Notch activation enhances mesenchymal stem cell sheet osteogenic potential by inhibition of cellular senescence.

Our previous studies have confirmed the therapeutic effects of mesenchymal stem cell (MSC) monolayer sheet transplantation on allograft repair. A limiting factor in their application is the loss of MSC multi-potency as a result of high density sheet culture-induced senescence. In the study reported in this article, we tested whether Notch activation could be used to prevent or delay sheet culture-induced cell aging. Our results showed that, during in vitro long-term (5-day) cell sheet culture, MSCs progressively lose their progenitor characteristics. In contrast, Notch activation by Jagged1 in MSC sheet culture showed reduced cellular senescence and cell cycle arrest compared with control MSCs without Notch activation. Importantly, knockdown of Notch target gene Hes1 totally blocked the inhibition effect of Jagged1 on cellular senescence. Finally, the in vivo allograft transplantation data showed a significant enhanced callus formation and biomechanical properties in Notch activation cultured long-term sheet groups when compared with long-term cultured sheet without Notch activation. Our results suggest that Notch activation by Jagged1 could be used to overcome the stem cell aging caused by high density sheet culture, thereby increasing the therapeutic potential of MSC sheets for tissue regeneration.

Notch signaling indirectly promotes chondrocyte hypertrophy via regulation of BMP signaling and cell cycle arrest.

Cell cycle regulation is critical for chondrocyte differentiation and hypertrophy. Recently we identified the Notch signaling pathway as an important regulator of chondrocyte proliferation and differentiation during mouse cartilage development. To investigate the underlying mechanisms, we assessed the role for Notch signaling regulation of the cell cycle during chondrocyte differentiation. Real-time RT-PCR data showed that over-expression of the Notch Intracellular Domain (NICD) significantly induced the expression of p57, a cell cycle inhibitor, in chondrocytes. Flow cytometric analyses further confirmed that over-expression of NICD in chondrocytes enhances the G0/G1 cell cycle transition and cell cycle arrest. In contrast, treatment of chondrocytes with the Notch inhibitor, DAPT, decreased both endogenous and BMP2-induced SMAD 1/5/8 phosphorylation and knockdown of SMAD 1/5/8 impaired NICD-induced chondrocyte differentiation and p57 expression. Co-immunoprecipitation using p-SMAD 1/5/8 and NICD antibodies further showed a strong interaction of these proteins during chondrocyte maturation. Finally, RT-PCR and Western blot results revealed a significant reduction in the expression of the SMAD-related phosphatase, PPM1A, following NICD over-expression. Taken together, our results demonstrate that Notch signaling induces cell cycle arrest and thereby initiates chondrocyte hypertrophy via BMP/SMAD-mediated up-regulation of p57.

Deletion of RBPJK in Mesenchymal Stem Cells Enhances Osteogenic Activity by Up-Regulation of BMP Signaling.

Recently we have demonstrated the importance of RBPjk-dependent Notch signaling in the regulation of mesenchymal stem cell (MSC) differentiation during skeletogenesis both in vivo and in vitro. Here we further performed RBPJK loss-of-function experiments to demonstrate for the first time that RBPJK deficient MSC shows enhanced differentiation and osteogenesis acts via up-regulation of the BMP signaling. In the present study, we first compared the spontaneous and osteogenic differentiation in normal and recombination signal binding protein for immunoglobulin kappa J region (RBPJK) deficient human bone marrow-derived mesenchymal stem cells (MSCs). It was found that RBPJK highly expressed in fresh isolated MSCs and its expression was progressing down-regulated during spontaneous differentiation and even greater in osteogenic media inducted differentiation. Deletion of RBPJK in MSCs not only enhances cell spontaneous differentiation, but also significantly accelerates condition media inducted osteogenic differentiation by showing enhanced alkaline phosphatase (ALP) activity, Alizarin red staining, gene expression of Runx2, Osteopontin (OPN), Type I collagen (COL1a1) in culture. Additionally, BMP signaling responsive reporter activity and phosphor-smad1/5/8 expression were also significantly increased upon removal of RBPJK in MSCs. These data proved that inhibition of Notch signaling in MSCs promotes cell osteogenic differentiation by up-regulation of BMP signaling, and RBPJK deficient MSC maybe a better cell population for cell-based bone tissue engineering.

Notch inhibits chondrogenic differentiation of mesenchymal progenitor cells by targeting Twist1.

