Impact of the functional status of saeRS on in vivo phenotypes of Staphylococcus aureus†sarA mutants.

We investigated the in vivo relevance of the impact of sarA and saeRS on protease production using derivatives of the USA300 strain LAC. The results confirmed that mutation of saeRS or sarA reduces virulence in a bacteremia model to a comparable degree. However, while eliminating protease production restored virulence in the sarA mutant, it had little impact in the saeRS mutant. Additionally, constitutive activation of saeRS (saeRS(C)) enhanced the virulence of LAC and largely restored virulence in the isogenic sarA mutant. Based on these results, together with our analysis of the representative virulence factors alpha toxin, protein A (Spa), and extracellular nucleases, we propose a model in which the attenuation of saeRS mutants is defined primarily by decreased production of such factors, while constitutive activation of saeRS increases virulence, and reverses the attenuation of sarA mutants, because it results in both increased production and decreased protease-mediated degradation of these same factors. This regulatory balance was also apparent in a murine model of catheter-associated infection, with the results suggesting that the impact of saeRS on nuclease production plays an important role during the early stages of these infections that is partially offset by increased protease production in sarA mutants.

BB0238, a presumed tetratricopeptide repeat-containing protein, is required during Borrelia burgdorferi mammalian infection.

The Lyme disease spirochete, Borrelia burgdorferi, occupies both a tick vector and mammalian host in nature. Considering the unique enzootic life cycle of B. burgdorferi, it is not surprising that a large proportion of its genome is composed of hypothetical proteins not found in other bacterial pathogens. bb0238 encodes a conserved hypothetical protein of unknown function that is predicted to contain a tetratricopeptide repeat (TPR) domain, a structural motif responsible for mediating protein-protein interactions. To evaluate the role of bb0238 during mammalian infection, a bb0238-deficient mutant was constructed. The bb0238 mutant was attenuated in mice infected via needle inoculation, and complementation of bb0238 expression restored infectivity to wild-type levels. bb0238 expression does not change in response to varying culture conditions, and thus, it appears to be constitutively expressed under in vitro conditions. bb0238 is expressed in murine tissues during infection, though there was no significant change in expression levels among different tissue types. Localization studies indicate that BB0238 is associated with the inner membrane of the spirochete and is therefore unlikely to promote interaction with host ligands during infection. B. burgdorferi clones containing point mutations in conserved residues of the putative TPR motif of BB0238 demonstrated attenuation in mice that was comparable to that in the bb0238 deletion mutant, suggesting that BB0238 may contain a functional TPR domain.

sarA-mediated repression of protease production plays a key role in the pathogenesis of Staphylococcus aureus USA300 isolates.

Mutation of staphylococcal accessory regulator (sarA) results in increased production of extracellular proteases in Staphylococcus aureus, which has been correlated with decreased biofilm formation and decreased accumulation of extracellular toxins. We used murine models of implant-associated biofilm infection and S. aureus bacteraemia (SAB) to compare virulence of USA300 strain LAC, its isogenic sarA mutant, and derivatives of each of these strains with mutations in all 10 of the genes encoding recognized extracellular proteases. The sarA mutant was attenuated in both models, and this was reversed by eliminating production of extracellular proteases. To examine the mechanistic basis, we identified proteins impacted by sarA in a protease-dependent manner. We identified 253 proteins where accumulation was reduced in the sarA mutant compared with the parent strain, and was restored in the sarA/protease mutant. Additionally, in SAB, the LAC protease mutant exhibited a hypervirulent phenotype by comparison with the isogenic parent strain, demonstrating that sarA also positively regulates production of virulence factors, some of which are subject to protease-mediated degradation. We propose a model in which attenuation of sarA mutants is defined by their inability to produce critical factors and simultaneously repress production of extracellular proteases that would otherwise limit accumulation of virulence factors.

Use of xylitol to enhance the therapeutic efficacy of polymethylmethacrylate-based antibiotic therapy in treatment of chronic osteomyelitis.

Using a rabbit model of postsurgical osteomyelitis, we demonstrate that incorporation of xylitol into polymethylmethacrylate (PMMA) bone cement enhances the elution of daptomycin under in vivo conditions. We also demonstrate that this can be correlated with an improved therapeutic outcome in the treatment of a chronic bone infection following surgical debridement.

