Syndesmosis fixation in supination-external rotation ankle fractures. Long-Term results of a prospective randomised study.

BACKROUND: The clinical relevance and treatment of syndesmosis injury in supination-external rotation (SER) ankle fractures are controversial. METHODS: After malleolar fixation 24 SER 4 ankle fracture patients with unstable syndesmosis in external rotation stress test were randomised to syndesmosis transfixation with a screw (13 patients) or no fixation (11 patients). Mean follow-up time was 9.7 years (range, 8.9-11.0). The primary outcome measure was the Olerud-Molander Ankle Outcome Score (OMAS). Secondary outcome measures included ankle mortise congruity and degenerative osteoarthritis, 100-mm visual analogue scale for function and pain, RAND 36-Item Health Survey, and range of motion. RESULTS: Mean OMAS in the syndesmosis transfixation group was 87.3 (SD 15.5) and in the no-syndesmosis-fixation group 89.0 (SD 16.0) (difference between means 1.8, 95% CI -10.4-14.0, P = 0.76). There were no differences between the two groups in secondary outcome measures. CONCLUSION: With the numbers available, SER 4 ankle fractures with unstable syndesmosis can be treated with malleolar fixation only, with good to excellent long-term functional outcome.

Suture button versus syndesmosis screw fixation in pronation-external rotation ankle fractures: A minimum 6-year follow-up of a randomised controlled trial.

OBJECTIVE: Randomised controlled trials (RCT) with short-term follow-ups have shown that, in treatment of syndesmosis injuries, a suture button device (SB) resulted in better radiographic and functional outcome compared to syndesmosis screw fixation (SS). However, only one RCT has reported long-term results; thus, the syndesmosis malreduction rates for both implants might increase during longer follow-up. The primary objective of this RCT was to evaluate the maintenance of syndesmosis reduction with the SS compared to the SB fixation in patients during a minimum follow-up of 6-years. The secondary objectives were to assess the post-traumatic osteoarthritis (OA) grade and the functional outcome. PATIENTS AND METHODS: At Oulu University Hospital, between January 2010 and December 2011, we enrolled 43 patients with Lauge-Hansen pronation-external rotation type 4/Weber C, ankle fractures with unstable syndesmosis. Patients were randomised to treatment with either a single 3.5-mm tricortical SS (22 patients) or an SB (21 patients). The mean follow-up was 7.1 years (range, 6.2-7.9). Syndesmosis reduction and OA grade was assessed with standing cone-beam computed tomography (CBCT) of both ankles. Malreduction was defined as >2 mm side-to-side difference in the mean width of the syndesmosis. OA was graded according to the Morrey & Wiedeman classification. The Olerud-Molander Ankle Outcome Score (OMAS) and a quality of life questionnaire (RAND 36-Item Health Survey) were used to evaluate functional outcome. RESULTS: Two syndesmoses in the SS group and one in the SB group were malreduced (P = 0.58). Moderate OA after a mean of 7 years post-injury was common. In the SS and SB groups, 9 of 16 and 11 of 13 patients, respectively, had one or more grades serious OA in the injured ankle than in the uninjured ankle (P = 0.11). The mean OMAS was 88 in the SS group and 78 in the SB group (difference between means 7.1, 95% CI: -7.0-21.1, P = 0.32). The RAND-36 results did not differ between groups. CONCLUSION: The SS and SB maintained syndesmosis reduction equally well during follow-up. Our study findings also suggest that both methods result in moderate OA rates and the functional outcome is comparable between these two syndesmosis fixation methods.

Surgery versus non-operative treatment for ER-stress unstable Weber-B unimalleolar fractures: a study protocol for a prospective randomized non-inferiority (Super-Fin) trial.

