Combined methylphenidate and fluoxetine treatment in adolescent rats significantly impairs weight gain with minimal effects on skeletal development.

Methylphenidate (MP) is frequently prescribed to treat Attention-Deficit/Hyperactivity Disorder (ADHD); however, many patients with ADHD experience depression and anxiety. As such, concomitant administration of selective serotonin reuptake inhibitors such as fluoxetine (FLX) is common. Our laboratory and others have shown that MP impairs skeletal development in preclinical and clinical settings, and FLX has also been linked to skeletal deficits. Unfortunately, little is known about the effects of combined MP and FLX treatment on skeletal development. The objective of this study was to investigate the effects of MP and FLX on bone morphology and biomechanical properties in adolescent rats. Four-week-old male Sprague-Dawley rats were randomly divided into the following 4 groups: Water, MP, FLX, and MP + FLX. As body weights in the MP, FLX, and MP + FLX groups were all lower than Water, the data were compared directly and after adjusting to body weight via linear regression. The direct comparison revealed that MP + FLX rats had significantly shorter (~12 %) and narrower femora and tibiae (~10 %) compared to most other groups, along with shorter (26-35 %), disorganized tibial growth plates. MicroCT analyses of the trabecular compartment of the proximal tibia identified reductions of 47 % for TV, 86 % for BV, 74 % for BV/TV, 68 % for Tb.N, 25 % in Tb.Th, and 74 % in vBMD concomitant with increases of 44 % for Tb.Sp for MP + FLX compared to Water. Similar analyses of femoral midshaft cortical bone identified reductions of 29 % for Ct.V, 30 % for Ps.V, 30 % for Ec. V, and 51 % for pMOI, as well as increases of 17 % for Ct.Th and 2 % for TMD for MP + FLX compared to Water. Biomechanically, MP + FLX femora were weaker, as indicated by a reduction in ultimate force (14 %) in MP + FLX compared to Water. The microstructural and biomechanical effects of MP + FLX were eliminated after adjustment for body weight, though the detrimental effects on growth plate morphology remained. We conclude that while the adverse microstructural and biomechanical effects of MP + FLX seen via direct comparison are predominantly attributable to reductions in body weight rather than direct effects on bone, MP and FLX, particularly in combination show detrimental effects on growth plate structure and chondrocyte morphology. These findings warrant further research into the effect of these drugs on weight gain, skeletal development and growth plate morphology, as well as consideration by physicians treating children and adolescents with ADHD.

Acute postoperative pain and dorsal root ganglia transcriptomic signatures following total knee arthroplasty (TKA) in rats: An experimental study.

Total knee arthroplasty (TKA) is the final treatment option for patients with advanced knee osteoarthritis (OA). Unfortunately, TKA surgery is accompanied by acute postoperative pain that is more severe than arthroplasty performed in other joints. Elucidating the molecular mechanisms specific to post-TKA pain necessitates an animal model that replicates clinical TKA procedures, induces acute postoperative pain, and leads to complete functional recovery. Here, we present a new preclinical TKA model in rats and report on functional and behavioral outcomes indicative of pain, analgesic efficacy, serum cytokine levels, and dorsal root ganglia (DRG) transcriptomes during the acute postoperative period. Following TKA, rats exhibited marked deficits in weight bearing that persisted for 28 days. Home cage locomotion, rearing, and gait were similarly impacted and recovered by day 14. Cytokine levels were elevated on postoperative days one and/or two. Treatment with morphine, ketorolac, or their combination improved weight bearing while gabapentin lacked efficacy. When TKA was performed in rats with OA, similar functional deficits and comparable recovery time courses were observed. Analysis of DRG transcriptomes revealed upregulation of transcripts linked to multiple molecular pathways including inflammation, MAPK signaling, and cytokine signaling and production. In summary, we developed a clinically relevant rat TKA model characterized by resolution of pain and functional recovery within five weeks and with pain-associated behavioral deficits that are partially alleviated by clinically administered analgesics, mirroring the postoperative experience of TKA patients.