Inhibin A is an endocrine stimulator of bone mass and strength.

Gonadal function plays a major role in bone homeostasis. It is widely held that the skeletal consequences of hypogonadism are solely due to a loss of sex steroids; however, increases in bone turnover begin during perimenopause before decreases in serum estradiol levels. These data and our demonstration that inhibins acutely regulate bone cell differentiation in vitro led us to test whether inhibin A (InhA) regulates bone mass in vivo. Using a transgenic model of inducible human InhA expression, InhA increased total body bone mineral density, increased bone volume, and improved biomechanical properties at the proximal tibia in intact mice and also prevented the loss of BMD and bone volume and strength associated with gonadectomy at both the spine and proximal tibia. In addition, InhA increased mineral apposition rate, double-labeled surface, and serum osteocalcin levels in vivo and osteoblastogenesis ex vivo without affecting osteoclast number or activity. Together these results demonstrate novel stimulatory effects of InhA on the skeleton in vivo. These studies provide in vivo evidence demonstrating that gonadal factors other than sex steroids play an important role in regulating bone mass and strength and, combined with our previous clinical data, suggest that gonadal InhA may be a component of the normal endocrine repertoire that regulates bone quality in both the axial and appendicular skeleton.

Tumor-derived interleukin-8 stimulates osteolysis independent of the receptor activator of nuclear factor-kappaB ligand pathway.

Bone is a common site of cancer metastasis. Breast, prostate, and lung cancers show a predilection to metastasize to bone. Recently, we reported that the chemokine interleukin 8 (IL-8) stimulates both human osteoclast formation and bone resorption. IL-8 mRNA expression was surveyed in a panel of human breast cancer lines MDA-MET, MDA-MB-231, MDA-MB-435, MCF-7, T47D, and ZR-75, and the human lung adenocarcinoma cell line A549. IL-8 mRNA expression was higher in cell lines with higher osteolytic potential in vivo. Human osteoclast formation was increased by MDA-MET or A549 cell-conditioned medium, but not by MDA-MB-231. Pharmacologic doses of receptor activator of nuclear factor-kappaB (RANK)-Fc or osteoprotogerin had no effect on the pro-osteoclastogenic activity of the conditioned medium; however, osteoclast formation stimulated by conditioned medium was inhibited 60% by an IL-8-specific neutralizing antibody. The data support a model in which tumor cells cause osteolytic bone destruction independently of the RANK ligand (RANKL) pathway. Tumor-produced IL-8 is a major contributor to this process. The role of secreted IL-8 isoforms was examined by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, which detected distinct IL-8 isoforms secreted by MDA-MET and MDA-231 cells, suggesting different pro-osteoclastogenic activities of the two IL-8-derived peptides. These data indicate that (a) osteoclast formation induced by MDA-MET breast cancer cells and A549 adenocarcinoma cells is primarily mediated by IL-8, (b) cell-specific isoforms of IL-8 with distinct osteoclastogenic activities are produced by tumor cells, and (c) tumor cells that support osteoclast formation independent of RANKL secrete other pro-osteoclastogenic factors in addition to IL-8.

Utility of an allograft tendon for scoliosis correction via the costo-transverse foreman.

Current convex tethering techniques for treatment of scoliosis have centered on anterior convex staples or polypropylene tethers. We hypothesized that an allograft tendon tether inserted via the costo-transverse foramen would correct an established spinal deformity. In the pilot study, six 8-week-old pigs underwent allograft tendon tethering via the costo-transverse foreman or sham to test the strength of the transplanted tendon to retard spine growth. After 4 months, spinal deformity in three planes was induced in all animals with allograft tendons. In the treatment study, the allograft tendon tether was used to treat established scoliosis in 11 8-week-old pigs (spinal deformity > 50°). Once the deformity was observed (4 months) animals were assigned to either no treatment group or allograft tendon tether group and progression assessed by monthly radiographs. At final follow-up, coronal Cobb angle and maximum vertebral axial rotation of the treatment group was significantly smaller than the non-treatment group, whereas sagittal kyphosis of the treatment group was significantly larger than the non-treatment group. In sum, a significant correction was achieved using a unilateral allograft tendon spinal tether, suggesting that an allograft tendon tethering approach may represent a novel fusion-less procedure to correct idiopathic scoliosis. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:183-192, 2017.