Roughly two-thirds of ankle fractures are unimalleolar injuries, the Weber B-type fibula fracture being by far the most common type. Depending on the trauma and the accompanying soft-tissue injury, these fractures are either stable or unstable. Current clinical practice guidelines recommend surgical treatment for unstable Weber B-type fibula fractures. An ongoing randomized, parallel group, non-inferiority trial comparing surgery and non-operative treatment for unstable Weber B-type ankle fractures with allocation ratio 1:1. The rationale for non-inferiority design is as follows: By being able to prove non-inferiority of non-operative treatment, we would be able to avoid complications related to surgery. However, the primary concern related to non-operative treatment is increased risks of ankle mortise incongruency, leading to secondary surgery, early post-traumatic osteoarthritis and poor function. After providing informed consent, 126 patients aged 16 years or older with an unimalleolar Weber B-type unstable fibula fracture were randomly assigned to surgery (open reduction and internal fixation) or non-operative treatment (6-week cast immobilization). We have completed the patient enrolment and are currently in the final stages of the 2-year follow-up. The primary, non-inferiority outcome is the Olerud-Molander Ankle Score (OMAS) at 2 years (primary time point). The predefined non-inferiority margin is set at 8 OMAS points. Secondary outcomes include the Foot and Ankle Score, a 100 mm Visual Analogue Scale for function and pain, the RAND-36-Item Health Survey for health-related quality-of-life, the range-of-motion of the injured ankle, malunion (ankle joint incongruity) and fracture union. Treatment-related complications and harms; symptomatic non-unions, loss of congruity of the ankle joint, reoperations and wound infections will also be recorded. We hypothesize that non-operative treatment yields non-inferior functional outcome to surgery, the current standard treatment, with no increased risk of harms.

Stability-Based Classification of Ankle Fractures-The Long-Term Outcome After 11-13 Years of Follow-up.

OBJECTIVES: To assess the long-term functional and radiological outcome, as well as complications and reoperations of ankle fracture patients treated based on stability classification. DESIGN: Retrospective. SETTING: Main Trauma Center, University Hospital. PATIENTS AND INTERVENTIONS: One hundred sixty patients were treated based on stability classification. After a mean follow-up of 12 years, 102 patients were assessed; 67 visited the outpatient clinic, and standard standing ankle radiographs were taken; osteoarthritis (OA) was graded according to Kellgren-Lawrence classification. The remaining patients (n = 35) were followed up via mail or telephone. The complications and reoperations of all 160 patients were collected from electronic patient records. MAIN OUTCOME MEASURES: Olerud-Molander ankle score, foot and ankle outcome score, visual analog scale, RAND-36 item health survey, range-of-motion measurements, and Kellgren-Lawrence OA classification. RESULTS: Very good to excellent ankle functional outcome was reported by 96% of the stable fracture group [mean Olerud-Molander ankle score (OMAS), 92; range, 20-100] and 82% of the unstable group (mean OMAS, 86; range, 30-100). For patients with an unstable fracture, OMAS and VAS pain scores significantly improved from 2 to 12 years, even though higher grades of radiologic OA were found. No patients with fractures deemed stable needed operative intervention even in the long-term follow-up. Instead, 30% of the operatively treated patients underwent reoperation during the long follow-up. CONCLUSIONS: The treatment of ankle fractures based on stability-based classification led to predictable, functionally good outcomes even during long-term follow-up. Ankle fractures can reliably be deemed stable based on this classification and treated without failures in the long term. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Fibular nailing for fixation of ankle fractures in patients at high risk of surgical wound infection.

BACKGROUND: Postoperative infection is a severe complication after operative treatment of ankle fractures, associated with age, comorbidities, and severe soft tissue injuries. We assessed the efficacy of intramedullary fibular nailing for treating ankle fractures in patients at high risk of wound complications. METHODS: 41 high-risk patients were included in the study. We retrospectively reviewed the medical records to assess the risk profile, the treatment data, and possible infections and re-operations. After a minimum of 2 years eight patients had died, three had advanced-staged dementia and two were lost to follow-up. Remaining 28 patients reported the functional outcome and QoL through patient-reported questionnaires. Radiographs and cone-beam computed tomography were performed, as well as range-of-motion was measured. RESULTS: No surgical wound infections were found. The mean Olerud-Molander score was 67 points (SD 28 [20-100]). The osteoarthritis stages and the range-of-motion were significantly different between the injured and uninjured ankles, but we detected no significant effect on the QoL. CONCLUSION: Intramedullary fibular fixation appeared to be a safe treatment choice for ankle fractures in high-risk patients. LEVEL OF EVIDENCE: IV.

The functional outcome and quality of life after treatment of isolated medial malleolar fractures.