Low-Intensity Continuous Ultrasound Therapies­A Systematic Review of Current State-of-the-Art and Future Perspectives.

Therapeutic ultrasound has been studied for over seven decades for different medical applications. The versatility of ultrasound applications are highly dependent on the frequency, intensity, duration, duty cycle, power, wavelength, and form. In this review article, we will focus on low-intensity continuous ultrasound (LICUS). LICUS has been well-studied for numerous clinical disorders, including tissue regeneration, pain management, neuromodulation, thrombosis, and cancer treatment. PubMed and Google Scholar databases were used to conduct a comprehensive review of all research studying the application of LICUS in pre-clinical and clinical studies. The review includes articles that specify intensity and duty cycle (continuous). Any studies that did not identify these parameters or used high-intensity and pulsed ultrasound were not included in the review. The literature review shows the vast implication of LICUS in many medical fields at the pre-clinical and clinical levels. Its applications depend on variables such as frequency, intensity, duration, and type of medical disorder. Overall, these studies show that LICUS has significant promise, but conflicting data remain regarding the parameters used, and further studies are required to fully realize the potential benefits of LICUS.

Therapeutic Potential Low-Intensity Pulsed Ultrasound for Osteoarthritis: Pre-clinical and Clinical Perspectives.

Osteoarthritis (OA), degeneration of cartilage associated with aging, lifestyle, and trauma, is one of the most common diseases that leads to lower quality of life and socioeconomic burden in the United States. Clinically, OA is initially managed by non-steroidal anti-inflammatory drugs, but eventually requires surgical intervention to reduce pain and increase function. Cartilage is a mechanotransductive tissue and requires a mechanical stimulus to sustain its mechanical and physiologic properties. Low-intensity pulsed ultrasound (LIPUS) is a cyclic acoustic wave that can provide essential mechanical stimuli to activate molecular and cellular pathways leading to chondrocyte proliferation, differentiation and activity, as well as to inhibit inflammatory pathways associated with OA. The activation of chondrocyte proliferation and inhibition of anti-inflammatory cytokines make LIPUS a potential therapy for mild to moderate OA. Although a few review articles have described the effects of ultrasound on chondrocytes and cartilage, there remains a need for a comprehensive analysis of our current understanding of the basic science and clinical status of the effects of low-intensity ultrasound on chondrocytes and cartilage and the implications of these studies on LIPUS as a therapeutic option for OA. This review analyzes recent literature describing the results of LIPUS using in vitro and in vivo pre-clinical models and clinical studies, as well as future directions for research.

Utilization of Finite Element Analysis for Articular Cartilage Tissue Engineering.

Scaffold design plays an essential role in tissue engineering of articular cartilage by providing the appropriate mechanical and biological environment for chondrocytes to proliferate and function. Optimization of scaffold design to generate tissue-engineered cartilage has traditionally been conducted using in-vitro and in-vivo models. Recent advances in computational analysis allow us to significantly decrease the time and cost of scaffold optimization using finite element analysis (FEA). FEA is an in-silico analysis technique that allows for scaffold design optimization by predicting mechanical responses of cells and scaffolds under applied loads. Finite element analyses can potentially mimic the morphology of cartilage using mesh elements (tetrahedral, hexahedral), material properties (elastic, hyperelastic, poroelastic, composite), physiological loads by applying loading conditions (static, dynamic), and constitutive stress-strain equations (linear, porous-elastic, biphasic). Furthermore, FEA can be applied to the study of the effects of dynamic loading, material properties cell differentiation, cell activity, scaffold structure optimization, and interstitial fluid flow, in isolated or combined multi-scale models. This review covers recent studies and trends in the use of FEA for cartilage tissue engineering and scaffold design.

ADAMTS9 and ADAMTS20 are differentially affected by loss of B3GLCT in mouse model of Peters plus syndrome.

Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ß3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose ß1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.

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.

Dynamic acoustic radiation force retains bone structural and mechanical integrity in a functional disuse osteopenia model.

Disuse osteopenia and bone loss have been extensively reported in long duration space mission and long term bed rest. The pathology of the bone loss is similar to osteoporosis but highly confined to weight bearing bones. The current anabolic and/or anti-resorptive drugs have systemic effects and are costly over extended time, with concerns of long term fracture risk. This study use Low Intensity Pulsed Ultrasound (LIPUS) as a non-invasive acoustic force and anabolic stimulus to countermeasure disuse induced bone loss. Four-month old C57BL/6 mice were randomized into five groups, 1) age-matched (AM), 2) non-suspended sham (NS), 3) non-suspended-LIPUS (NU), 4) suspended sham (SS), and 5) suspended-LIPUS (SU) groups. After four weeks of suspension, µCT analyses showed significant decreases in trabecular bone volume fraction (BV/TV) (-36%, p<0.005), bone tissue mineral density (TMD) (-3%, p<0.05), trabecular thickness (Tb.Th) (-12.5%, p<0.005), and increase in bone surface/bone volume (+BS/BV) (+16%, p<0.005), relative to age-matched (AM). The application of LIPUS for 20 min/day for 5 days/week, significantly increased TMD (+3%, p<0.05), Tb.Th (+6%, p<0.05), and decreased BS/BV (-10%, p<0.005), relative to suspension alone (SS) mice. Histomorphometry analyses showed a breakdown of bone microstructure under disuse conditions consist with µCT results. In comparison to SS mice, LIPUS treated bone showed increased structural integrity with increased bone formation rates at metaphysical endosteal and trabecular surfaces (+0.104±0.07 vs 0.031±0.30 µm(3)/µm(2)/day) relative to SS. Four-point bending mechanical tests of disused SS femurs showed reduced elastic modulus (-53%, p<0.05), yield (-33%, p<0.05) and ultimate strength (-45%, p<0.05) at the femoral diaphysis relative to AM bone. LIPUS stimulation mitigated the adverse effects of disuse on bone elastic modulus (+42%, p<0.05), yield strength (+29%, p<0.05), and ultimate strength (+39%, p<0.05) relative to SS femurs. LIPUS provides the essential mechanical stimulus to retain bone morphological and mechanical integrity in disuse conditions. This study demonstrates LIPUS potential as regional therapeutic agent to countermeasure disuse induced bone loss while maintaining bone's integrity.

Three TNFR-binding domains of PGRN act independently in inhibition of TNF-alpha binding and activity.

PGRN was previously reported to bind to TNF receptors (TNFR) and is therapeutic against inflammatory arthritis. Here we present further evidences demonstrating the PGRN inhibition of TNF-alpha binding and activity, and clarifying the distinct mechanisms underlying TNF-alpha inhibition between PGRN and classic TNF-alpha-binding inhibitors. In addition, we present evidences indicating that three TNFR binding domains of PGRN act independently in binding to TNFR. Furthermore, changing the order of three TNFR-binding domains in Atsttrin, a PGRN-derived molecule composed of these TNFR-binding domains, does not affect its anti-inflammatory and anti-TNF activities in both collagen-induced inflammatory arthritis and human TNF-alpha transgenic mouse model. Taken together, these findings provide the additional molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various inflammatory diseases and conditions.

Enhancement of osteogenic differentiation and proliferation in human mesenchymal stem cells by a modified low intensity ultrasound stimulation under simulated microgravity.

