Osteogenic potential of postnatal skeletal muscle-derived stem cells is influenced by donor sex.

This study compared the osteogenic differentiation of F-MDSCs and M-MDSCs. Interestingly, M-MDSCs expressed osteogenic markers and underwent mineralization more readily than F-MDSCs; a characteristic likely caused by more osteoprogenitor cells within the M-MDSCs than the F-MDSCs and/or an accelerated osteogenic differentiation of M-MDSCs. INTRODUCTION: Although therapies involving stem cells will require both female and male cells, few studies have investigated whether sex-related differences exist in their osteogenic potential. Here, we compared the osteogenic differentiation of female and male mouse skeletal muscle-derived stem cells (F- and M-MDSCs, respectively), a potential cell source for orthopedic tissue engineering. MATERIALS AND METHODS: F- and M-MDSCs were stimulated with bone morphogenetic protein (BMP)4, followed by quantification of alkaline phosphatase (ALP) activity and expression of osteogenic genes. F- and M-MDSCs were also cultured as pellets in osteogenic medium to evaluate mineralization. Single cell-derived colonies of F- and M-MDSCs were stimulated with BMP4, stained for ALP, and scored as either Low ALP+ or High ALP+ to detect the presence of osteoprogenitor cells. F- and M-MDSCs were transduced with a BMP4 retrovirus (MDSC-BMP4 cells) and used for the pellet culture and single cell-derived colony formation assays. As well, F- and M-MDSC-BMP4 cells were implanted in the intramuscular pocket of sex-matched and sex-mismatched hosts, and bone formation was monitored radiographically. RESULTS AND CONCLUSIONS: When stimulated with BMP4, both F- and M-MDSCs underwent osteogenic differentiation, although M-MDSCs had a significantly greater ALP activity and a larger increase in the expression of osteogenic genes than F-MDSCs. In the pellet culture assay, M-MDSCs showed greater mineralization than F-MDSCs. BMP4 stimulation of single cell-derived colonies from M-MDSCs showed higher levels of ALP than those from F-MDSCs. Similar results were obtained with the MDSC-BMP4 cells. In vivo, F-MDSC-BMP4 cells displayed variability in bone area and density, whereas M-MDSC-BMP4 cells showed a more consistent and denser ectopic bone formation. More bone formation was also seen in male hosts compared with female hosts, regardless of the sex of the implanted cells. These results suggest that M-MDSCs may contain more osteoprogenitor cells than F-MDSCs, which may have implications in the development of cellular therapies for bone healing.     

Adequacy of "Hot Biopsy" for the Treatment of Diminutive Polyps: A Prospective Randomized Trial

Patients with diminutive polyps in the rectum or sigmoid colon were randomized to "hot biopsy" treatment for either 1) electrocautery for 2 s (fixed duration cautery) or 2) cautery until visible necrosis of the polyp base was evident (variable duration cautery). Sigmoidoscopy was performed 4 wk after treatment to determine the adequacy of polyp eradication. In the fixed duration cautery group, 11 of 21 polyps (52%) were eradicated, compared with 12 of 14 polyps (86%) in the variable duration cautery group (p = 0.04). When analyzed according to whether or not visible necrosis was achieved (some of the polyps in the fixed duration cautery group showed necrosis with 2 s cautery), 19 of 23 polyps (83%) were eradicated when necrosis was evident, compared to 5 of 12 (42%) without necrosis (p = 0.004). We conclude that hot biopsy treatment for diminutive polyps is significantly more effective when visible necrosis is achieved during cautery. Furthermore, even with visible necrosis, there is a 17% failure rate of polyp eradication.     

Investigating the role of DNA damage in tobacco smoking-induced spine degeneration

Background contextTobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage.PurposeTo determine if DNA damage plays a causal role in smoking-induced spine degeneration.Study designTo compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes.MethodsTwo-month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1^?/Δ mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis (³⁵S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis), and vertebral bone morphology (microcomputed tomography) were measured.ResultsExposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and an 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1^?/Δ mice.ConclusionsShort-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice, indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.     

Improvement of muscle healing through enhancement of muscle regeneration and prevention of fibrosis

Skeletal muscle is able to repair itself through regeneration. However, an injured muscle often does not fully recover its strength because complete muscle regeneration is hindered by the development of fibrosis. Biological approaches to improve muscle healing by enhancing muscle regeneration and reducing the formation of fibrosis are being investigated. Previously, we have determined that insulin-like growth factor-1 (IGF-1) can improve muscle regeneration in injured muscle. We also have investigated the use of an antifibrotic agent, decorin, to reduce muscle fibrosis following injury. The aim of this study was to combine these two therapeutic methods in an attempt to develop a new biological approach to promote efficient healing and recovery of strength after muscle injuries. Our findings indicate that further improvement in the healing of muscle lacerations is attained histologically by the combined administration of IGF-1 to enhance muscle regeneration and decorin to reduce the formation of fibrosis. This improvement was not associated with improved responses to physiological testing, at least at the time-points tested in this study.     

