Mechanotransduction in striated muscle via focal adhesion kinase

Contractile tissues demonstrate a pronounced capacity to remodel their composition in response to mechanical challenges. Descriptive evidence suggests the upstream involvement of the phosphotransfer enzyme FAK (focal adhesion kinase) in the molecular control of load-dependent muscle plasticity. Thereby FAK evolves as a myocellular transducer of mechanical signals towards downstream transcript expression in myofibres. Recent advances in somatic gene therapy now allow the exploration of the functional involvement of this enzyme in mechanotransduction in intact muscle.

Sensitivity of osteoblasts, fibroblasts, bone marrow cells, and dendritic cells to 5-aminolevulinic acid based photodynamic therapy

INTRODUCTION: Photodynamic therapy with 5-aminolevulinic acid (5-ALA-PDT) exerts cell type specific effects on target cells. Since chondrocytes were found to be more resistant than osteoblasts to 5-ALA-PDT, the pre-treatment of osteochondral grafts with 5-ALA-PDT may represent a means to devitalize the osseous portion while maintaining functional cartilage. The present study was designed to determine the effects of 5-ALA-PDT in vitro on cell populations residing in skeletal tissues. METHODS: Osteoblasts, fibroblasts, bone marrow cells, and dendritic cells were incubated with 0.5 mM 5-ALA for 4 h. Protoporphyrin IX (PpIX) accumulation and after exposure to light cellular functions were assessed for up to 6 days. RESULTS: Accumulation of PpIX reached a plateau at 0.5 mM in osteoblasts, fibroblasts, and dendritic cells, and at 2.0 mM in bone marrow cells. At 0.5 mM 5-ALA, similar responses to illumination were observed in all cells with a survival rate of less than 12% at a light dose of 20 J/cm(2). The function of osteoblasts (proliferation, levels of mRNA encoding collagen type I, alkaline phosphatase activity) and fibroblasts (proliferation, levels of mRNAs encoding collagens type I and III) was not affected, when the cells were treated with 5-ALA and light doses of < or =10 J/cm(2). Paralleling the reduction of viable cells after 5-ALA-PDT, the capacity of dendritic cells to stimulate T cells in a mixed leukocyte reaction decreased to 4+/-2% at 20 J/cm(2). CONCLUSION: The investigated cell types were sensitive to 5-ALA-PDT and the residual cell debris did not elicit an allogenic response. These findings, together with the resistance of chondrocytes to 5-ALA-PDT, encourage the further investigation of this protocol in the pretreatment of osteochondral allografts.

Impact of pore size on the vascularization and osseointegration of ceramic bone substitutes in vivo

The repair of bone defects with biomaterials depends on a sufficient vascularization of the implantation site. We analyzed the effect of pore size on the vascularization and osseointegration of biphasic calcium phosphate particles, which were implanted into critical-sized cranial defects in Balb/c mice. Dense particles and particles with pore sizes in the ranges 40-70, 70-140, 140-210, and 210-280 mum were tested (n = 6 animals per group). Angiogenesis, vascularization, and leukocyte-endothelium interactions were monitored for 28 days by intravital microscopy. The formation of new bone and the bone-interface contact (BIC) were determined histomorphometrically. Twenty-eight days after implantation, the functional capillary density was significantly higher with ceramic particles whose pore sizes exceeded 140 mum [140-210 mum: 6.6 (+/-0.8) mm/mm(2); 210-280 mum: 7.3 (+/-0.6) mm/mm(2)] than with those whose pore sizes were lesser than 140 mum [40-70 mum: 5.3 (+/-0.4) mm/mm(2); 70-140 mum: 5.6 (+/-0.3) mm/mm(2)] or with dense particles [5.7 (+/-0.8) mm/mm(2)]. The volume of newly-formed bone deposited within the implants increased as the pore size increased [40-70 mum: 0.07 (+/-0.02) mm(3); 70-140 mum: 0.10 (+/-0.06) mm(3); 140-210 mum: 0.13 (+/-0.05) mm(3); 210-280 mum: 0.15 (+/-0.06) mm(3)]. Similar results were observed for the BIC. The data demonstrates pore size to be a critical parameter governing the dynamic processes of vascularization and osseointegration of bone substitutes. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2007.