BACKGROUND: The optimal treatment of isolated medial malleolar fractures is widely debated. The aim of this study is to analyse the different treatment methods. METHODS: The study included 137 patients with an isolated medial malleolar fracture treated at our hospital between 2000 and 2010. Functional outcome was assessed using Olerud-Molander score and health-related quality-of-life (HRQoL) was measured with RAND36 item health survey. Patients were sent follow-up questionnaires after an average of 9.5 years (4.3-15.2) from the date of treatment. RESULTS: The overall improvement in function was equivalent in both operative and non-operative treatment groups. However, the outcome scores declined in both groups as the primary displacement increased, regardless of the treatment method. HRQoL was similar in both groups. CONCLUSIONS: If maximal fracture displacement is ≤2mm, isolated medial malleolar fractures can be treated non-operatively with good outcome, but the operative treatment may also be performed without serious complications. The degree of fracture displacement is an independent risk factor for inferior functional result, regardless of the treatment method. LEVEL OF EVIDENCE: Therapeutic level of evidence: IV, retrospective cohort study.

Dynamic Stress Testing Is Unnecessary for Unimalleolar Supination-External Rotation Ankle Fractures with Minimal Fracture Displacement on Lateral Radiographs.

BACKGROUND: This study aimed to identify factors from standard radiographs that contributed to the stability of the ankle mortise in patients with isolated supination-external rotation fractures of the lateral malleolus (OTA/AO 44-B). METHODS: Non-stress radiographs of the mortise and lateral views, without medial clear space widening or incongruity, were prospectively collected for 286 consecutive patients (mean age, 45 years [range, 16 to 85 years]), including 144 female patients (mean age, 50 years [range, 17 to 85 years]) and 142 male patients (mean age, 40 years [range, 16 to 84 years]) from 2 trauma centers. The radiographs were analyzed for fracture morphology by 2 orthopaedic surgeons, who were blinded to each other's measurements and to the results of external rotation stress radiographs (the reference for stability). Factors significantly associated with ankle mortise stability were tested in multiple logistic regression. Receiver operating characteristic analyses were performed for continuous variables to determine optimal thresholds. A sensitivity of >90% was used as the criterion for an optimal threshold. RESULTS: According to external rotation stress radiographs, 217 patients (75.9%) had a stable injury, defined as that with a medial clear space of <5 mm. Independent factors that predicted stable ankle mortise were female sex (odds ratio [OR], 2.5 [95% confidence interval (CI), 1.4 to 4.6]), a posterior diastasis of <2 mm (corresponding with a sensitivity of 0.94 and specificity of 0.39) on lateral radiographs (OR, 10.8 [95% CI, 3.7 to 31.5]), and only 2 fracture fragments (OR, 7.3 [95% CI, 2.1 to 26.3]). When the posterior diastasis was <2 mm and only 2 fracture fragments were present, the probability of a stable ankle mortise was 0.98 for 48 female patients (16.8%) and 0.94 for 37 male patients (12.9%). CONCLUSIONS: Patients with noncomminuted lateral malleolar fractures (85 patients [29.7%]) could be diagnosed with a stable ankle mortise without further stress testing, when the fracture line widths were <2 mm on lateral radiographs. LEVEL OF EVIDENCE: Prognostic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Rotational Dynamics of the Talus in a Normal Tibiotalar Joint as Shown by Weight-Bearing Computed Tomography.

BACKGROUND: The aim of this study was to investigate the normal anatomy and rotational dynamics of the talus in the tibiotalar joint using weight-bearing cone-beam computed tomography (WBCT). METHODS: In a cross-sectional study of thirty-two healthy subjects divided into two age groups (twenty-six to thirty-six years of age and sixty to sixty-four years of age), low-dose WBCT scans of both uninjured ankles were obtained. The rotation of the talus, medial clear space, anterior and posterior widths of the tibiotalar joint, translation of the talus, and talar tilt were measured. The primary outcome measures were intersubject and intrasubject (bilateral) variation of the talar movements between maximal internal and external rotation. The secondary outcome measures were the effect of sex and age on the movements of the talus. RESULTS: When the ankle is rotated, the talus rotates a mean of 10° with no substantial widening of the medial clear space. All of the measured values changed subtly but statistically significantly between maximal internal and maximal external rotation, with mean changes of 10° (standard deviation [SD] = 5.8°) in talar rotation (p = 0.006), 2.0° (SD = 1.5°) in talar tilt (p = 0.0015), -0.2 mm (SD = 0.5 mm) in the medial clear space (p = 0.01), 0.9 mm (SD = 0.8 mm) in the anterior width of the tibiotalar joint (p = 0.003), -0.4 mm (SD = 0.9 mm) in the posterior width of the tibiotalar joint (p = 0.011), and 2.9 mm (SD = 2.2 mm) in translation of the talus (p = 0.002). Intersubject variation was large, but there was very little intrasubject variation in the total rotational range of motion. There were no differences between men and women with regard to any of the measurements. CONCLUSIONS: This study provides reference values with which to evaluate the dynamics of the normal tibiotalar joint in order to clarify rotational stability of the ankle mortise. The internal control of the contralateral ankle seems to be a better reference than population-based normal values.

