In vivo effects of cell seeding technique in an ex vivo regional gene therapy model for bone regeneration.

When delivering cells on a scaffold to treat a bone defect, the cell seeding technique determines the number and distribution of cells within a scaffold, however the optimal technique has not been established. This study investigated if human adipose-derived stem cells (ASCs) transduced with a lentiviral vector to overexpress bone morphogenetic protein 2 (BMP-2) and loaded on a scaffold using dynamic orbital shaker could reduce the total cell dose required to heal a critical sized bone defect when compared with static seeding. Human ASCs were loaded onto a collagen/biphasic ceramic scaffold using static loading and dynamic orbital shaker techniques, compared with our labs standard loading technique, and implanted into femoral defects of nude rats. Both a low dose and standard dose of transduced cells were evaluated. Outcomes investigated included BMP-2 production, radiographic healing, micro-computerized tomography, histologic assessment, and biomechanical torsional testing. BMP-2 production was higher in the orbital shaker cohort compared with the static seeding cohort. No statistically significant differences were noted in radiographic, histomorphometric, and biomechanical outcomes between the low-dose static and dynamic seeding groups, however the standard-dose static seeding cohort had superior biomechanical properties. The standard-dose 5 million cell dose standard loading cohort had superior maximum torque and torsional stiffness on biomechanical testing. The use of orbital shaker technique was labor intensive and did not provide equivalent biomechanical results with the use of fewer cells.

Biodistribution of lentiviral transduced adipose-derived stem cells for "ex-vivo" regional gene therapy for bone repair.

Ex-vivo gene therapy has been shown to be an effective method for treating bone defects in pre-clinical models. As gene therapy is explored as a potential treatment option in humans, an assessment of the safety profile becomes an important next step. The purpose of this study was to evaluate the biodistribution of viral particles at the defect site and various internal organs in a rat femoral defect model after implantation of human ASCs transduced with lentivirus (LV) with two-step transcriptional activation (TSTA) of bone morphogenetic protein-2 (LV-TSTA-BMP-2). Animals were sacrificed at 4-, 14-, 56-, and 84-days post implantation. The defects were treated with either a standard dose (SD) of 5 million cells or a high dose (HD) of 15 million cells to simulate a supratherapeutic dose. Treatment groups included (1) SD LV-TSTA-BMP-2 (2) HD LV-TSTA-BMP-2, (3) SD LV-TSTA-GFP (4) HD LV-TSTA-GFP and (5) SD nontransduced cells. The viral load at the defect site and ten organs was assessed at each timepoint. Histology of all organs, ipsilateral tibia, and femur were evaluated at each timepoint. There were nearly undetectable levels of LV-TSTA-BMP-2 transduced cells at the defect site at 84-days and no pathologic changes in any organ at all timepoints. In conclusion, human ASCs transduced with a lentiviral vector were both safe and effective in treating critical size bone defects in a pre-clinical model. These results suggest that regional gene therapy using lentiviral vector to treat bone defects has the potential to be a safe and effective treatment in humans.

Driving performance and turning reaction time following hip arthroscopy for FAIS: does capsular repair matter?

PURPOSE: (1) To compare the pre- and postoperative driving performance in patients undergoing hip arthroscopy for femoroacetabular impingement syndrome (FAIS); (2) to examine the differences in driving performance between patients with versus without capsular repair. METHODS: Patients who underwent arthroscopic hip surgery for FAIS were included. Driving performance of participating patients was collected using a driving simulator preoperatively and at 2 weeks, 4-6 weeks and 8-12 weeks postoperatively. Data collected included demographics, surgery laterality, intraoperative procedures, left and right turn reaction time, total turn reaction time, gas off time (GOF), and break reaction time (BRT). Repeated measures analysis of variance (ANOVA) was used for statistical analysis. RESULTS: 21 subjects (9 males, 12 females) with a mean age of 30 ± 9 years were included and 57.1% of the subjects had right-sided surgery. There was no difference between the mean preoperative and the 2-week postoperative left (0.72 seconds and 0.75 seconds, respectively) right (0.77 seconds and 0.75 seconds, respectively), and total (0.74 seconds and 0.75 seconds, respectively) turn reaction times as well as GOF (0.62 seconds and 0.60 seconds, respectively) and BRT (0.92 seconds and 0.93 seconds, respectively), indicating that the patients' driving performance returned to the preoperative level as early as 2 weeks following hip arthroscopy for FAIS. There was no significant difference amongst any of the driving variables between patients who underwent capsular repair (50%) and those who did not. There was no significant difference amongst any of the driving variable s between patients who underwent left versus right hip arthroscopy. CONCLUSIONS: Patients' driving performance returns to the preoperative level as early as 2 weeks after hip arthroscopy for FAIS. Surgery laterality nor capsular repair make any significant difference in the time for driving abilities to return to baseline. The impact of intraoperative procedures performed, and the analgesic medications used postoperatively on the driving ability of patients undergoing hip arthroscopy warrants further investigation in larger patient populations.