Different molecular mechanisms underlie ethanol-induced bone loss in cycling and pregnant rats.

Chronic ethanol (EtOH) consumption can result in osteopenia. In the current study, we examined the modulation of EtOH-induced bone loss during pregnancy. Nonpregnant and pregnant dams were intragastrically infused either control or EtOH-containing diets throughout gestation (gestation d 5 through 20 or an equivalent period of 15 d) by total enteral nutrition. The effects of EtOH (8.5 to 14 g/kg/d) on tibial bone mineral density (BMD), mineral content (BMC), and bone mineral area were assessed at gestation d 20 via peripheral quantitative computerized tomography. EtOH caused a dose-dependent decrease in BMD and BMC without affecting bone mineral area. Trabecular BMD and BMC were significantly lower in EtOH-treated, nonpregnant dams, compared with pregnant cohorts at the same infused dose of EtOH and urinary ethanol concentrations. Static histomorphometric analysis of tibiae from pregnant rats after EtOH treatment showed decreased osteoblast and osteoid surface, indicating inhibited bone formation, whereas EtOH-treated cycling rats showed higher osteoclast and eroded surface, indicative of increased bone resorption. Circulating osteocalcin and 1,25-dihydroxyvitamin D3 were lower in both EtOH-fed nonpregnant and pregnant rats. Gene expression of osteoclast markers, 70 kDa v-ATPase, and tartrate-resistant acid phosphatase were increased selectively in nonpregnant EtOH-treated rats but not pregnant rats. Moreover, only nonpregnant EtOH-fed rats showed induction in bone marrow receptor activator of nuclear factor-kappaB ligand mRNA and decreased circulating 17beta-estradiol levels. Our data suggest that EtOH-induced bone loss in pregnant rats is mainly due to inhibited bone formation, whereas in nonpregnant rats, the data are consistent with increased osteoclast activation and bone resorption concomitant with decreased estradiol levels.

Bone formation is impaired in a model of type 1 diabetes.

The effects of type 1 diabetes on de novo bone formation during tibial distraction osteogenesis (DO) and on intact trabecular and cortical bone were studied using nonobese diabetic (NOD) mice and comparably aged nondiabetic NOD mice. Diabetic mice received treatment with insulin, vehicle, or no treatment during a 14-day DO procedure. Distracted tibiae were analyzed radiographically, histologically, and by microcomputed tomography (microCT). Contralateral tibiae were analyzed using microCT. Serum levels of insulin, osteocalcin, and cross-linked C-telopeptide of type I collagen were measured. Total new bone in the DO gap was reduced histologically (P < or = 0.001) and radiographically (P < or = 0.05) in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice (P < or = 0.001) and normalized with insulin treatment. Evaluation of the contralateral tibiae by microCT and mechanical testing demonstrated reductions in trabecular bone volume and thickness, cortical thickness, cortical strength, and an increase in endosteal perimeter in diabetic animals, which were prevented by insulin treatment. These studies demonstrate that bone formation during DO is impaired in a model of type 1 diabetes and preserved by systemic insulin administration.

A novel mouse model for the study of the inhibitory effects of chronic ethanol exposure on direct bone formation.