Rotational Dynamics of the Normal Distal Tibiofibular Joint With Weight-Bearing Computed Tomography.

BACKGROUND: The normal distal tibiofibular joint is strongly stabilized by the syndesmosis, where previous cadaveric, biomechanical studies demonstrated only minimal widening and posterior translation of the fibula in external rotation of the ankle. However, little is known about normal rotational dynamics of the distal tibiofibular joint in upright weight-bearing conditions. The purpose of this study was to investigate the normal anatomy and rotational dynamics of the distal tibiofibular joint under physiological conditions on weight-bearing cone beam computed tomography (WBCT). METHODS: In a cross-sectional study of 32 subjects, low-dose WBCT scans of uninjured bilateral ankles were performed. Normal intersubject and intrasubject variation in neutral position and changes in maximal internal and external rotation of the ankle were studied. Sagittal translation of the fibula, anterior and posterior widths of the distal tibiofibular syndesmosis, tibiofibular clear space (TFCS), and rotation of the fibula were measured. RESULTS: In the neutrally loaded ankle, the fibula was located anteriorly in the tibial incisura in 88% of the subjects. When the ankle was rotated, mean anteroposterior motion was 1.5 mm and mean rotation of the fibula was 3 degrees. There was no significant change in TFCS between internal and external rotation. Large intersubject variation was detected, but intrasubject variation between ankles was less than 1 mm and 1 degree. CONCLUSIONS: This study provides reference values to evaluate the dynamics of the normal distal tibiofibular joint. The internal control of the contralateral ankle seemed to be a better reference than the population-based normal values. CLINICAL RELEVANCE: The current study provides the reference values to evaluate the rotational dynamics of a normal distal tibiofibular joint.

The mechanism of action of induced membranes in bone repair.

BACKGROUND: Inducement of foreign-body granulation tissue is a relatively novel therapeutic modality in bone repair. A two-stage bone reconstruction method, known as the Masquelet technique, combines inducement of a granulation tissue membrane and subsequent bone autografting as a biphasic technique allowing reconstruction of large bone defects. In light of their already well-characterized osteogenesis-improving capabilities in animals, we performed this translational study to investigate these membranes in patients. METHODS: Fourteen patients with complicated fractures and bone defects were randomly selected for this study. Biopsy samples of foreign-body-induced membranes were collected at different time points during scheduled surgical procedures. The membranes were co-cultured with mesenchymal stromal cells, and differentiation into the osteoblastic lineage was assessed by measuring alkaline phosphatase activity, aminoterminal propeptide of type-I procollagen (PINP) production, and Ca2+ concentration. Histological characteristics were evaluated with image analysis. Quantitative reverse transcription polymerase chain reaction was used to measure vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and type-I collagen (Col-1) expression. RESULTS: The induced membranes were characterized histologically by maturating vascularized fibrous tissue. The vascularization was greatest in one-month-old samples and decreased to <60% in three-month-old samples. One-month-old membrane samples had the highest expression of VEGF, IL-6, and Col-1, whereas two-month-old membranes expressed <40% of the levels of the one-month-old membranes. Specific alkaline phosphatase activity, PINP production, and Ca2+ concentration were increased in co-cultures when a membrane sample was present. In cultures of one-month-old membranes, PINP production was more than two times and Ca2+ deposition was four times higher than that in cultures of two-month-old membranes. CONCLUSIONS: The induced membranes have osteogenesis-improving capabilities. These capabilities, however, appear to decrease over time. We speculate that the optimal time for performing second-stage surgery may be within a month after implantation of foreign material.

Monitoring mitochondrial inner membrane potential for detecting early changes in viability of bacterium-infected human bone marrow-derived mesenchymal stem cells.