Influence of donor age and comorbidities on transduced human adipose-derived stem cell in vitro osteogenic potential.

Human adipose-derived mesenchymal stem cells (ASCs) transduced with a lentiviral vector system to express bone morphogenetic protein 2 (LV-BMP-2) have been shown to reliably heal bone defects in animal models. However, the influence of donor characteristics such as age, sex, race, and medical co-morbidities on ASC yield, growth and bone regenerative capacity, while critical to the successful clinical translation of stem cell-based therapies, are not well understood. Human ASCs isolated from the infrapatellar fat pads in 122 ASC donors were evaluated for cell growth characteristics; 44 underwent additional analyses to evaluate in vitro osteogenic potential, with and without LV-BMP-2 transduction. We found that while female donors demonstrated significantly higher cell yield and ASC growth rates, age, race, and the presence of co-morbid conditions were not associated with differences in proliferation. Donor demographics or the presence of comorbidities were not associated with differences in in vitro osteogenic potential or stem cell differentiation, except that transduced ASCs from healthy donors produced more BMP-2 at day 2. Overall, donor age, sex, race, and the presence of co-morbid conditions had a limited influence on cell yield, proliferation, self-renewal capacity, and osteogenic potential for non-transduced and transduced (LV-BMP-2) ASCs. These results suggest that ASCs are a promising resource for both autologous and allogeneic cell-based gene therapy applications.

Effects of cell seeding technique and cell density on BMP-2 production in transduced human mesenchymal stem cells.

Small animal models have demonstrated the efficacy of ex vivo regional gene therapy using scaffolds loaded with BMP-2-expressing mesenchymal stem cells (MSCs). Prior to clinical translation, optimization of seeding techniques of the transduced cells will be important to minimize time and resource expenditure, while maximizing cell delivery and BMP-2 production. No prior studies have investigated cell-seeding techniques in the setting of transduced cells for gene therapy applications. Using BMP-2-expressing transduced adipose-derived MSCs and a porous ceramic scaffold, this study compared previously described static and dynamic seeding techniques with respect to cell seeding efficiency, uniformity of cell distribution, and in vitro BMP-2 production. Static and negative pressure seeding techniques demonstrated the highest seeding efficiency, while orbital shaking was associated with the greatest increases in BMP-2 production per cell. Low density cell suspensions were associated with the highest seeding efficiency and uniformity of cell distribution, and the greatest increases in BMP-2 production from 2 to 7 days after seeding. Our results highlight the potential for development of an optimized cell density and seeding technique that could greatly reduce the number of MSCs needed to produce therapeutic BMP-2 levels in clinical situations. Further studies are needed to investigate in vivo effects of cell seeding techniques on bone healing.

Improving Lentiviral Transduction of Human Adipose-Derived Mesenchymal Stem Cells.

Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34+ hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.

Allogeneic Cell Combination Therapy Ameliorates Chronic Kidney Disease-Induced Heart Failure with Preserved Ejection Fraction.