Excessive alcohol consumption has been reported to interfere with human bone homeostasis and repair in multiple ways. Previous studies have demonstrated that chronic ethanol exposure in the rat via an intragastric dietary delivery system inhibits direct bone formation during distraction osteogenesis (DO, limb lengthening). The opportunity to extend the rat ethanol studies to mice is now possible due to the development of mouse models of DO. This study employed a novel combination of liquid ethanol diet delivery and a murine DO model to test the hypothesis that chronic ethanol exposure would result in deficits in direct bone formation during DO in contrast to the pair-fed controls. Twenty-eight 12-month-old C57BL/6 male mice were acclimated to the Lieber-DeCarli liquid control diet #710027 (Dyets Inc.) over a 1-week period. The mice were separated into two diet groups (n=14/group): pair-fed control and ethanol (diet #710260). After being on diet for 82 days, all mice underwent placement of an external fixator and osteotomy on the left tibia. Following a 6-day latency period, distraction began at a rate of 0.075 mm twice a day (b.i.d.) for 14 days. The weight changes were equivalent for both groups. The hypothesis that chronic ethanol exposure would inhibit direct bone formation and produce skeletal toxicity was supported by radiographic (P=.011) and histologic (P=.002) analyses of the % new bone formation in the DO gaps, by peripheral quantitative computed tomography analysis of the total volumetric bone mineral density of the contralateral proximal tibias (P<.001) and contralateral femoral necks (P=.012), by three-point bending on the contralateral tibias (P<.001 energy to break), by pin site bone formation measures (P<.001), and by ethanol-associated increased adipocyte area (adjacent to the gap) percentages (P<.002). We conclude that this model can be used to study the mechanisms underlying inhibition of bone formation by chronic ethanol exposure and to test preclinical interventions.

Expression of interleukin 8 and not parathyroid hormone-related protein by human breast cancer cells correlates with bone metastasis in vivo.

Metastasis is the process by which tumor cells spread from their site of origin to distant sites after gaining access to the circulatory system. An understanding of the factors contributing to the metastatic potential of breast cancer cells to bone will enhance the prospect of developing new therapies that impede metastasis. In this study, we have used an in vivo selection scheme involving left cardiac ventricle injection into nude mice to identify a highly metastatic human breast cancer cell line (MDA-MET) from a less metastatic (MDA-231) parental cell line. In this model, tumor-bearing mice exhibit features similar to those associated with human metastatic bone disease such as osteolytic bone destruction. After inoculation, MDA-MET cells form devastating lesions within 4 weeks, whereas the parental cells do not, even after 10 weeks. In vitro, the MDA-MET cells have a similar growth rate to the parental MDA-231 cells yet demonstrate distinct adhesive and invasive phenotypes. MDA-MET cells show increased early adhesion to type IV collagen and are significantly more invasive through Matrigel than MDA-231 cells. Analysis of the gene expression profile in the metastatic MDA-MET versus poorly metastatic MDA-231 cells identified relatively few genes whose expression was altered >2-fold. Of particular interest was the lack of parathyroid hormone-related protein (PTHrP) mRNA expression, which was supported at the protein level by immunoradiometric assay. These data support the idea that PTHrP is not predictive of the metastasis of human breast cancer to bone. Another important difference between the two cell lines was the elevated expression by MDA-MET cells of the cytokine IL-8. Reverse transcriptase-PCR and ELISA confirmed the increased expression of IL-8 in MDA-MET cells. In addition, IL-8 mRNA expression is also elevated in a variety of human cancer cell lines with different metastatic potential in vivo. These experiments suggest that the elevated expression of IL-8 (and not PTHrP) by MDA-MET cells is a phenotypic change that may be related to their enhanced ability to metastasize to the skeleton.

Antibiotic-loaded phosphatidylcholine inhibits staphylococcal bone infection.

AIM: To test antibiotic-loaded coating for efficacy in reducing bacterial biofilm and development of osteomyelitis in an orthopaedic model of implant infection. METHODS: Phosphatidylcholine coatings loaded with 25% vancomycin were applied to washed and sterilized titanium wires 20 mm in length. A 10 mm segment was removed from rabbit radius (total = 9; 5 coated, 4 uncoated), and the segment was injected with 1 × 10(6) colony forming units (CFUs) of Staphylococcus aureus (UAMS-1 strain). Titanium wires were inserted through the intramedullary canal of the removed segment and into the proximal radial segment and the segment was placed back into the defect. After 7 d, limbs were removed, X-rayed, swabbed for tissue contamination. Wires were removed and processed to determine attached CFUs. Tissue was swabbed and streaked on agar plates to determine bacteriological score. RESULTS: Antibiotic-loaded coatings resulted in significantly reduced biofilm formation (4.7 fold reduction in CFUs; P < 0.001) on titanium wires and reduced bacteriological score in surrounding tissue (4.0 ± 0 for uncoated, 1.25 ± 0.5 for coated; P = 0.01). Swelling and pus formation was evident in uncoated controls at the 7 d time point both visually and radiographically, but not in antibiotic-loaded coatings. CONCLUSION: Active antibiotic was released from coated implants and significantly reduced signs of osteomyelitic symptoms. Implant coatings were well tolerated in bone. Further studies with additional control groups and longer time periods are warranted. Antibiotic-loaded phosphatidylcholine coatings applied at the point of care could prevent implant-associated infection in orthopaedic defects.