INTRODUCTION: One of the most challenging safety issues in the manufacture of cell based medicinal products is the control of microbial risk as cell-based products cannot undergo terminal sterilization. Accordingly, sensitive and reliable methods for detection of microbial contamination are called for. As mitochondrial function has been shown to correlate with the viability and functionality of human mesenchymal stem cells (hMSCs) we have studied the use of a mitochondrial inner membrane potential sensitive dye for detecting changes in the function of mitochondria following infection by bacteria. METHODS: The effect of bacterial contamination on the viability of bone marrow-derived mesenchymal stem cells (BMMSCs) was studied. BMMSC lines were infected with three different bacterial species, namely two strains of Pseudomonas aeruginosa, three strains of Staphylococcus aureus, and three strains of Staphylococcus epidermidis. The changes in viability of the BMMSCs after bacterial infection were studied by staining with Trypan blue, by morphological analysis and by monitoring of the mitochondrial inner membrane potential. RESULTS: Microscopy and viability assessment by Trypan blue staining showed that even the lowest bacterial inocula caused total dissipation of BMMSCs within 24 hours of infection, similar to the effects seen with bacterial loads which were several magnitudes higher. The first significant signs of damage induced by the pathogens became evident after 6 hours of infection. Early changes in mitochondrial inner membrane potential of BMMSCs were evident after 4 hours of infection even though no visible changes in viability of the BMMSCs could be seen. CONCLUSIONS: Even low levels of bacterial contamination can cause a significant change in the viability of BMMSCs. Moreover, monitoring the depolarization of the mitochondrial inner membrane potential may provide a rapid tool for early detection of cellular damage induced by microbial infection. Accordingly, mitochondrial analyses offer sensitive tools for quality control and monitoring of safety and efficacy of cellular therapy products.

Tezosentan inhibits uptake of proinflammatory endothelin-1 in stenotic aortic valves.

BACKGROUND AND AIM OF THE STUDY: Aortic valve stenosis (AS) is an actively regulated pathobiological process which has an inflammation origin, and manifests as an accumulation of lipids and, ultimately, calcification of the aortic valve tissue. Increased plasma levels of the proinflammatory factor endothelin-1 (ET-1) have been reported in AS. Moreover, increased tissue levels of ET-1 and its ET(A) receptor, which mediates the fibrotic and proliferative effects of ET-1, have been reported in stenotic aortic valves. The study aim was to determine whether endothelin receptor antagonism has an effect on the supposed receptor-mediated uptake of ET-1 to aortic valves when ET-1 may be involved in the pathogenesis of AS. METHODS: By using valve tissue explants in culture, it was determined whether the ET(A)-ET(B) receptor antagonist tezosentan was capable of reducing the uptake of 125I-labeled ET-1 to human aortic valves. Aortic valves were obtained from 16 patients (11 males, five females; mean age 71 +/- 11.2 years) and from two donors without AS (as controls) at the time of aortic valve or aortic root surgery. Valve tissue samples were cultured in ET-1 (10 nmol/l), in the presence or absence of tezosentan (10 nmol/l). RESULTS: ET-1 uptake was found to be pronounced in the calcified areas of the valve, and tezosentan markedly reduced the receptor-mediated uptake of 125I-labeled ET-1. The inhibitory effect was most evident in the well-calcified part of the valve. The gene expression levels of the ET receptors ET(A) and ET(B) were unaltered in human aortic valves during a four-day exposure to the antagonist. CONCLUSION: The ability of the ET(A)-ET(B) receptor antagonist tezosentan to inhibit ET-1 uptake in valve tissue suggests that continuous ET antagonist therapy might serve as new strategy to slow down the pathophysiological processes of AS.

CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells.

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.

Attempt to treat congenital pseudarthrosis of the tibia with mesenchymal stromal cell transplantation.