Adult stem cells can differentiate into multiple lineages depending on their exposure to differing biochemical and biomechanical inductive factors. Lack of mechanical signals due to disuse can inhibit osteogenesis and induce adipogenesis of mesenchymal stem cells (MSCs). Long-term bed rest due to both brain/spinal cord injury and space travel can lead to disuse osteoporosis that is in part caused by a reduced number of osteoblasts. Thus, it is essential to provide proper mechanical stimulation for cellular viability and osteogenesis, particularly under disuse conditions. The objective of this study was to examine the effects of low intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of adipose-derived human stem cells (Ad-hMSC) in simulated microgravity conditions. Cells were cultured in a 1D clinostat to simulate microgravity (SMG) and treated with LIPUS at 30mW/cm(2) for 20 min/day. It was hypothesized that the application of LIPUS to SMG cultures would restore osteogenesis in Ad-hMSCs. The results showed significant increases in ALP, OSX, RANKL, RUNX2, and decreases in OPG in LIPUS treated SMG cultures of Ad-MSC compared to non-treated cultures. LIPUS also restored OSX, RUNX2 and RANKL expression in osteoblast cells. SMG significantly reduced ALP positive cells by 70% (p<0.01) and ALP activity by 22% (p<0.01), while LIPUS treatment restored ALP positive cell number and activity to equivalence with normal gravity controls. Extracellular matrix collagen and mineralization was assessed by Sirius red and Alizarin red staining, respectively. SMG cultures showed little or no collagen or mineralization, but LIPUS treatment restored collagen content to 50% (p<0.001) and mineralization by 45% (p<0.001) in LIPUS treated-SMG cultures relative to SMG-only cultures. The data suggest that LIPUS treatment can restore normal osteogenic differentiation of MSCs from disuse by daily short duration stimulation.

Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains.

We previously reported that PGRN directly bound to TNF receptors (TNFR) in vitro and in chondrocytes (Tang, et al., Science, 2011). Here we report that PGRN also associated with TNFR in splenocytes, and inhibited the binding of TNFα to immune cells. Proper folding of PGRN is essential for its binding to TNFR, as DTT treatment abolished its binding to TNFR. In contrast, the binding of PGRN to Sortilin was enhanced by DTT. Protein interaction assays with mutants of the TNFR extracellular domain demonstrated that CRD2 and CRD3 of TNFR are important for the interaction with PGRN, similar to the binding to TNFα. Taken together, these findings provide the molecular basis underlying PGRN/TNFR interaction and PGRN-mediated anti-inflammatory activity in various autoimmune diseases and conditions.

Reversal of the detrimental effects of simulated microgravity on human osteoblasts by modified low intensity pulsed ultrasound.

Microgravity (MG) is known to induce bone loss in astronauts during long-duration space mission because of a lack of sufficient mechanical stimulation under MG. It has been demonstrated that mechanical signals are essential for maintaining cell viability and motility, and they possibly serve as a countermeasure to the catabolic effects of MG. The objective of this study was to examine the effects of high-frequency acoustic wave signals on osteoblasts in a simulated microgravity (SMG) environment (created using 1-D clinostat bioreactor) using a modified low-intensity pulsed ultrasound (mLIPUS). Specifically, we evaluated the hypothesis that osteoblasts (human fetal osteoblastic cell line) exposure to mLIPUS for 20 min/d at 30 mW/cm(2) will significantly reduce the detrimental effects of SMG. Effects of SMG with mLIPUS were analyzed using the MTS proliferation assay for proliferation, phalloidin for F-actin staining, Sirius red stain for collagen, and Alizarin red for mineralization. Our data showed that osteoblast exposure to SMG results in significant decreases in proliferation (∼ -38% and ∼ -44% on days 4 and 6, respectively; p < 0.01), collagen content (∼ -22%; p < 0.05) and mineralization (∼ -37%; p < 0.05) and actin stress fibers. In contrast, mLIPUS stimulation in SMG condition significantly increases the rate of proliferation (∼24% by day 6; p < 0.05), collagen content (∼52%; p < 0.05) and matrix mineralization (∼25%; p < 0.001) along with restoring formation of actin stress fibers in the SMG-exposed osteoblasts. These data suggest that the acoustic wave can potentially be used as a countermeasure for disuse osteopenia.