Nutlin-3 treatment spares cisplatin-induced inhibition of bone healing while maintaining osteosarcoma toxicity.

The majority of Osteosarcoma (OS) patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. These protocols include distraction osteogenesis (DO), which is characterized by direct new bone formation. Cisplatin (CDP) is extensively used for OS chemotherapy and recent studies, using a mouse DO model, have demonstrated that CDP has profound negative effects on bone repair. Recent oncological therapeutic strategies are based on the use of standard cytotoxic drugs plus an assortment of biologic agents. Here we demonstrate that the previously reported CDP-associated inhibition of bone repair can be modulated by the administration of a small molecule p53 inducer (nutlin-3). The effects of nutlin-3 on CDP osteotoxicity were studied using both pre- and post-operative treatment models. In both cases the addition of nutlin-3, bracketing CDP exposure, demonstrated robust and significant bone sparing activity (p < 0.01-0.001). In addition the combination of nutlin-3 and CDP induced equivalent OS tumor killing in a xenograft model. Collectively, these results demonstrate that the induction of p53 peri-operatively protects bone healing from the toxic effects of CDP, while maintaining OS toxicity. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1716-1724, 2016.

Early detection of bone infection and differentiation from post-surgical inflammation using 2-deoxy-2-[18F]-fluoro-D-glucose positron emission tomography (FDG-PET) in an animal model.

Diagnosing bone infection in the context of post-surgical inflammation is problematic since many of the early signs of infection are similar to normal post-surgical changes. We used a rabbit osteomyelitis model to evaluate the use of 2-deoxy-2-[(18)F]-fluoro-d-glucose positron emission tomography (FDG-PET) as a means of detecting post-operative infection in the context of post-surgical inflammation. Comparisons were made between infected and non-infected rabbits in which infection with Staphylococcus aureus was initiated at the time of surgery. Weekly PET scans were obtained 30 and 60 min after the introduction of FDG and analyzed based on standardized uptake values (SUV) at the surgical site and visual assessment of the presence or absence of infection. Concurrent X-rays were taken immediately prior to scanning. At 4weeks post-operatively, animals were sacrificed for histologic and bacteriologic confirmation of infection. Uptake of FDG was evident in the bone of all rabbits on day 1 post-surgery, however, SUV comparisons from the surgical site could not be used to distinguish between the infected and uninfected groups until day 15. Visual analysis of FDG-PET scans revealed a significant difference (p<0.01) between the infected and uninfected groups as early as day 8. This was due in part to the ability to visualize regional lymph nodes by FDG-PET.

Immunohistochemical study of osteopontin expression during distraction osteogenesis in the rat.

Distraction osteogenesis (DO) is a limb-lengthening procedure that combines mechanical tension stress with fracture healing to provide a unique opportunity for detailed histological examination of bone formation. Osteopontin (OPN) is a multifunctional matricellular protein believed to play a key role in wound healing and cellular response to mechanical stress. We studied the expression of OPN during DO using standard immunohistochemical (IHC) staining techniques. In addition, we compared the expression of OPN to proliferation (PCNA-positive cells) in the DO gap. After 14 days of distraction in the rat, these stains revealed variations in OPN expression and its relationship to proliferation according to the cell type, tissue type, and mode of ossification examined. Fibroblast-like cells within the central fibrous area exhibited intermittent low levels of OPN, but no relationship was observed between OPN and proliferation. In areas of transchondral ossification, OPN expression was very high in the morphologically intermediate oval cells. During intramembranous ossification, osteoblasts appeared to exhibit a bimodal expression of OPN. Specifically, proliferating pre-osteoblasts expressed osteopontin, but OPN was not detected in the post-proliferative pre-osteoblasts/osteoblasts that border the new bone columns. Finally, intracellular OPN was detected in virtually all of the mature osteoblasts/osteocytes within the new bone columns, while detection of OPN in the matrix of the developing bone columns may increase with the maturity of the new bone. These results imply that the expression of OPN during DO may be more similar to that seen during embryogenesis than would be expected from other studies. Furthermore, the biphasic expression of OPN during intramembranous ossification may exemplify the protein's multi-functional role. Early expression may facilitate pre-osteoblastic proliferation and migration, while the latter downregulation may be necessary for hydroxyapatite crystal formation.