BACKGROUND AIMS: Congenital pseudarthrosis of the tibia (CPT) caused by neurofibromatosis type 1 (NF1) is a refractory disease occurring in childhood. We present two cases that had failed all earlier treatment attempts and, as a last treatment attempt, the patients were chosen to receive mesenchymal stromal cell (MSC) transplantation prior to amputation. METHODS: The MSC from bone marrow (BM) were harvested from the iliac crest and cultured in osteoinductive medium for 3 weeks. The cultured MSC were injected in solution into BM canals of the tibia and around the resection line or bone defect in a 3-dimensional collagen sponge scaffold. After the MSC transplantation, the patients were monitored during a 10-month follow-up period. In both cases, bone formation at the pseudarthrosis site was observed and two of three treated bone defects healed. For clinical reasons not related to cell transplantation, such as new infection and pseudarthrosis and severe shortening of the leg, both extremities were finally amputated and bone samples were analyzed to evaluate MSC therapy effect and safety. RESULTS: MSC transplantation normalized bone remodeling, promoted bone resorption and improved the overall structure of bone. The number of osteoclasts in the cortical bone was 2-fold higher compared with the monitored situation before MSC transfer. In addition, the mineral content of the bone improved after transplantation. We could see no sign of aberrant bone formation or malignant transformation. CONCLUSIONS: Our data suggest that MSC transplantation is a possibility for treatment of CPT caused by NF1 in less severe cases without adjunct defects.

Mitochondrial function determines the viability and osteogenic potency of human mesenchymal stem cells.

Advanced therapies medicinal products (ATMPs) have introduced innovative cell-based products. However, the regulatory demands for characterization of ATMPs are currently unable to adequately address the safety of such products. As recent studies have emphasized the role of mitochondria in the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we have studied in detail the viability and osteogenic differentiation potency of the hMSCs intended for use as ATMPs based on analyses of the mitochondrial inner membrane potential (DeltaPsi(m)). Flow cytometric measurement of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), propidium iodide fluorescence, and AnnexinV was employed to determine DeltaPsi(m), plasma membrane integrity, and organization of phosphatidylserine in plasma membrane, respectively, in cultured hMSCs. Apoptosis was induced by incubating cells at critical concentration (20 muM) of menadione. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as an indicator for cell proliferation and alkaline phosphatase activity and calcium deposition as indicators of osteogenic differentiation. Based on JC-1 fluorescence, cell morphology, organization of phosphatidylserine, and plasma membrane integrity, we could sort cells into four categories that represented different cell quality. A strong correlation between JC-1 and osteogenic differentiation was demonstrated for the first time and thus this analytical tool is suitable not only to determine cell viability but also to predict osteogenic differentiation of hMSC.

Congenital pseudarthrosis of neurofibromatosis type 1: impaired osteoblast differentiation and function and altered NF1 gene expression.

Three patients with neurofibromatosis 1 (NF1) were operated for congenital pseudarthrosis (PA) of the tibia. Three non-NF1 patients served as reference. Both NF1 mRNA and protein were detected in the PAs and in rows of osteoblasts and numerous osteoclasts next to the NF1-related PA arguing against inactivation of both NF1 alleles in the resident cells. Analyses on mesenchymal stem cells (MSCs) cultured from the red bone marrow of 1) next to PA of the affected NF1 tibiae, 2) the non-affected NF1 iliac crest of the same patients, and from 3) non-NF1 bone marrow demonstrated that the potential to form bone in vitro was the lowest in cells from the affected NF1-tibiae. The latter cells also displayed reduced levels of NF1 mRNA and protein, and upregulated phosphorylated p44/42 MAPK levels, consistent with an upregulated Ras-pathway. An exhaustive NF1 gene analysis detected constitutional mutation in each case, but no second hits or loss of heterozygosity were found. However, one patient displayed a mutation resulting in two potential active splice sites ultimately affecting exon 6. Interestingly, only one of the respective transcripts was detected in cells from the iliac crest, but two novel transcripts were detected in MSCs cultured from site next to PA. This finding may identify a novel mechanism how a single NF1 gene mutation may exert distinct effects on separate anatomical locations. The molecular pathogenesis of NF1-related PA apparently may not be entirely explained by second mutations or loss of heterozygosity of the NF1 gene.