BACKGROUND: Left ventricular hypertrophy and heart failure with preserved ejection fraction (HFpEF) are primary manifestations of the cardiorenal syndrome in patients with chronic kidney disease (CKD). Therapies that improve morbidity and mortality in HFpEF are lacking. Cell-based therapies promote cardiac repair in ischemic and non-ischemic cardiomyopathies. We hypothesized that cell-based therapy ameliorates CKD-induced HFpEF. METHODS AND RESULTS: Yorkshire pigs (n = 26) underwent 5/6 embolization-mediated nephrectomy. CKD was confirmed by increased creatinine and decreased glomerular filtration rate (GFR). Mean arterial pressure (MAP) was not different between groups from baseline to 4 weeks. HFpEF was evident at 4 weeks by increased LV mass, relative wall thickening, end-diastolic pressure, and end-diastolic pressure-volume relationship, with no change in ejection fraction (EF). Four weeks post-embolization, allogeneic (allo) bone marrow-derived mesenchymal stem cells (MSC; 1 × 107 cells), allo-kidney-derived stem cells (KSC; 1 × 107 cells), allo-cell combination therapy (ACCT; MSC + KSC; 1:1 ratio; total = 1 × 107 cells), or placebo (Plasma-Lyte) was delivered via intra-renal artery. Eight weeks post-treatment, there was a significant increase in MAP in the placebo group (21.89 ± 6.05 mmHg) compared to the ACCT group. GFR significantly improved in the ACCT group. EF, relative wall thickness, and LV mass did not differ between groups at 12 weeks. EDPVR improved in the ACCT group, indicating decreased ventricular stiffness. CONCLUSIONS: Intra-renal artery allogeneic cell therapy was safe in a CKD swine model manifesting the characteristics of HFpEF. The beneficial effect on renal function and ventricular compliance in the ACCT group supports further research of cell therapy for cardiorenal syndrome.

Update on injury epidemiology in rowing: our experience with female NCAA Division I athletes and a systematic review of the literature.

OBJECTIVES: (1) To report the distribution of injuries per body site and time lost from participation in female NCAA Division I rowers and (2) to present an updated summary of the existing evidence regarding the analysis of injuries per body site in rowers. METHODS: Case series: The distribution of injuries per body site in female NCAA Division I rowers and the time lost from participation were retrieved from an institutional registry. Injuries per body site were reported as number of athletes with site-specific injury per total number of injured athletes. Systematic review: Based on the PRISMA guidelines for systematic reviews, three electronic databases were searched for studies reporting the epidemiology of injuries per body site in rowers and analyzed. RESULTS: Case series: One-hundred and thirty-seven injuries were recorded in 92 female rowers over 5 years. The risk of injury (number of injured athletes over total number of athletes on roaster) was 52% (92/176). Among the 92 injured athletes, 38% sustained injury to the lower back, while 23% and 16% of these athletes sustained injury to the ribs and hip-groin area, respectively. Systematic review: Ten studies were included. The level of evidence was IV and the mean MINORS score was 13.2(9-15) (fair quality). Most studies reported injuries to the lower spine (8/10,80%) whereas injuries to the ribs or other anatomic sites were reported in ≤4 (40%) studies. Significant heterogeneity of the injury reporting methodologies (injury definition; measures of injury occurrence; description of the injury site) was detected and precluded meta-analysis. CONCLUSIONS: Most injuries recorded in a single team of female NCAA Division I athletes occurred in the lower back followed by injuries to the ribs and the hip. The existing injury epidemiology literature in mostly focuses on injuries to the lower back, while injuries to other anatomic sites were less frequently analyzed. No conclusions can be made regarding the most common injuries in rowers based on the current evidence due to substantial heterogeneity of injury reported methodologies which warrants further investigation.

Musculoskeletal tissue engineering: Regional gene therapy for bone repair.

Bone loss associated with fracture nonunion, revision total joint arthroplasty (TJA), and pseudoarthrosis of the spine presents a challenging clinical scenario for the orthopaedic surgeon. Current treatment options including autograft, allograft, bone graft substitutes, and bone transport techniques are associated with significant morbidity, high costs, and prolonged treatment regimens. Unfortunately, these treatment strategies have proven insufficient to safely and consistently heal bone defects in the stringent biological environments often encountered in clinical cases of bone loss. The application of tissue engineering (TE) to musculoskeletal pathology has uncovered exciting potential treatment strategies for challenging bone loss scenarios in orthopaedic surgery. Regional gene therapy involves the local implantation of nucleic acids or genetically modified cells to direct specific protein expression, and has shown promise as a potential TE technique for the regeneration of bone. Preclinical studies in animal models have demonstrated the ability of regional gene therapy to safely and effectively heal critical sized bone defects which otherwise do not heal. The purpose of the present review is to provide a comprehensive overview of the current status of gene therapy applications for TE in challenging bone loss scenarios, with an emphasis on gene delivery methods and models, scaffold biomaterials, preclinical results, and future directions.

