodomains) present in BRPF1-3 in bone maintenance. We report three potent and selective inhibitors: one (PFI-4) with high selectivity for the BRPF1B isoform and two pan-BRPF bromodomain inhibitors (OF-1, NI-57). The developed inhibitors displaced BRPF bromodomains from chromatin and did not inhibit cell growth and proliferation. Intriguingly, the inhibitors impaired RANKL-induced differentiation of primary murine bone marrow cells and human primary monocytes into bone resorbing osteoclasts by specifically repressing transcriptional programs required for osteoclastogenesis. The data suggest a key role of BRPF in regulating gene expression during osteoclastogenesis, and the excellent druggability of these bromodomains may lead to new treatment strategies for patients suffering from bone loss or osteolytic malignant bone lesions.

Studies of OC-STAMP in Osteoclast Fusion: A New Knockout Mouse Model, Rescue of Cell Fusion, and Transmembrane Topology.

The fusion of monocyte/macrophage lineage cells into fully active, multinucleated, bone resorbing osteoclasts is a complex cell biological phenomenon that utilizes specialized proteins. OC-STAMP, a multi-pass transmembrane protein, has been shown to be required for pre-osteoclast fusion and for optimal bone resorption activity. A previously reported knockout mouse model had only mononuclear osteoclasts with markedly reduced resorption activity in vitro, but with paradoxically normal skeletal micro-CT parameters. To further explore this and related questions, we used mouse ES cells carrying a gene trap allele to generate a second OC-STAMP null mouse strain. Bone histology showed overall normal bone form with large numbers of TRAP-positive, mononuclear osteoclasts. Micro-CT parameters were not significantly different between knockout and wild type mice at 2 or 6 weeks old. At 6 weeks, metaphyseal TRAP-positive areas were lower and mean size of the areas were smaller in knockout femora, but bone turnover markers in serum were normal. Bone marrow mononuclear cells became TRAP-positive when cultured with CSF-1 and RANKL, but they did not fuse. Expression levels of other osteoclast markers, such as cathepsin K, carbonic anhydrase II, and NFATc1, were not significantly different compared to wild type. Actin rings were present, but small, and pit assays showed a 3.5-fold decrease in area resorbed. Restoring OC-STAMP in knockout cells by lentiviral transduction rescued fusion and resorption. N- and C-termini of OC-STAMP were intracellular, and a predicted glycosylation site was shown to be utilized and to lie on an extracellular loop. The site is conserved in all terrestrial vertebrates and appears to be required for protein stability, but not for fusion. Based on this and other results, we present a topological model of OC-STAMP as a 6-transmembrane domain protein. We also contrast the osteoclast-specific roles of OC- and DC-STAMP with more generalized cell fusion mechanisms.

Role of apoptosis in glucocorticoid-induced osteoporosis and osteonecrosis.

Bone disease is a side effect of concern regarding chronic glucocorticoid (GC) administration. Most GC-treated patients exhibit a process of bone loss, frequently leading to osteoporosis, with increased fracture risk, especially in spinal vertebrae. Some GC-treated patients will develop osteonecrosis, a disease with distinct clinical and histopathological features, most often occurring underneath the articular surface of the femoral head. Remarkably, both of these GC-induced bone diseases are associated with osteoblast and osteocyte apoptosis, which is emerging as a potential primary pathogenic mechanism. Here, we review the evidence for osteoblast and osteocyte apoptosis in GC-induced bone disease and highlight current debates: (1) With recent reports describing the antiapoptotic effect of GCs in some in vitro osteoblast models, and with the known adverse effects of GCs on osteoblast cell cycle and differentiation, could the in vivo osteoblast apoptosis be an indirect rather than a direct effect of GCs? (2) What is the pathogenic relationship between GC-induced osteoporosis and osteonecrosis? Could the latter be a mere manifestation of the former? and (3) How does apoptosis fit into the traditional concept of ischemia as a key etiology in osteonecrosis? Regardless of the answers, recent studies with cells, animals, and humans point out bone cell apoptosis as a potential target in the design of treatment for GC-induced bone disease.

Using osteoclast differentiation as a model for gene discovery in an undergraduate cell biology laboratory.

