7 research outputs found
LUMiC(A (R)) Endoprosthetic Reconstruction After Periacetabular Tumor Resection:Short-term Results
Reconstruction of periacetabular defects after pelvic tumor resection ranks among the most challenging procedures in orthopaedic oncology, and reconstructive techniques are generally associated with dissatisfying mechanical and nonmechanical complication rates. In an attempt to reduce the risk of dislocation, aseptic loosening, and infection, we introduced the LUMiC(A (R)) prosthesis (implantcast, Buxtehude, Germany) in 2008. The LUMiC(A (R)) prosthesis is a modular device, built of a separate stem (hydroxyapatite-coated uncemented or cemented) and acetabular cup. The stem and cup are available in different sizes (the latter of which is also available with silver coating for infection prevention) and are equipped with sawteeth at the junction to allow for rotational adjustment of cup position after implantation of the stem. Whether this implant indeed is durable at short-term followup has not been evaluated. (1) What proportion of patients experience mechanical complications and what are the associated risk factors of periacetabular reconstruction with the LUMiC(A (R)) after pelvic tumor resection? (2) What proportion of patients experience nonmechanical complications and what are the associated risk factors of periacetabular reconstruction with the LUMiC(A (R)) after pelvic tumor resection? (3) What is the cumulative incidence of implant failure at 2 and 5 years and what are the mechanisms of reconstruction failure? (4) What is the functional outcome as assessed by Musculoskeletal Tumor Society (MSTS) score at final followup? We performed a retrospective chart review of every patient in whom a LUMiC(A (R)) prosthesis was used to reconstruct a periacetabular defect after internal hemipelvectomy for a pelvic tumor from July 2008 to June 2014 in eight centers of orthopaedic oncology with a minimum followup of 24 months. Forty-seven patients (26 men [55%]) with a mean age of 50 years (range, 12-78 years) were included. At review, 32 patients (68%) were alive. The reverse Kaplan-Meier method was used to calculate median followup, which was equal to 3.9 years (95% confidence interval [CI], 3.4-4.3). During the period under study, our general indications for using this implant were reconstruction of periacetabular defects after pelvic tumor resections in which the medial ilium adjacent to the sacroiliac joint was preserved; alternative treatments included hip transposition and saddle or custom-made prostheses in some of the contributing centers; these were generally used when the medial ilium was involved in the tumorous process or if the LUMiC(A (R)) was not yet available in the specific country at that time. Conventional chondrosarcoma was the predominant diagnosis (n = 22 [47%]); five patients (11%) had osseous metastases of a distant carcinoma and three (6%) had multiple myeloma. Uncemented fixation (n = 43 [91%]) was preferred. Dual-mobility cups (n = 24 [51%]) were mainly used in case of a higher presumed risk of dislocation in the early period of our study; later, dual-mobility cups became the standard for the majority of the reconstructions. Silver-coated acetabular cups were used in 29 reconstructions (62%); because only the largest cup size was available with silver coating, its use depended on the cup size that was chosen. We used a competing risk model to estimate the cumulative incidence of implant failure. Six patients (13%) had a single dislocation; four (9%) had recurrent dislocations. The risk of dislocation was lower in reconstructions with a dual-mobility cup (one of 24 [4%]) than in those without (nine of 23 [39%]) (hazard ratio, 0.11; 95% CI, 0.01-0.89; p = 0.038). Three patients (6%; one with a preceding structural allograft reconstruction, one with poor initial fixation as a result of an intraoperative fracture, and one with a cemented stem) had loosening and underwent revision. Infections occurred in 13 reconstructions (28%). Median duration of surgery was 6.5 hours (range, 4.0-13.6 hours) for patients with an infection and 5.3 hours (range, 2.8-9.9 hours) for those without (p = 0.060); blood loss was 2.3 L (range, 0.8-8.2 L) for patients with an infection and 1.5 L (range, 0.4-3.8 L) for those without (p = 0.039). The cumulative incidences of implant failure at 2 and 5 years were 2.1% (95% CI, 0-6.3) and 17.3% (95% CI, 0.7-33.9) for mechanical reasons and 6.4% (95% CI, 0-13.4) and 9.2% (95% CI, 0.5-17.9) for infection, respectively. Reasons for reconstruction failure were instability (n = 1 [2%]), loosening (n = 3 [6%]), and infection (n = 4 [9%]). Mean MSTS functional outcome score at followup was 70% (range, 33%-93%). At short-term followup, the LUMiC(A (R)) prosthesis demonstrated a low frequency of mechanical complications and failure when used to reconstruct the acetabulum in patients who underwent major pelvic tumor resections, and we believe this is a useful reconstruction for periacetabular resections for tumor or failed prior reconstructions. Still, infection and dislocation are relatively common after these complex reconstructions. Dual-mobility articulation in our experience is associated with a lower risk of dislocation. Future, larger studies will need to further control for factors such as dual-mobility articulation and silver coating. We will continue to follow our patients over the longer term to ascertain the role of this implant in this setting. Level IV, therapeutic study
Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells
<div><p>The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.</p></div
CRISPR/Cas9 baculovirus mediated Cas9 expression in U-2 OS cells.