Inhibition of cyclooxygenase-2 down-regulates osteoclast and osteoblast differentiation and favours adipocyte formation in vitro.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenases (COX) and are widely used for post-trauma musculoskeletal analgesia. In animal models, NSAIDs have been reported to delay fracture healing and cause non-union, possibly due to the drug-induced inhibition of osteoblast recruitment and differentiation. To further investigate the cellular effects of these drugs in the context of bone healing, we examined the effects of COX-1 inhibitor indomethacin and COX-2 inhibitors, parecoxib and NS398 on osteoclast and osteoblast differentiation and activity in vitro. We discovered that all tested COX-inhibitors significantly inhibited osteoclast differentiation, by 93%, 94% and 74% of control for 100 microM indomethacin, 100 microM parecoxib and 3 microM NS398, respectively. Furthermore, inhibition of COX-2 reduced also the resorption activity of mature osteoclasts. All tested COX-inhibitors also significantly inhibited osteoblast differentiation from human mesenchymal stem cells. Simultaneously, the number of adipocytes was significantly increased. The adipocyte covered areas in the cultures with 1 microM indomethacin, 1 microM parecoxib and 3 microM NS398 were 9%, 29% and 24%, respectively, as compared with 6% in the control group. This data suggests that COX-2 inhibition disturbs bone remodelling by inhibiting osteoclast differentiation and diverting stem cell differentiation towards adipocyte lineage instead of osteoblast lineage. In conclusion, our results further suggest cautious use of COX-2 inhibitors after osseous trauma.

Calcification and cellularity in human aortic heart valve tissue determine the differentiation of bone-marrow-derived cells.

Human bone-marrow-derived mesenchymal stem cells (MSC) are responsible the remodeling of human tissue. However, damaged aortic valves are lack the ability to regenerate which is an active cell-mediated process. Diseased aortic valve remodeling has similarities even to bone formation. In this study, the prerequisites for cultured MSCs to undergo osteoblastic differentiation on aortic valves were explored. An ex vivo model using a human aortic valve microenvironment was developed. The expression of type I procollagen, alkaline phosphatase activity, osteocalcin secretion and osteocalcin immunostaining were studied to evaluate the induction of osteogenesis of the MSCs on noncalcified and calcified human aortic valves. Aortic valves were exposed to freeze-thaw injury to devitalize valves in order to separately study the role of valve matrix vs. endothelial cells in the explants. Thus, valves were assigned to 1 of 4 treatment groups: noncalcified uninjured valves, calcified uninjured valves, noncalcified injured and calcified injured. Finally, valves were decalcified to separately explore the effect of a calcified matrix on the osteogenesis. In this co-culture system, the noncalcified uninjured valves inhibited osteogenesis of MSCs, whereas the calcified valves promoted differentiation towards osteoblastic lineage. Devitalization of the valve matrix inflicted a significant increase in the osteogenesis of co-cultured MSCs. Calcified matrix in the valves seemed to have a role in the spontaneous osteogenesis of the MSCs. This spontaneous matrix induced differentiation of MSCs into osteoblast lineage could not be inhibited by pravastatin, indomethacin or tetracycline. In conclusion, these results suggest that interactions between MSCs and aortic valve matrix components and cells modulate MSC phenotype in this environment. Further studies are required to characterize this interesting phenomenon in greater detail.

Estrogen receptor alpha genotype confers interindividual variability of response to estrogen and testosterone in mesenchymal-stem-cell-derived osteoblasts.

Hormone replacement therapy is effectively used to prevent postmenopausal bone loss. Variation in response to the therapy is, however, frequently seen. In addition, the direct effects of sex steroids on isolated human bone marrow stromal cells have been reported to vary depending on the donor, but the biological mechanisms are not understood. The aim of this study was to investigate the effects of 17beta-estradiol (E2) and testosterone in human-bone-marrow-derived mesenchymal stem cell (MSC) cultures from both female and male donors of various ages. The osteoblast differentiation capacity and activity of the MSCs were quantified in vitro by measuring alkaline phosphatase activity and calcium deposition. We show here that also the osteoblast responses of MSCs to sex hormones vary widely depending on the donor. When the results from all donors were analyzed together, treatment with E2 increased calcium deposition significantly by MSCs of both sexes but ALP activity only in the male MSCs. Testosterone had no effect on ALP activity nor calcium deposition in either sex. To further characterize the individual variation, we investigated estrogen receptor alpha PvuII restriction site polymorphism with PCR. Restriction fragment-length polymorphism was assigned as P or non-P, P signifying the absence of the restriction site. Our results indicate that higher basal osteoblast differentiation capacity of MSCs is associated with the presence of the P allele in females, whereas higher response to sex steroids treatment is associated with the non-P allele. These results could help explain the contradictory effects of E2 on osteoblasts in vitro and might also provide new insights to understanding the differences in responses to hormone replacement therapy.