David E. Komatsu

Enhanced Bone Regeneration Associated With Decreased Apoptosis in Mice With Partial HIF-1α Deficiency

HIF‐1α activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HIF‐1α+/− fracture calluses are larger, stronger, and stiffer than HIF‐1α+/+ calluses because of decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration.

Molecular cloning and characterization of Mustang, a novel nuclear protein expressed during skeletal development and regeneration

Bone regeneration occurs as a series of events that requires temporal and spatial orchestration of numerous cell types guided by the transcriptional activity of thousands of genes, as recently demonstrated by our laboratory. Using the rat femoral fracture model, bioinformatics, cloning, expression assays, fusion proteins, and transfection, we report on the identification and characterization of one such differentially expressed gene, termed Mustang (musculoskeletal temporally activated novel gene). Mustang encodes for an 82 amino acid nuclear protein with no homology to any known protein family. However, other species homologues (mouse, human, cow) were identified within EST (expressed sequence tag) databases. Nuclear localization was confirmed using a GFP‐Mustang fusion protein. Using in situ hybridization, Mustang expression was localized to differentiating periosteal osteogenic cells, proliferating chondrocytes, and osteoblasts of the fracture callus. Unlike adult tissues, developing embryos abundantly express Mustang, especially in mesenchymal condensations of limbs, vertebral perichondrium, and mesenchymal cells of the intervertebral discs. Although the precise function of Mustang is unknown, its unique pattern of expression during bone development and regeneration, absence in adult tissues (except skeletal muscle and tendon), and nuclear localization suggest that Mustang is involved in the development and regeneration of the mammalian musculoskeletal system.—Lombardo, F., Komatsu, D., Hadjiargyrou, M. Molecular cloning and characterization of Mustang, a novel nuclear protein expressed during skeletal development and regeneration.—Lombardo, F., Komatsu, D., Hadjiargyrou, M. Molecular cloning and characterization of Mustang, a novel nuclear protein expressed during skeletal development and regeneration. FASEB J. 18, 52–61 (2004)

A pharmacokinetic model of oral methylphenidate in the rat and effects on behavior.

Most animal studies using methylphenidate (MP) do not administer it the same way it is administered clinically (orally), but rather by injection, resulting in an altered pharmacokinetic profile (quicker and higher peak concentrations). We evaluated several oral-dosing regimens in rats, including dual-dose drinking, to mimic clinical drug delivery. Using an 8-hour-limited-access-drinking-paradigm, MP solutions were delivered at different doses (20, 30, or 60mg/kg/day; as well as dual-dosages of 4 and 10mg/kg/day, 20 and 30mg/kg/day, or 30 and 60mg/kg/day, in which the low dose was administered in the first hour of drinking followed by 7 h of drinking the high dose). Plasma was assayed for MP levels at many time points. Results showed that an 8-hour limited drinking of a dual-dosage 30/60mg/kg MP solution achieved a pharmacokinetic profile similar to clinically administered doses of MP at the high end of the spectrum (peaking at ~30ng/mL), while the 4/10mg/kg MP dual-dosage produced plasma levels in the range produced by typically prescribed clinical doses of MP (peaking at ~8ng/mL). Treatment with the higher dual-dosage (HD: 30/60mg/kg) resulted in hyperactivity, while the lower (LD: 4/10mg/kg) had no effect. Chronic effects of these dual-dosages were assessed throughout three months of treatment and one month of abstinence, beginning in adolescence. MP dose-dependently decreased body weight, which remained attenuated throughout abstinence. MP decreased food intake during early treatment, suggesting that MP may be an appetite suppressant and may also speed metabolism and/or suppress growth. Chronic HD MP resulted in hyperactivity limited during the dark cycle, decreased exploratory behavior, and increased anxiolytic behavior. Findings suggest that these dual-dosage-drinking-paradigms can be used to examine the effects of clinically relevant pharmacokinetic doses of MP and that chronic treatment with such dosages can result in long-lasting developmental and behavioral changes.

Chronic exposure to methylphenidate impairs appendicular bone quality in young rats

Methylphenidate (MP) is a psychostimulant widely prescribed to treat Attention Deficit Hyperactivity Disorder (ADHD). Although generally well tolerated, growth deficits have been reported in children and adolescents undergoing MP treatment. This study was designed to elucidate the skeletal effects of chronic MP administration in adolescent rats. Male, 4-week-old rats received one of two doses of MP (MP-Low or MP-High) delivered for 8 h a day via drinking water, or were untreated (water only). After 13 weeks, half were sacrificed (N=12/group) and the remaining rats were left to recover, untreated for 5 additional weeks. Femora, tibiae, and L5 vertebra were analyzed using calipers, DXA, and mechanical testing. Immediately following treatment, MP decreased femoral anterior-posterior diameter (5% and 9% for MP-Low and MP-High, respectively), femoral and tibial bone mineral density (BMD) (6% and 5% for MP-High femora and tibiae, respectively), and bone mineral content (BMC) (9% for MP-High femora and tibiae). In addition, femora from MP treated rats had reduced ultimate force (20% for MP-High) and energy to failure (20% and 33% for MP-Low and MP-High, respectively). However, after recovery, there were no statistically significant differences for any measured parameters. Despite these effects on the appendicular skeleton, no differences were identified between vertebral samples at either time-point. In summary, MP treatment resulted in smaller, less mineralized, and weaker bones at appendicular sites, but did not affect the axial site. Although these effects were ameliorated within 5 weeks, these data suggest that adolescents undergoing MP treatment may be at an increased risk for long bone fractures.