Enhancement of tendon-bone integration of anterior cruciate ligament grafts with bone morphogenetic protein-2 gene transfer: a histological and biomechanical study

BACKGROUND: The integration of tendon grafts used for replacement of the anterior cruciate ligament is still sometimes unsatisfactory and may be associated with postoperative anterior-posterior laxity. The goal of this study was to examine the capacity of bone morphogenetic protein-2 (BMP-2) gene transfer to improve the integration of semitendinosus tendon grafts at the tendon-bone interface after reconstruction of the anterior cruciate ligament in rabbits. METHODS: The anterior cruciate ligaments of adult New Zealand White rabbits were replaced with autologous double-bundle semitendinosus tendon grafts. The semitendinosus tendon grafts had been infected in vitro with adenovirus-luciferase, adenovirus-LacZ (AdLacZ), or adenovirus-BMP-2 (AdBMP-2); untreated grafts served as controls. The grafts were examined histologically at two, four, six, and eight weeks after surgery. In additional experiments, the structural properties of the femur-anterior cruciate ligament graft-tibia complexes, from animals killed eight weeks postoperatively, were determined from uniaxial tests. The stiffness (N/mm) and ultimate load to failure (N) were determined from the resulting load-elongation curves. RESULTS: Genetically engineered semitendinosus tendon grafts expressed reporter genes as well as BMP-2 in vitro. The AdLacZ-infected grafts showed two different histological patterns of transduction. Intra-articularly, infected cells were mostly aligned along the surface, and they decreased in number between two and eight weeks after surgery. In the intra-tunnel portions of the grafts, the number of infected cells did not decrease during the observation period. Moreover, a high number of transduced cells was found in the deeper layers of the tendons. In the control group, granulation-type tissue at the tendon-bone interface showed progressive reorganization into a dense connective tissue, and a later establishment of fibers resembling Sharpey fibers. In the specimens with an AdBMP-2-infected anterior cruciate ligament graft, a broad zone of newly formed matrix resembling chondro-osteoid had formed at the tendon-bone interface at four weeks after surgery. This area was increased at six weeks, showing a transition from bone to mineralized cartilage and nonmineralized fibrocartilage. In addition, in the AdBMP-2-treated specimens, the tendon-bone interface in the osseous tunnel was similar to that of a normal anterior cruciate ligament insertion. The stiffness (29.0 +/- 7.1 N/mm compared with 16.7 +/- 8.3 N/mm) and the ultimate load to failure (108.8 +/- 50.8 N compared with 45.0 +/- 18.0 N) were significantly enhanced in the specimens with an AdBMP-2-transduced graft when compared with the control values (p < 0.05). CONCLUSION: This study demonstrates that BMP-2 gene transfer significantly improves the integration of semitendinosus tendon grafts in bone tunnels after reconstruction of the anterior cruciate ligament in rabbits. Clinical Relevance: Novel technologies including gene therapy and tissue engineering, such as those described in this study, may provide useful therapeutic procedures to enhance biological healing after reconstruction of the anterior cruciate ligament.     

Ex-vivo-Gentherapie mit BMP4 bei kritischen Substanzdefekten und Frakturen im osteoporotischen Tiermodell

Fractures in osteoporotic bones or segment defects are problematic bone lesions with a reduced biological capability of regeneration. We tested the hypothesis that cell-mediated ex vivo gene therapy to deliver BMP4 can heal critically sized defects and improve bone healing in osteoporotic rats. Primary muscle-derived cells were isolated from the hindlimb muscle of rats and retrovirally transduced to express bone morphogenic protein 4 (BMP4). The bone formation was evaluated following local application of these cells in critically sized defects and in fractures of osteoporotic bones. Radiographic analysis revealed bridging callus formation in a critically sized defect in all specimens using muscle-derived cells expressing BMP4 at 12 weeks. These findings were confirmed by histological evaluation, which revealed callus bone formation with good integration to the distal and proximal bone. Following treatment with muscle-derived cells expressing BMP4, the bone healing process in the osteoporotic bone was improved to the level similar to that of normal bone. The ex vivo gene therapy could be a promising tool for the treatment of osteoporotic fractures and critically sized defects. The reduced number of complications (nonunions, loss of reduction, and fragment dislocation), shortening of hospitalization period, and improvement of bone strength are decisive advocates for this treatment option.     

Ablation of Atrial-Ventricular Junction Tissues via the Coronary Sinus Using Cryo Balloon Technology

Introduction: The coronary sinus (CS) can provide access to targets across and within the atrioventricular (AV) junction.Methods: In 12 dogs (32 ± 3 Kg), cryo balloons (10–19 mm) were applied to regions of the AV junction for 3 minutes at a temperature of –75.9 ± 9C (ranging –57 to –83). Electrical activity and pacing within the CS were assessed pre and post ablation and at least 3 months later in 9 dogs. In the 3 other dogs, hearts were examined immediately after cryo ablation. CS and circumflex angiography was performed pre and post ablation. The hearts, CS, and Cx were then examined for structural injury. The AV junction was sectioned and the hearts were immersed in Tetrazolium, and the lesions were inspected for transmurality across the AV groove.Results: In 3/12 dogs the distal CS cryo lesions resulted in inferior ST segment depression that resolved within 5 minutes. There was no arrhythmia or hemodynamic changes. No CS electrical activity was noted post ablation. The pacing threshold increased from 2 ± 2.3 mA to 7.4 ± 3.6 mA (p < 0.001). Pathological examination of 3 acute hearts revealed hematomas.There was no pericardial effusion. No evidence of stenosis or thrombosis was seen within the CS and the circumflex artery. After 3 months of recovery, transmural lesions across the AV groove were present in all of the targeted AV regions.Conclusion: Intra-CS cryo balloon ablation is safe and can potentially replace endocardial RF ablation targeting the AV junction and the CS muscular sleeve.This research was partially funded by: VA Merit grant, Boston Scientific Corporation/EP Technologies, Scimed.