Management Options for Shoulder Impingement Syndrome in Athletes: Insights and Future Directions.

Athletes participating in overhead sports are at particularly high risk of shoulder impingement syndrome. Subcoracoid impingement is defined as impingement of the anterior soft tissues of the shoulder between the coracoid process and the lesser tuberosity. Subacromial impingement syndrome (SIS) occurs due to extrinsic compression of the rotator cuff between the humeral head and coracoacromial structures or intrinsic degeneration of the supraspinatus tendon and subsequent superior migration of the humerus. Internal impingement is a major cause of shoulder pain in overhead athletes, and it occurs due to repetitive impingement of the articular surface of the rotator cuff with the glenoid during maximum abduction and external rotation of the arm. When examining athletes with suspected impingement of the shoulder, it is important to discuss the sport-specific motion that regenerates the symptoms and perform a combination of physical examination tests to improve the diagnostic accuracy. Radiographic evaluation is recommended, and the extent of soft tissue abnormalities can be assessed on ultrasound or magnetic resonance imaging of the shoulder. Management of shoulder impingement syndrome can be conservative or operative, based on the severity and chronicity of symptoms and the associated structural abnormalities. This review provides an update on the management of SIS, subcoracoid impingement, and internal impingement in the athletic population.

Filling Open Screw Holes in the Area of Metaphyseal Comminution Does Not Affect Fatigue Life of the Synthes Variable Angle Distal Femoral Locking Plate in the AO/OTA 33-A3 Fracture Model.

INTRODUCTION: The aim of this study is to evaluate the biomechanical effect of filling locking variable angle (VA) screw holes at the area of metaphyseal fracture comminution in a Sawbones® (Sawbones USA, Vashon, Washington) model (AO/OTA 33A-3 fracture) using a Synthes VA locking compression plate (LCP) (Depuy Synthes, Warsaw, Indiana). MATERIALS AND METHODS: Seven Sawbones® femur models had a Synthes VA-LCP placed as indicated by the manufacturers technique. A 4cm osteotomy was then created to simulate an AO/OTA 33-A3 femoral fracture pattern with metaphyseal comminution. The control group consisted of four constructs in which the open screw holes at the area of comminution were left unfilled; the experimental group consisted of three constructs in which the VA screw holes were filled with locking screws. One of the control constructs was statically loaded to failure at a rate of 5mm/min. A value equal to 75% of the ultimate load to failure was used as the loading force for fatigue testing of 250,000 cycles at 3Hz. Cycles to failure was recorded for each construct and averages were compared between groups. RESULTS: The average number of cycles to failure in the control and experimental groups were 37524±8187 and 43304±23835, respectively (p=0.72). No significant difference was observed with respect to cycles to failure or mechanism of failure between groups. In all constructs in both the control and experimental groups, plate failure reproducibly occurred with cracks through the variable angle holes in the area of bridged comminution. CONCLUSIONS: The Synthes VA-LCP in a simulated AO/OTA 33-A3 comminuted metaphyseal femoral fracture fails in a reproducible manner at the area of comminution through the "honeycomb" VA screw holes. Filling open VA screw holes at the site of comminution with locking screws does not increase fatigue life of the Synthes VA-LCP in a simulated AO/OTA 33-A3 distal femoral fracture. Further studies are necessary to determine whether use of this particular plate is contraindicated when bridging distal femoral fractures with metaphyseal comminution.

A Combination of Allogeneic Stem Cells Promotes Cardiac Regeneration.