Use of a B cell marker (B220) to discriminate between allergens and irritants in the local lymph node assay.

It has been shown that exposure of mice to contact allergens induces B cell activation in the draining lymph nodes (DLN), as seen by an increase in the percentage of B220+ or IgG/IgM+ cells. We have now examined whether the measurement of the percentage of B220+ cells could be used as an alternative or supplementary endpoint for the local lymph node assay (LLNA) to differentiate between allergenic responses and those few irritants that induce low-level proliferation in the DLN. Mice were treated on the ears, daily for 3 consecutive days, with various allergens (1-chloro-2,4-dinitrobenzene, alpha-hexylcinnamaldehyde, trinitrochlorobenzene, isoeugenol, and eugenol) or irritants (benzalkonium chloride, methyl salicylate, salicylic acid, and sodium lauryl sulfate). The DLN were excised 72 h following the final topical treatment, and the cells were prepared for B220 analysis using flow cytometry. The percentage of B220+ cells in lymph nodes derived from test and vehicle-treated animals was determined for 5 allergens and 4 irritants tested in multiple experiments (n = 3 to 17). As expected, the percentage of B220+ B cells was increased with each of the allergens tested, whereas irritant treatment did not cause similar increases. Moreover, the method was reproducible. For example, the strong allergen, 1-chloro-2,4-dinitrobenzene and the weak allergen, alpha-hexylcinnamaldehyde were identified as allergens in 17 of 17 and in 12 of 13 experiments, respectively. The percentage of B220 values for each chemical treatment (41 observations for allergens; 28 observations for irritants) versus the percentage of B220 values for the concurrent vehicle controls were plotted, and a classification tree model was developed that defined a B220 test:vehicle ratio cutoff of 1.25 for discriminating between allergens (>1.25) and irritants (<1.25). Using this B220 test:vehicle ratio of 1.25 in 93% of the 69 independent observations made, the allergens and irritants tested were identified correctly. Finally, to evaluate the performance of this model in a second independent laboratory, 3 allergens and 2 irritants were tested. Each of the allergens and irritants were classified correctly using the B220 test:vehicle ratio cutoff of 1.25. These data demonstrate that analysis of B220 expression in DLN may be useful in differentiating between allergen and irritant responses induced in chemically treated mice.

The treatment of experimental osteomyelitis by surgical debridement and the implantation of calcium sulfate tobramycin pellets.

Calcium sulfate was used as a biodegradable delivery system for the administration of antibiotics in musculoskeletal infection. New Zealand white rabbits were infected with Staplylococcus aureus, debrided, and randomized to one of four treatment groups: calcium sulfate pellets with 10% tobramycin sulfate, placebo calcium sulfate pellets and IM tobramycin, placebo calcium sulfate pellets, or debridement. Serum and wound exudate tobramycin concentrations and serum calcium levels were measured. Radiographs, cultures, and histology were analyzed for efficacy and treatment. Rabbits treated with 10% tobramycin sulfate pellets showed a significantly higher eradication of infection (11/13) than rabbits treated with debridement only (5/12), placebo pellets and IM tobramycin (5/14). or placebo pellets (3/13). In the group receiving 10% tobramycin sulfate pellets, serum tobramycin concentrations peaked 3 h post-operatively at 5.87 microg/ml and were non-detectable after day 1. In the group receiving placebo pellets and IM tobramycin, serum concentrations peaked at 7.82 microg/ml 1 h post-operatively, fell to 6.12 microg/ml on day 2, and averaged 4.18 microg/ ml for the remainder of the treatment period. The wound exudate tobramycin concentrations in the animals treated with tobramycin sulfate pellets peaked at 11.9 mg/ml on day 1 and dropped to 2.5 microg/ml on day 7. There was no significant difference in the serum calcium levels in any of the treatment groups. Calcium sulfate containing tobramycin sulfate has potential utility as a biodegradable local antibiotic delivery system in the treatment of musculoskeletal infections.