While Notch signaling plays a critical role in the regulation of cartilage formation, its downstream targets are unknown. To address this we performed gain and losses of function experiments and demonstrate that Notch inhibition of chondrogenesis acts via up-regulation of the transcription factor Twist1. Upon Notch activation, murine limb bud mesenchymal progenitor cells in micromass culture displayed an inhibition of chondrogenesis. Twist1 was found to be exclusively expressed in mesenchymal progenitor cells at the onset stage of chondrogenesis during Notch activation. Inhibition of Notch signaling in these cells significantly reduced protein expression of Twist1. Furthermore, the inhibition effect of NICD1 on MPC chondrogenesis was markedly reduced by knocking down of Twist1. Constitutively active Notch signaling significantly enhanced Twist1 promoter activity; whereas mutation studies indicated that a putative NICD/RBPjK binding element in the promoter region is required for the Notch-responsiveness of the Twist1 promoter. Finally, chromatin immunoprecipitation assays further confirmed that the Notch intracellular domain influences Twist1 by directly binding to the Twist1 promoter. These data provide a novel insight into understanding the molecular mechanisms behind Notch inhibition of the onset of chondrogenesis.

A quantitative analysis of tension band plating of the femur diaphysis.

OBJECTIVES: To measure quantitatively the strains in the concave and convex sides of a femur to substantiate the Arbeitsgemeinschaft für Osteosynthesefragen (AO) tension band principle. METHODS: Eight cadaveric fresh-frozen femurs were cleaned of soft tissue and prepared for mounting the strain gages (Vishay, Raleigh, NC). The student strain gages were mounted at the anterior, lateral and medial surfaces of a femur. They were loaded at 100, 250, 500, 1,000 and 1,500 N axial forces in a mechanical testing machine (Instron Model 4202, Norwood, MA) and the micro strains were measured for intact and after fixation of a simulated fracture by an eight hole broad 4.5 mm stainless steel compression plate (Synthes LCP). In order to simulate the fracture conditions, where all eight screw holes might not be filled, three different arrangements were tested: 8 screws, 4 screws and 2 screws. The microstrains under different loads were analyzed by Analysis of Variance (ANOVA) with Holm-Sidak multiple comparison method. RESULTS: At the same gage location micro strains were not significantly different between the intact, and the femur fixated by 8, 4 and 2 screws. For intact bones at 1,500 N the strain ratio between the tension to compression sides was -0.21 proximally and -0.04 distally. The comparison of these strain ratios at different loads did not show any significant differences at p = 0.05 and power of 0.8. CONCLUSIONS: The data showed a trend validating the tension band principle as tensile strains lowered and compressive strains increased after placement of the plate.

Biomechanics of humerus fracture fixation by locking, cortical, and hybrid plating systems in a cadaver model.

The goal of this study was to discover how locking or cortical screws or a hybrid of both would perform in stabilizing a simulated humerus fracture. We simulated stripping of screw threads or poor bone quality by overdrilling the screw hole, and also studied a control group with no overdrilling. A total of 38 fresh frozen cadaver humeri were divided into 2 groups: 16 undergoing overdrilling with a drill bit 0.3 mm less than the diameter of the screw and 22 undergoing no overdrilling. A 4-point bending test followed torsional fatigue of 1000 cycles with an amplitude of +/-10 degrees. The post-fatigued samples were retested in the same way after tightening the loose screws if necessary. Finally, each fatigued specimen was tested for failure in torsion at 0.5 Hz by applying a maximum rotational displacement of 60 degrees .The bending stiffness values (Nm/mm) of cortical and locking screws, with the exception of the hybrid system, were significantly higher for the overdrilled group than the non-overdrilled (cortical, 6.9 vs 5.6; locking, 9.1 vs 6.3; hybrid, 8.4 vs 6.8). Fatigue had no effect on the bending stiffness of all the screw/plate systems (cortical, 6.9 vs 7.4; locking, 9.1 vs 8.8; hybrid, 8.5 vs 8.1). The overdrilling had no effect on the failure loads and displacements for all the screws except cortical screws, where the failure displacement was significantly higher for the overdrilling group. The torsional stiffness retentions after 1000 cycles were significantly different for overdrilled specimens in the cortical screws group only.Overdrilling had a minimal effect on bending and torsional properties. The results of the locking and hybrid were close, and the cortical screw had only slightly lower bending stiffness.

Screw versus plate fixation of proximal first metatarsal crescentic osteotomy.