Identification of the microRNA transcriptome during the early phases of mammalian fracture repair.

Fracture repair is a complex process that involves multiple biological processes requiring spatiotemporal expression of thousands of genes. The molecular regulation of this process is not completely understood. MicroRNAs (miRNAs) regulate gene expression by promoting mRNA degradation or blocking translation. To identify miRNAs expressed during fracture repair, we generated murine bone fractures and isolated miRNA-enriched RNA from intact and post-fracture day (PFD) 1, 3, 5, 7, 11, and 14 femurs. RNA samples were individually hybridized to mouse miRNA microarrays. Results indicated that 959 (51%) miRNAs were absent while 922 (49%) displayed expression in at least one sample. Of the 922 miRNAs, 306 (33.2%) and 374 (40.6%) were up- and down-regulated, respectively, in the calluses in comparison to intact bone. Additionally, 20 (2.2%) miRNAs displayed combined up- and down-regulated expression within the time course and the remaining 222 (24%) miRNAs did not exhibit any changes between calluses and intact bone. Quantitative-PCR validated the expression of several miRNAs. Further, we identified 2048 and 4782 target genes that were unique to the up- and down-regulated miRNAs, respectively. Gene ontology and pathway enrichment analyses indicated relevant biological processes. These data provide the first complete analysis of the miRNA transcriptome during the early phases of fracture repair.

Biomechanical Comparison of Spinopelvic Fixation Constructs: Iliac Screw Versus S2-Alar-Iliac Screw

STUDY DESIGN Biomechanical cadaveric study. OBJECTIVE To compare the biomechanical properties of the iliac and S2-Alar-Iliac (S2AI) screw in a similar spinopelvic fixation construct. SUMMARY OF BACKGROUND DATA Spinopelvic fixation is used in the correction of pelvic obliquity, high-grade spondylolisthesis, and long spinal fusions. With the development of pedicle screw fixation, the iliac screw has been used as an anchor point to the pelvis. The associated morbidity with this fixation has led to the development of the S2AI screw. Many studies have examined the biomechanical properties of iliac and S2AI screws; however, a direct comparison has not been performed. METHODS Eight cadaveric spines were instrumented with pedicle screws bilaterally at L5 and S1. Four specimens were further instrumented with iliac screws placed with a starting point at the posterior superior iliac spine, and four specimens were instrumented with S2AI screws placed with a starting point 1 mm inferolateral to the S1 foramen. Screws were connected with 6.35 mm rods. Subfailure testing was performed by loading at 1°/second to a torque of 10 Nm in four directions: left bending, right bending, extension, and flexion. Specimens then underwent a monotonic load to failure under flexion at a rate of 1°/second. RESULTS There were no significant differences for torsional stiffness in extension, flexion, left bending, or right bending between S2AI and iliac screw constructs. There were no significant differences in S2AI versus iliac screws for failure torque (30.9 ± 12.00 Nm vs. 22.61 ± 6.25 Nm) and yield torque (11.86 ± 0.41 Nm vs. 12.01 ± 1.70 Nm). CONCLUSION Iliac screws have been associated with increased dissection, wound complications, an additional construct failure point, and hardware prominence. The S2AI screw was developed as an alternative and has been associated with less morbidity. The iliac and S2AI screw demonstrate no statistical difference in stiffness and load-to-failure in a spinopelvic fixation model. LEVEL OF EVIDENCE Level V.

Microsurgical Simulation Exercise for Surgical Training

OBJECTIVE Initial training for orthopedic surgical residents (postgraduate years 1-5) in microsurgery using the turkey wing model and evaluation of their proficiency. DESIGN Residents were given a questionnaire on their comfort level with microsurgery and microsurgical knowledge, followed by a lecture on the subject. They watched a surgical dissection and repair of the turkey wings neurovasculature. Residents performed the dissection and repairs of the artery, vein, and nerve. A postquestionnaire was administered following the simulation exercise. Their performances on repairs were graded and results compared by academic year. SETTING AND PARTICIPANTS A total of 21 orthopedic surgery residents were recruited from Stony Brook University Medical Center, Stony Brook, NK. RESULTS This training activity resulted in significant improvements in both microsurgical knowledge (41%) and comfort (37%). Senior residents scored significantly higher than juniors on 6 microsurgical parameters. The largest effect was in nerve repair showing 4 parameters that differed significantly between groups. CONCLUSION Microsurgical techniques require extensive training to master. The turkey wing model for repair of the artery, vein, and nerve represents a realistic simulation of a human hand artery, vein, and nerve. It provides an inexpensive method for residents to practice on real tissue for improving microsurgical technique.