Chitosan coating to enhance the therapeutic efficacy of calcium sulfate-based antibiotic therapy in the treatment of chronic osteomyelitis.

We demonstrate that coating calcium sulfate with deacetylated chitosan enhances the elution profile of daptomycin by prolonging the period during which high concentrations of antibiotic are released. Coatings reduced initial bolus release of daptomycin by a factor of 10 to approximately 1000 µg/ml, and levels remained above 100 µg/ml for up to 10 days. Chitosan-coated and uncoated calcium sulfate implants with and without 15% daptomycin were evaluated in an experimental model of staphylococcal osteomyelitis through bacteriology scores, radiology, histopathology, and Gram staining. Significant reduction in bacteriology scores was observed for implants containing daptomycin and coated with chitosan compared with all the other groups. We confirm that the use of chitosan-coated calcium sulfate beads for local antibiotic delivery can be correlated with an improved therapeutic outcome following surgical debridement in the treatment of chronic osteomyelitis.

Sc65 is a novel endoplasmic reticulum protein that regulates bone mass homeostasis.

Members of the Leprecan family of proteins include enzymes, prolyl 3-hydroxylase 1 (P3h1), P3h2, and P3h3, and nonenzymatic proteins, Crtap and Sc65. Mutations in CRTAP and LEPRE1 (encoding P3H1) have been associated with human disease such as recessive osteogenesis imperfecta; however, the function of Sc65, which is closely related and highly homologous to Crtap, is unknown. Sc65 has been described as a synaptonemal complex protein, a nucleolar protein, and a cytoplasmic adapter protein. In light of its high sequence similarity with Crtap, an endoplasmic reticulum (ER)-associated protein, and the importance of post-translational modifications such as collagen prolyl 3-hydroxylation in bone metabolism, we hypothesized that Sc65 was an ER-resident protein that would have an important role in bone homeostasis. In this study, we demonstrate that Sc65 is a previously unrecognized ER protein and that it does not localize in the nucleus of somatic cells. Moreover, Sc65 is expressed and functional during skeletal development because loss of Sc65 results in a progressive osteopenia that affects both trabecular and cortical bone. Bone loss is the result of increased bone resorption mediated by a non-cell-autonomous effect on osteoclasts. Therefore, Sc65, like its related family member Crtap, is an important modulator of bone homeostasis, acting as a negative regulator of osteoclastogenesis.

Cisplatin inhibits bone healing during distraction osteogenesis.

Osteosarcoma (OS) is the most common malignant bone tumor affecting children and adolescents. Many patients are treated with a combination of chemotherapy, resection, and limb salvage protocols. Surgical reconstructions after tumor resection include structural allografts, non-cemented endoprostheses, and distraction osteogenesis (DO), which require direct bone formation. Although cisplatin (CDP) is extensively used for OS chemotherapy, the effects on bone regeneration are not well studied. The effects of CDP on direct bone formation in DO were compared using two dosing regimens and both C57BL/6 (B6) and tumor necrosis factor receptor 1 knockout (TNFR1KO) mice, as CDP toxicity is associated with elevated TNF levels. Detailed evaluation of the five-dose CDP regimen (2 mg/kg/day), demonstrated significant decreases in new bone formation in the DO gaps of CDP treated versus vehicle treated mice (p < 0.001). Further, no significant inhibitory effects from the five-dose CDP regimen were observed in TNFR1KO mice. The two-dose regimen significantly inhibited new bone formation in B6 mice. These results demonstrate that CDP has profound short term negative effects on the process of bone repair in DO. These data provide the mechanistic basis for modeling peri-operative chemotherapy doses and schedules and may provide new opportunities to identify molecules that spare normal cells from the inhibitory effects of CDP.