Influence of preoperative functional status on outcome after total hip arthroplasty

BACKGROUND: International registries with large, heterogeneous patient populations provide excellent research opportunities for studying factors that influence treatment outcomes after total hip arthroplasty. In the present study, we used a European multinational database to investigate whether there is an association between three functional variables (preoperative pain, mobility, and motion) and functional outcome. METHODS: We performed a retrospective cohort study on preoperative and follow-up clinical data that were prospectively entered into the International Documentation and Evaluation System European hip registry between 1967 and 2002. The inclusion criteria for this study were an age of more than twenty years, an underlying diagnosis of osteoarthritis, and a Charnley class-A functional designation at the time of surgery. A total of 12,925 patients (13,766 total hip arthroplasties) who met these criteria were entered into the analysis. Three functional variables (pain, mobility, and motion) that were assessed preoperatively were evaluated postoperatively at various follow-up examinations for a maximum of ten years. RESULTS: Six thousand four hundred and one patients could walk longer than ten minutes preoperatively; of these, 57.1% had a walking capacity of more than sixty minutes at the time of the most recent follow-up. In comparison, 6896 patients had a preoperative walking capacity of less than ten minutes and only 38.9% of these patients could walk more than sixty minutes at the time of the most recent follow-up. The difference was significant (p < 0.01). Similarly, 10,375 patients had a preoperative hip flexion range of >70 degrees ; of these, 74.7% had a flexion range of >90 degrees at the time of the most recent follow-up. In comparison, 2793 patients had a preoperative hip flexion range of <70 degrees and only 62.6% of these patients had a flexion range of >90 degrees at the time of the most recent follow-up. The difference was also significant (p < 0.01). Lasting, complete, or almost complete pain relief was achieved by >80% of the patients following total hip arthroplasty regardless of their preoperative categorization of pain. CONCLUSIONS: Patients with poor preoperative walking capacity and hip flexion are less likely to achieve an optimal outcome with regard to walking and motion. In contrast, there is no correlation between the preoperative pain level and pain alleviation, which is generally good and long-lasting after total hip arthroplasty.

Reattachment of the tuberosities with cable wires and bone graft in hemiarthroplasties done for proximal humeral fractures with cable wire and bone graft: 58 patients with a 22-month minimum follow-up

OBJECTIVE: The stability of 2 fixation techniques for the tuberosities in patients with 3- or 4-part proximal humerus fractures treated with hemiarthroplasties was compared. DESIGN: Retrospective review of a nonrandomized sequential series of patients. SETTING: Level I university orthopaedic surgery department. PATIENTS: A consecutive series of 58 patients (average age, 64 years) from 1990 to 1999 with 3- and 4-part fractures of the proximal humerus. INTERVENTION: In group 1, 31 patients were treated with either a Neer or Aequalis shoulder prosthesis using nonabsorbable sutures and no bone graft for the reattachment of the tuberosities. In group 2, 27 patients were treated with either an Aequalis or Epoca shoulder prosthesis and a combination of cable fixation and bone grafting. MAIN OUTCOME MEASUREMENTS: At follow-up (average, 32 months), radiographs were taken to confirm tuberosity fixation or degree of displacement or resorption. Functional outcome was assessed by the Constant-Murley Score. RESULTS: Significantly more dislocated tuberosities were found radiographically in group 1 (10 of 13 in total, P = 0.011), and significantly more tuberosities were resorbed in group 1 (9 of 12 in total, P = 0.012). Significant differences in functional results among healed versus failed tuberosity fixation were observed for activity of daily living (P = 0.05), range of motion (P = 0.002), strength (P = 0.01), the total score (P = 0.008), and the passive rotation amplitude (P = 0.04). CONCLUSION: In hemiarthroplasties for proximal humeral fractures, the reattachment of the tuberosities with cable wire and bone grafting gives consistently better radiographic and functional results than with suture fixation alone.