BACKGROUND: The combination of autologous mesenchymal stem cells (MSCs) and cardiac stem cells (CSCs) synergistically reduces scar size and improves cardiac function in ischemic cardiomyopathy. Whereas allogeneic (allo-)MSCs are immunoevasive, the capacity of CSCs to similarly elude the immune system remains controversial, potentially limiting the success of allogeneic cell combination therapy (ACCT). OBJECTIVES: This study sought to test the hypothesis that ACCT synergistically promotes cardiac regeneration without provoking immunologic reactions. METHODS: Göttingen swine with experimental ischemic cardiomyopathy were randomized to receive transendocardial injections of allo-MSCs + allo-CSCs (ACCT: 200 million MSCs/1 million CSCs, n = 7), 200 million allo-MSCs (n = 8), 1 million allo-CSCs (n = 4), or placebo (Plasma-Lyte A, n = 6). Swine were assessed by cardiac magnetic resonance imaging and pressure volume catheterization. Immune response was tested by histologic analyses. RESULTS: Both ACCT and allo-MSCs reduced scar size by -11.1 ± 4.8% (p = 0.012) and -9.5 ± 4.8% (p = 0.047), respectively. Only ACCT, but not MSCs or CSCs, prevented ongoing negative remodeling by offsetting increases in chamber volumes. Importantly, ACCT exerted the greatest effect on systolic function, improving the end-systolic pressure-volume relation (+0.98 ± 0.41 mm Hg/ml; p = 0.016). The ACCT group had more phospho-histone H3+ (a marker of mitosis) cardiomyocytes (p = 0.04), and noncardiomyocytes (p = 0.0002) than did the placebo group in some regions of the heart. Inflammatory sites in ACCT and MSC-treated swine contained immunotolerant CD3+/CD25+/FoxP3+ regulatory T cells (p < 0.0001). Histologic analysis showed absent to low-grade inflammatory infiltrates without cardiomyocyte necrosis. CONCLUSIONS: ACCT demonstrates synergistic effects to enhance cardiac regeneration and left ventricular functional recovery in a swine model of chronic ischemic cardiomyopathy without adverse immunologic reaction. Clinical translation to humans is warranted.

Positive Psychology and Physical Health: Research and Applications.

Positive psychology is the scientific study of a healthy and flourishing life. The goal of positive psychology is to complement and extend the traditional problem-focused psychology that has proliferated in recent decades. Positive psychology is concerned with positive psychological states (eg, happiness), positive psychological traits (eg, talents, interests, strengths of character), positive relationships, and positive institutions. We describe evidences of how topics of positive psychology apply to physical health. Research has shown that psychological health assets (eg, positive emotions, life satisfaction, optimism, life purpose, social support) are prospectively associated with good health measured in a variety of ways. Not yet known is whether positive psychology interventions improve physical health. Future directions for the application of positive psychology to health are discussed. We conclude that the application of positive psychology to health is promising, although much work remains to be done.

Differential neuroprotective effects of 5'-deoxy-5'-methylthioadenosine.

BACKGROUND: 5'-deoxy-5'-methylthioadenosine (MTA) is an endogenous compound produced through the metabolism of polyamines. The therapeutic potential of MTA has been assayed mainly in liver diseases and, more recently, in animal models of multiple sclerosis. The aim of this study was to determine the neuroprotective effect of this molecule in vitro and to assess whether MTA can cross the blood brain barrier (BBB) in order to also analyze its potential neuroprotective efficacy in vivo. METHODS: Neuroprotection was assessed in vitro using models of excitotoxicity in primary neurons, mixed astrocyte-neuron and primary oligodendrocyte cultures. The capacity of MTA to cross the BBB was measured in an artificial membrane assay and using an in vitro cell model. Finally, in vivo tests were performed in models of hypoxic brain damage, Parkinson's disease and epilepsy. RESULTS: MTA displays a wide array of neuroprotective activities against different insults in vitro. While the data from the two complementary approaches adopted indicate that MTA is likely to cross the BBB, the in vivo data showed that MTA may provide therapeutic benefits in specific circumstances. Whereas MTA reduced the neuronal cell death in pilocarpine-induced status epilepticus and the size of the lesion in global but not focal ischemic brain damage, it was ineffective in preserving dopaminergic neurons of the substantia nigra in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine (MPTP)-mice model. However, in this model of Parkinson's disease the combined administration of MTA and an A2A adenosine receptor antagonist did produce significant neuroprotection in this brain region. CONCLUSION: MTA may potentially offer therapeutic neuroprotection.