A key goal of molecular/cell biology/biotechnology is to identify essential genes in virtually every physiological process to uncover basic mechanisms of cell function and to establish potential targets of drug therapy combating human disease. This article describes a semester-long, project-oriented molecular/cellular/biotechnology laboratory providing students, within a framework of bone cell biology, with a modern approach to gene discovery. Students are introduced to the topics of bone cells, bone synthesis, bone resorption, and osteoporosis. They then review the theory of microchip gene arrays, and study microchip array data generated during the differentiation of bone-resorbing osteoclasts in vitro. The class selects genes whose expression increases during osteoclastogenesis, and researches them in small groups using web-based bioinformatics tools. Students then go to a biotechnology company website to find and order small inhibitory RNAs (siRNAs) designed to "knockdown" expression of the gene of interest. Students then learn to transfect these siRNAs into osteoclasts, stimulate the cells to differentiate, assay osteoclast differentiation in vitro, and measure specific gene expression using real-time PCR and immunoblotting. Specific siRNA knockdown resulting in a decrease in osteoclastogenesis is indicative of a gene's physiological relevance. The results are analyzed statistically and presented to the class in groups. In the past 2 years, students identified several genes essential for optimal osteoclast differentiation, including Myo1d. The students hypothesize that the myo1d protein functions in osteoclasts to deliver important proteins to the cell surface via vesicular transport along microfilaments. Student response to the new course was overwhelmingly positive.

False-positive beta-galactosidase staining in osteoclasts by endogenous enzyme: studies in neonatal and month-old wild-type mice.

Escherichia coli beta-galactosidase (beta-gal), encoded by the lacZ gene, has become an essential tool in studies of gene expression and function in higher eukaryotes. lac-Z is widely used as a marker gene to detect expression of transgenes or Cre recombinase driven by tissue-specific promoters. The timing and location of promoter activity is easily visualized in whole embryos or specific tissues using the cleavable, chromogenic substrate, 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-gal). The tissue specificity of promoters in transgenic constructs is routinely tested by using a promoter of choice to drive lacZ. Alternatively, the targeted expression of Cre recombinase to perform in vivo recombination of loxP sites can be visualized by beta-gal staining in mice carrying a Cre-activated lacZ transgene, such as the ROSA26 strain. In the course of our investigations, we examined beta-gal activity in bone tissue from genetically normal mice using standard detection methodology and found very high endogenous activity in bone-resorbing osteoclasts. This was true in frozen, paraffin, and glycol methacrylate sections. X-gal staining colocalized with the osteoclast marker, tartrate-resistant acid phosphatase (TRAP). beta-gal activity was present in osteoclasts in long bones, in the mandible, and in both neonatal and more mature animals. We present this brief article as a caution to those testing genetic models of skeletal gene expression using beta-gal as a marker gene.

Expression of and role for ovarian cancer G-protein-coupled receptor 1 (OGR1) during osteoclastogenesis.

Osteoclasts differentiate from hematopoietic mononuclear precursor cells under the control of both colony stimulating factor-1 (CSF-1, or M-CSF) and receptor activator of NF-kappaB ligand (RANKL, or TRANCE, TNFSF11) to carry out bone resorption. Using high density gene microarrays, we followed gene expression changes in long bone RNA when CSF-1 injections were used to restore osteoclast populations in the CSF-1-null toothless (csf1(tl)/csf1(tl)) osteopetrotic rat. We found that ovarian cancer G-protein-coupled receptor 1 (OGR1, or GPR68) was strongly up-regulated, rising >6-fold in vivo after 2 days of CSF-1 treatments. OGR1 is a dual membrane receptor for both protons (extracellular pH) and lysolipids. Strong induction of OGR1 mRNA was also observed by microarray, real-time RT-PCR, and immunoblotting when mouse bone marrow mononuclear cells and RAW 264.7 pre-osteoclast-like cells were treated with RANKL to induce osteoclast differentiation. Anti-OGR1 immunofluorescence showed intense labeling of RANKL-treated RAW cells. The time course of OGR1 mRNA expression suggests that OGR1 induction is early but not immediate, peaking 2 days after inducing osteoclast differentiation both in vivo and in vitro. Specific inhibition of OGR1 by anti-OGR1 antibody and by small inhibitory RNA inhibited RANKL-induced differentiation of both mouse bone marrow mononuclear cells and RAW cells in vitro, as evidenced by a decrease in tartrate-resistant acid phosphatase-positive osteoclasts. Taken together, these data indicate that OGR1 is expressed early during osteoclastogenesis both in vivo and in vitro and plays a role in osteoclast differentiation.