BACKGROUND: Hallux valgus associated with metatarus primus varus is a deformity that disrupts normal foot function. Standard treatment has often included distal or proximal metatarsal osteotomy with or without a distal soft tissue procedure. The intrinsically unstable proximal crescentic osteotomy relies on operative fixation for stability. This study examined the strength of fixation of a single screw versus a plate for stabilization of proximal first metatarsal crescentic osteotomies for correction of hallux valgus. MATERIALS AND METHODS: A crescentic osteotomy was performed on nine pairs of fresh/frozen cadaveric feet. The distal fragment was rotated laterally and the osteotomy was temporarily stabilized with a Kirschner wire. The osteotomized metatarsal fragments were fixed with a cancellous screw on one side and single plate on the contralateral side. Each specimen was loaded in a mechanical testing machine, and its response was measured by monitoring the opening of the osteotomy and change in alignment of the fragments. Position and alignment of the proximal and distal fragments were calculated. RESULTS: All of the measures of fixation strength were statistically greater in specimens treated with a plate rather than with screw fixation. Plate fixation provided approximately twice the resistance to disruption of the osteotomy under cyclic loading conditions. CONCLUSION: The dorsal plate is biomechanically more stable than a single cancellous screw when applied to proximal crescentic osteotomies. CLINICAL RELEVANCE: The greater stability of the plate construct may be helpful in selecting the fixation device for these osteotomies.

Lateral talar process fracture and peroneal tendon dislocation: a previously unrecognized injury complex.

BACKGROUND: Lateral talar process fractures and peroneal tendon dislocations are frequently unrecognized at the time of injury. Lateral process fractures were initially classified by Hawkins as three types. Type II injuries are comminuted fractures involving both the talofibular and talocalcaneal articular surfaces. The purpose of this retrospective chart review was to describe an injury complex of Type II lateral talar process fracture with peroneal tendon dislocation. MATERIALS AND METHODS: Between January of 1995 and December 2006, 13 patients were seen for a lateral talar process fracture. Patients' charts were reviewed for fracture classification, mechanism of injury, radiographic studies, treatment, secondary procedures, length of followup and return to previous activity level. Concurrent peroneal tendon dislocations were identified in a subset of these patients. RESULTS: Thirteen patients were identified with lateral talar process fractures all of which were classified as a Hawkins Type II. Six patients (46%) had a simultaneous peroneal tendon dislocation. All patients underwent operative excision of the comminuted lateral process. Patients with the injury complex were more likely to undergo additional operative procedures, and were more likely to develop subtalar arthritis. At final followup, 71% of patients with isolated lateral process fractures and 33% of injury complex patients had returned to their previous level of activity CONCLUSION: An injury complex of Hawkins Type II lateral talar process fractures and peroneal tendon dislocation exists. Patients with comminuted lateral talar process fractures, especially those resulting from high-energy injuries, should be carefully evaluated for the possibility of concurrent peroneal tendon dislocation.

Mechanical strain promotes fibroblast gene expression in presence of corticosteroid.

Posterior tibial tendon (PTT) dysfunction has commonly been treated with local corticosteroid injections to reduce inflammation. However, a concern with this treatment is potential degeneration and spontaneous rupture of the PTT. This study set out to determine whether mechanical strain may counteract the potentially deleterious effect of corticosteroid treatment on fibroblasts and therefore improve outcomes during recovery from tendinitis. In this study, PTT fibroblasts in vitro were treated with 0 M, 10(-7) M, 10(-6) M, and 10(-5) M triamcinolone acetonide (TA) while incubated under cyclic strains of 0% or 5% for 24 hr. Type I collagen and decorin mRNA expressions were determined by RT-PCR. The results indicated that mechanical strain significantly increased type I collagen and decorin gene expression in the PTT fibroblasts and TA decreased type I collagen and decorin gene expression. Therefore, mechanical strain might be beneficial to PTT after corticosteroid treatment by direct stimulation of fibroblast synthesis.

Computerized matching of autologous femoral grafts for the treatment of medial talar osteochondral defects.

BACKGROUND: Cored autologous graft from the distal ipsilateral femur has been used to fill osteochondral defects in the talus. There are no studies that compare the articular morphology of potential donor sites on the distal femur with recipient sites on the talus. METHODS: Using coronal MRI of the talus and distal femur of five matched cadaver, computer reconstructions of the articular surfaces were prepared. From these, six 10-mm in diameter donor sites from the nonweightbearing surfaces of the medial and lateral aspects of the femoral condyles were matched to three recipient sites on the anterior, middle, and posterior aspects of the corresponding medial talus using customized computer software that minimized differences between the articular surfaces of the graft and the talus. After matching the femoral to the talar graft, the average and maximal distances between the surfaces (surface contour) and the average and maximal distances of the offset at the outer 1 mm of the graft periphery (step-off) were determined. RESULTS: For all graft combinations, the average step-off was 0.24 +/- 0.03 mm and the maximum 0.60 mm. The average surface contour was 0.32 +/- 0.04 mm and the maximum was 1.16 mm. In all cases, the best donor site was from the superolateral femur for any medial talar lesion. CONCLUSION: In this study of grafts from the femoral condyles, the superolateral femur was the optimal location for an osteochondral graft for any medial talar lesion.

Chronic anterior midtibial stress fractures in athletes treated with reamed intramedullary nailing.

