Synthetic CRISPR/Cas9 reagents facilitate genome editing and homology directed repair.

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

Fluoroscopically guided caudal epidural steroid injections for axial low back pain associated with central disc protrusions: a prospective outcome study.

PURPOSE: To determine if axial low back pain (LBP) associated with central disc protrusions can be improved by caudal epidural steroid injections (ESIs). METHODS: Adults with chronic (> 3 months) moderate-to-severe axial LBP with L4-5 and/or L5-S1 central disc protrusions were enrolled in this prospective study. Participants underwent caudal ESIs under standard-of-care practice. The numerical rating scale (NRS) pain score, modified North American Spine Society satisfaction, and Roland Morris Disability Questionnaire (RMDQ) were collected at one week, one month, three months, six months, and one year post-injection. Pre-injection magnetic resonance images were assessed by a musculoskeletal radiologist. RESULTS: Sixty-eight participants (42 males, 26 females) were analyzed. There were statistically significant improvements in all outcome measures at all follow-up time points, with the exception of NRS best pain at six months. Clinically significant improvements in outcomes were observed at various time points: at three months and one year for current pain; at one week, one month, three months, six months, and one year for worst pain; and at one month and one year for RMDQ. The proportion of satisfied participants ranged from 57 to 69% throughout the study. No adverse events were observed. CONCLUSIONS: This study demonstrated significant improvements in pain and function following caudal ESIs in a cohort of axial LBP with associated central disc protrusions. Further studies, including the use of randomized controlled trials, are needed to determine the ideal subset of candidates for this treatment and to explore additional applications that caudal ESIs may have for chronic LBP.

Co-diagnoses of spondylolysis and femoroacetabular impingement: a case series of adolescent athletes.

Locating the source of lumbopelvic-hip pain requires the consideration of multiple clinical pathways. Although low back pain has an incidence of 50% in the adolescent population, the pathophysiology in this population typically differs from that of other age groups. Dynamic mechanical impairments of the hip, such as femoroacetabular impingement, may contribute to the pathogenesis of adolescent low back pain. Eight adolescent male athletes who presented to a single provider with a primary complaint of low back pain with hip pain or motion loss on exam and were ultimately diagnosed with lumbar spondylolysis and dynamic mechanical hip issues between 2009 and 2011 were included. The age at spondylolysis diagnosis ranged from 15 to 19 years (mean ± standard deviation: 16.3 ± 1.3 years). Seven patients had cam-type impingement, whereas one presented with pincer-type impingement. All patients demonstrated either decreased internal rotation at 90 degrees of hip flexion and neutral abduction or pain on the Flexion Adduction Internal Rotation test on at least one of hip. All eight patients were treated initially with 6 weeks of physical therapy consisting of attempted restoration of hip motion and the graduated progression of hip and spine stabilization exercises. Five patients (62.5%) returned to sport at an average of 11.2 weeks (range: 6-16 weeks). For three patients (37.5%), hip pain and motion loss persisted, thus requiring surgery. All subjects had symptoms for at least 6 weeks, with 6 months as the longest duration. This report is the first documented series of adolescent athletes with co-diagnoses of spondylolysis and femoroacetabular impingement. Study Information: This retrospective case series was approved by the Institutional Review Board at Hospital for Special Surgery.

Relationship of Radiographic and Clinical Parameters With Hallux Valgus and Second Ray Pathology.

BACKGROUND: Hallux valgus is frequently associated with additional forefoot pathologies, including hammertoes and midfoot osteoarthritis (OA). However, the pathogenesis of these concurrent pathologies remains to be elucidated. We sought to determine whether there is a relationship between demographic and radiographic parameters and the incidence of secondary pathologies in the setting of a bunion, with an emphasis on second tarsometatarsal (TMT) OA and hammertoes. METHODS: A total of 153 patients (172 feet) who underwent reconstruction for hallux valgus were divided into 3 groups: (1) bunion only (61 patients), (2) bunion with hammertoe without second TMT joint OA (78 patients), and (3) bunion with second TMT joint OA (14 patients). Preoperative age, sex, and body mass index (BMI) as well as hallux valgus angle (HVA), intermetatarsal angle (IMA), metatarsus adductus angle (MAA), ratio of second to first metatarsal length, and Meary's angle were recorded. One-way analysis of variance (normality demonstrated) and Kruskal-Wallis (normality not demonstrated) tests were used to assess differences in continuous variables. Post hoc tests were conducted with the Bonferroni technique. Associations between discrete variables and the study groups were analyzed using χ2 tests. Following the univariate analysis, multinomial logistic regression models were built to determine potential risk factors for hammertoe or TMT OA group placement. RESULTS: Patients in the hammertoe and TMT OA groups were significantly older than patients in the bunion only group ( P < .001 for both pairwise comparisons) and had significantly higher BMIs ( P = .024 and P < .001, respectively). Patients in the TMT OA group had a significantly higher mean HVA than patients in the bunion-only group ( P = .004) and a significantly higher mean MAA relative to both other study groups ( P ≤ .001 for both comparisons). IMA, Meary's angle, and the ratio of second to first metatarsal length did not differ significantly between groups. In the multivariate analysis, hammertoe group assignment was predicted only by age and HVA, while midfoot OA group assignment was predicted by age, HVA, BMI, and MAA. CONCLUSION: Our data show that older age and increased HVA were predictors of both second ray pathologies studied. Higher BMI and MAA were predictive only of TMT joint OA. These data may help identify patients with hallux valgus who are at greater risk for developing secondary pathologies. LEVEL OF EVIDENCE: Level III, retrospective comparative series.

Lumbar Intradiskal Platelet-Rich Plasma (PRP) Injections: A Prospective, Double-Blind, Randomized Controlled Study.

OBJECTIVE: To determine whether single injections of autologous platelet-rich plasma (PRP) into symptomatic degenerative intervertebral disks will improve participant-reported pain and function. DESIGN: Prospective, double-blind, randomized controlled study. SETTING: Outpatient physiatric spine practice. PARTICIPANTS: Adults with chronic (≥6 months), moderate-to-severe lumbar diskogenic pain that was unresponsive to conservative treatment. METHODS: Participants were randomized to receive intradiskal PRP or contrast agent after provocative diskography. Data on pain, physical function, and participant satisfaction were collected at 1 week, 4 weeks, 8 weeks, 6 months, and 1 year. Participants in the control group who did not improve at 8 weeks were offered the option to receive PRP and subsequently followed. MAIN OUTCOME MEASURES: Functional Rating Index (FRI), Numeric Rating Scale (NRS) for pain, the pain and physical function domains of the 36-item Short Form Health Survey, and the modified North American Spine Society (NASS) Outcome Questionnaire were used. RESULTS: Forty-seven participants (29 in the treatment group, 18 in the control group) were analyzed by an independent observer with a 92% follow-up rate. Over 8 weeks of follow-up, there were statistically significant improvements in participants who received intradiskal PRP with regards to pain (NRS Best Pain) (P = .02), function (FRI) (P = .03), and patient satisfaction (NASS Outcome Questionnaire) (P = .01) compared with controls. No adverse events of disk space infection, neurologic injury, or progressive herniation were reported following the injection of PRP. CONCLUSION: Participants who received intradiskal PRP showed significant improvements in FRI, NRS Best Pain, and NASS patient satisfaction scores over 8 weeks compared with controls. Those who received PRP maintained significant improvements in FRI scores through at least 1 year of follow-up. Although these results are promising, further studies are needed to define the subset of participants most likely to respond to biologic intradiskal treatment and the ideal cellular characteristics of the intradiskal PRP injectate.