<p>A) Schematic representation of the recombination of a pAceBac-Cas9 plasmid with a bacmid in EmBacY cells. The resulting bacmids were used for CRISPR/Cas9 baculovirus production in Sf9 cells. B) Representative FACS-profile showing GFP expression in U-2 OS cells treated with CRISPR/Cas9 baculovirus (MOI: 25). C) Western blot showing expression of Cas9 in U-2 OS cells treated with CRISPR/Cas9 baculoviruses (MOI: 25). α-tubulin was used as a loading control.</p
Effect of CRISPR/Cas9 baculoviral transduction in a panel of cell lines.
<p>A) Representative FACS-profiles showing GFP expression in cells treated with Cas9-GFP baculovirus (MOI: 75). The markers are set such that 2% of the cells treated with Cas9-puro baculovirus are included in this region. The percentage of cells treated with Cas9-GFP baculovirus within the marker region is indicated. B) Immunofluorescence images of mitotic cells treated with the indicated Cas9-GFP baculoviruses (MOI: 75) were scored by eye for the presence or absence of centromeric Aurora B. The bars represent the mean ± SD of 2 experiments. At least 24 cells were analyzed per experiment per condition.</p
Introduction of the H250Y mutation in Aurora B.
<p>A) Schematic representation of the introduction of an HDR template carrying point mutations in the pAceBac-Cas9 plasmid. B) Colony formation of HCT116 cells in ZM447439 with cells carrying either wildtype Aurora B alleles, or alleles with homozygous H250Y mutations, or with a heterozygous mutation for H250Y combined with an indel in the other allele. The numbers indicate colony outgrowth in the ZM447439 treated conditions relative to the untreated conditions. Clones were also tested for their capacity to form colonies in the presence of puromycin. C) Sequence chromatograms of the Aurora B locus in the vicinity of H250. Depicted is the Aurora B sequence trace of wildtype HCT116 cells or clones carrying either the homozygous Aurora B H250Y mutation or the heterozygous Aurora B H250Y mutation combined with an indel. The PAM site is indicated and the arrows point out the base substitutions that were introduced. The corresponding amino acid sequence is shown in the top graph. D) Representative immunofluorescence images of prometaphase cells that have either retained or lost histone H3 Serine 10 (H3S10) phosphorylation after treatment with ZM447439 (0.5 μM). E) Quantification of immunofluorescence images of prometaphase cells depicted in (D) Cells were scored for H3S10ph loss following treatment with ZM447439 (0.5 μM). F) Immunofluorescence images of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75) were arrested in mitosis and scored for loss of centromeric Aurora B. G) Representative FACS-profiles showing the DNA content of wildtype HCT116 cells and clones harboring homozygous Aurora B H250Y mutations that were treated with the indicated CRISPR/Cas9 baculoviruses (MOI: 75). Puromycin treatment was used to select for transduced cells and samples were harvested 4 days after transduction.</p
Endogenous tagging of Haspin using CRISPR/Cas9 baculovirus.
<p>A) Schematic representation of the introduction of an HDR template for endogenous tagging of <i>GSG2</i> in the pAceBac-Cas9 plasmid. B) Integration of the YFP-tag at the C-terminus of the gene encoding Haspin was confirmed by PCR using the indicated primer pairs, schematically depicted in Fig 5A. The PCR product obtained using primers 1 and 2 is of the untagged allele, based on its size. C) Representative live cell images of U-2 OS-LacO Haspin-YFP cells and RPE-1 Haspin-YFP cells in prometaphase and interphase. D) Schematic overview depicting the binding of LacI-tagRFP-Pds5B to the LacO repeats. Upon interaction between Pds5B and Haspin an YFP signal can be detected at this ectopic locus. E) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B and stained for DAPI, YFP and RFP. F) Quantification of Haspin-YFP at the LacO locus. Depicted is the mean of Haspin-YFP normalized over RFP ± SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. G) Immunofluorescence images of metaphase spreads of U-2 OS-LacO Haspin-YFP cells expressing LacI-tagRFP or LacI-tagRFP-Pds5B stained for DAPI, H3T3ph and RFP. H) Quantifications of H3T3ph at the LacO locus. Depicted is the mean of H3T3ph levels normalized over RFP ± the SD. Each dot represents a single cell. The data was analyzed using an un-paired Student’s t-test. A minimum of 23 cells was analyzed per experiment.</p