BACKGROUND: A chronic anterior midtibial stress fracture is a serious, difficult-to-treat injury that can adversely affect an athlete's career. HYPOTHESIS: The use of a reamed intramedullary nail for a chronic anterior tibial stress fracture is a safe and effective treatment for an athlete. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Seven collegiate-level athletes with 11 chronic anterior midtibial stress fractures were treated with reamed intramedullary nailing between 1997 and 2000. These patients were followed for a mean duration of 17 months. RESULTS: The mean age of the patients at the time of stress fracture diagnosis was 17 years. Seven of the fractures occurred in male athletes, whereas 4 occurred in female athletes. All patients had failed nonoperative treatment, including rest, activity modification, use of an orthosis, and low-intensity ultrasound stimulation, for a minimum of 4 months. Patients had experienced symptoms for a mean duration of 12 months. Clinical and radiological union occurred at a mean of 2.7 and 3 months, respectively. The mean duration for return to sports after surgery was 4 months. At last follow-up, all patients had full range of motion at the knee and ankle joints and were satisfied with the results. One patient developed bursitis at the tibial nail insertion site that was resolved with a steroid injection. Another patient sustained a traumatic fracture of the distal tibia 1 year after intramedullary nailing of the tibial stress fracture. This fracture healed with nonoperative treatment. No other complications were observed. CONCLUSION: Intramedullary nailing of the tibia for chronic stress fracture has a high union rate, allows for a low complication rate, and allows for an early return to competitive sports. This procedure is an excellent alternative treatment for those fractures that have failed nonsurgical treatment.

Evaluation and treatment of chronic ankle pain.

The evaluation and treatment of chronic ankle pain presents a challenge to the orthopaedic surgeon. A detailed history helps to determine causative factors resulting from earlier trauma or surgery. A careful physical examination and radiographic studies also are helpful in making an accurate diagnosis, which is the basis for choosing a specific and effective treatment regimen.

Immediate, full weightbearing cast treatment of acute Achilles tendon ruptures: a long-term follow-up study.

Thirty-two patients with 33 ruptures were available for comprehensive evaluation, which included a questionnaire, physical examination, and manual as well as objective (Kin-Com) strength testing. Data excluding Kin-Com strength testing were available in an additional seven patients with seven ruptures; hence, the data are based on these 39 patients with 40 ruptures. Five patients with eight ruptures were excluded from strength testing but were included in our rerupture data, which totaled 44 patients with 48 ruptures. All patients were treated using a standard protocol by a single surgeon. All patients were treated within 11 days of injury, and the average follow-up was 55 months (range, 24-133). Ninety-five percent (38/39) of patients were completely satisfied with their treatment. The American Orthopaedic Foot and Ankle Society (AOFAS) ankle hindfoot scores were >80 in 97.4% of patients, and the rerupture rate was 6.25% (3/48). Calf circumference and ankle range of motion were not statistically different between the two legs. The injured leg tended to be slightly weaker than the uninjured leg, but the difference was not significant for most of the quantitative strength measurements. This study provides data for the Achilles rupture patient who asks, "Is there a treatment option other than surgery?" In addition, the standardized protocol can be utilized to obtain successful outcomes in those who choose nonoperative treatment or are poor surgical candidates.

Variations on the insertion of the posterior tibialis tendon: a cadaveric study.

This cadaveric study specifically investigates the variations on the insertion of the posterior tibialis tendon (PTT) in the foot, a topic which is not well defined in anatomy discussions. The PTT insertion sites from 11 fresh-frozen cadaver feet (10 subjects) were evaluated. There were three distinct bands of the PTT seen in all specimens with variations on the insertion to the spring ligament (4/11), fifth metatarsal base (7/11), flexor hallucis brevis (9/11), and peroneus longus (4/11). This study also found a distinct slip to the abductor hallucis in five of the specimens.

Surgical treatment for posterior tibial tendonitis in young competitive athletes.

The surgical treatment and outcome of eight competitive athletes with posterior tibial tendonitis refractory to nonsurgical treatment was retrospectively reviewed. There were one professional, five collegiate, and two high school athletes with an average age of 22 years. All athletes had failed nonsurgical treatments including nonsteroidal anti-inflammatory drugs, physical therapy, custom shoe inserts, walking boots, or casts. Surgical debridement of the posterior tibial tendon was performed in all eight. Postoperatively, the athletes were treated in a cast or walking boot for 4 weeks. At the most recent follow-up, averaging 22 months, seven athletes had returned to full sports participation without difficulty. One athlete indicated that he had occasional pain with medial to lateral stressing maneuvers but could participate in his sport. Overall, each athlete reported satisfaction with his or her outcome with seven rating it as "excellent" and one as "good."