Sex Differences in the Physiological and Behavioral Effects of Chronic Oral Methylphenidate Treatment in Rats

Methylphenidate (MP) is a psychostimulant prescribed for Attention Deficit Hyperactivity Disorder. Previously, we developed a dual bottle 8-h-limited-access-drinking-paradigm for oral MP treatment of rats that mimics the pharmacokinetic profile of treated patients. This study assessed sex differences in response to this treatment. Male and female Sprague Dawley rats were assigned to one of three treatment groups at 4 weeks of age (n = 12/group): Control (water), low dose (LD) MP, and high dose (HD) MP. Rats drank 4 mg/kg MP (LD) or 30 mg/kg MP (HD) during the first hour, and 10 mg/kg (LD) or 60 mg/kg MP (HD) for the remaining 7 h each day. Throughout 3 months of treatment, rats were monitored for body weight, food intake, and fluid intake; as well as tested for open field behavior, circadian activity, novel object recognition, and social interaction. Chronic MP treated rats exhibited reduced fluid intake during distinct treatment weeks to a greater extent in males, and reduced total fluid intake in males only. HD MP treatment decreased body weight in both sexes, while HD MP increased total food intake in females only, likely to offset energy deficits resulting from MP-induced hyperactivity. LD and HD MP increased locomotor activity in the open field, particularly in females and during later treatment weeks. MP dose-dependently increased activity during the dark cycle of circadian testing in females, while in males hyperactivity was only exhibited by HD rats. HD MP increased center activity to a greater extent in males, while MP increased rearing behavior in females only. MP had no effect on social behavior or novel object recognition in either sex. This study concludes that chronic oral MP treatment at clinically-relevant dosages has significant effects on food intake, body weight, open field behavior, and wake cycle activity. Particularly marked sex differences were apparent for locomotor activity, with females being significantly more sensitive to the hyperactivating effects of the drug. These findings suggest that chronic MP exposure beginning in adolescence can have significant behavioral effects that are both dose- and sex-dependent, and raise concerns regarding the reversibility of these effects post-discontinuation of treatment.

Methylphenidate regulation of osteoclasts in a dose- and sex-dependent manner adversely affects skeletal mechanical integrity

Methylphenidate (MP) is the most prescribed psychostimulant for ADHD patients, with clinically demonstrated detrimental effects on bone quality, potentially leading to early onset osteoporosis and higher fracture risk. The underlying mechanism for the effects of MP on bone remains elusive. This study demonstrates that sex- and dose-dependent effects of MP on bone quality and quantity are mediated by osteoclast activity. Four-week-old male and female rats were treated with low and high dose MP for 13 weeks. Bone quality and quantity were analyzed using microCT, mechanical testing, histomorphometry, and TRAP staining. Male and female rat bone marrow-derived osteoclasts were treated in a dose-dependent manner (0–1000 ng/ml) and osteoclast activity was determined at days 5, 7, and 14 using TRAP staining, as well as a pit formation assay at day 18. Animal studies showed a dose- and a sex-dependent decrease in mechanical integrity in femora and increased TRAP staining in MP-treated rats. Primary cultures revealed that MP had direct dose- and sex-dependent effects on osteoclast activity, as seen by increased differentiation, activity, and resorption. This study demonstrates for the first time that osteoclasts are differentially regulated by MP in adolescent male and female rats, resulting in sex-dependent effects on the skeleton.

Recovery from behavior and developmental effects of chronic oral methylphenidate following an abstinence period

ABSTRACT Chronic oral methylphenidate (MP) exposure in rats is associated with numerous developmental and behavioral consequences. The present study investigated the persistence of the effects of chronic oral MP exposure after abstinence from MP use. Male and female rats were exposed to daily orally self‐administered water, low dose MP (LD), or high dose (HD) MP for 13 weeks, followed by a 4‐week abstinence period. Fluid, food consumption and bodyweights were monitored and animals were tested for locomotor activity, anxiety‐ and depressive‐like symptoms, learning and memory, and social behavior during both the treatment and abstinence phases of the experiment. During treatment, MP attenuated bodyweight regardless of sex, but increased food and fluid consumption in females and males by 20.7% and 30.1%, respectively. MP also increased locomotor activity in both males and females observed as increased distance travelled in an open field. (59.1% and 95.9%, respectively) and increased locomotor activity in the home cage over a 24‐hour circadian cycle (45.5% and 63.0%). Additionally, MP exerted an anxiolytic effect observed as increased time spent in the open arms of an elevated plus maze (31.1% in HD males, 59.2% in HD females), and an increased latency to immobility in a forced swim test (330% in HD males, 418% in HD females). The effects of MP (bodyweight, consumption, locomotion, anxiolytic, and anti‐depressive) were, almost without exception, eliminated during the abstinence period. MP had no impact on learning and memory performance as measured by a T‐maze, or social behavior during treatment. These findings suggest that the behavioral consequences of chronic oral MP treatment in our preclinical model are reversible in rats following an abstinence period from use of the drug.