Functional imaging of the parotid glands using blood oxygenation level dependent (BOLD)-MRI at 1.5T and 3T

PURPOSE: To evaluate the function of the parotid glands before and during gustatory stimulation, using an intrinsic susceptibility-weighted MRI method (blood oxygenation level dependent, BOLD-MRI) at 1.5T and 3T. MATERIALS AND METHODS: A total of 10 and 13 volunteers were investigated at 1.5T and 3T, respectively. Measurements were performed before and during gustatory stimulation using ascorbate. Circular regions of interest (ROIs) were delineated in the left and right parotid glands, and in the masseter muscle for comparison. The effects of stimulation were evaluated by calculating the difference between the relaxation rates, DeltaR(2)*. Baseline and stimulation were statistically compared (Student's t-tests), merging both parotid glands. RESULTS: The averaged DeltaR(2)* values prestimulation obtained in all parotid glands were stable (-0.61 to 0.38 x 10(-3) seconds(-1)). At 3T, these values were characterized by an initial drop (to -2.7 x 10(-3) seconds(-1)) followed by a progressive increase toward the baseline. No significant difference was observed between baseline and parotid gland stimulation at 1.5T, neither for the masseter muscle at both field strengths. A considerable interindividual variability (over 76%) was noticed at both magnetic fields. CONCLUSION: BOLD-MRI at 3T was able to detect DeltaR(2)* changes in the parotid glands during gustatory stimulation, consistent with an increase in oxygen consumption during saliva production.

Expression of CD95 on mature leukocytes of MRL/lpr mice after transplantation of genetically modified bone marrow stem cells

Bone marrow transplantation (BMT) is commonly used for the treatment of severe haematological and immunological diseases. For instance, the autoimmune lymphoproliferative syndrome (ALPS) caused by a complete expression defect of CD95 (Fas, APO-1) can be cured by allogeneic BMT. However, since this therapy may not generate satisfactory results when only partially compatible donors are available, we were interested in the development of a potential alternative treatment by using lentiviral gene transfer of a normal copy of CD95 cDNA in hematopoietic stem cells. Here, we show that this approach applied to MRL/lpr mice results in the expression of functional CD95 receptors on the surface of lymphocytes, monocytes, and granulocytes. This suggests that correction of CD95 deficiency can be achieved by gene therapy.

Hydrogel-based engineered skeletal muscle grafts normalize heart function early after myocardial infarction

Tissue engineering represents an attractive approach for the treatment of congestive heart failure. The influence of the differentiation of myogenic graft for functional recovery is not defined. We engineered a biodegradable skeletal muscle graft (ESMG) tissue and investigated its functional effect after implantation on the epicardium of an infarcted heart segment. ESMGs were synthesized by mixing collagen (2 mg/mL), Matrigel (2 mg/mL), and rat skeletal muscle cells (10(6)). Qualitative and quantitative aspects of ESMGs were optimized. Two weeks following coronary ligation, the animals were randomized in three groups: ESMG glued to the epicardial surface with fibrin (ESMG, n = 7), fibrin alone (fibrin, n = 5), or sham operation (sham, n = 4). Echocardiography, histology, and immunostaining were performed 4 weeks later. A cohesive three-dimensional tissular structure formed in vitro within 1 week. Myoblasts differentiated into randomly oriented myotubes. Four weeks postimplantation, ESMGs were vascularized and invaded by granulation tissue. Mean fractional shortening (FS) was, however, significantly increased in the ESMG group as compared with preimplantation values (42 +/- 6 vs. 33 +/- 5%, P < 0.05) and reached the values of controlled noninfarcted animals (control, n = 5; 45 +/- 3%; not significant). Pre- and postimplantation FS did not change over these 4 weeks in the sham group and the fibrin-treated animals. This study showed that it is possible to improve systolic heart function following myocardial infarction through implantation of differentiated muscle fibers seeded on a gel-type scaffold despite a low rate of survival.

Infantile hypophosphatasia due to a new compound heterozygous TNSALP mutation - functional evidence for a hydrophobic side-chain?

BACKGROUND: Infantile hypophosphatasia (IH) is an inherited disorder characterized by defective bone mineralization and a deficiency of alkaline phosphatase activity. OBJECTIVE/DESIGN: The aim of the study was to evaluate a new compound heterozygous TNSALP mutation for its residual enzyme activity and localization of the comprised amino acid residues in a 3D-modeling. PATIENT: We report on a 4-week old girl with craniotabes, severe defects of ossification, and failure to thrive. Typical clinical features as low serum alkaline phosphatase, high serum calcium concentration, increased urinary calcium excretion, and nephrocalcinosis were observed. Vitamin D was withdrawn and the patient was started on calcitonin and hydrochlorothiazide. Nonetheless, the girl died at the age of 5 months from respiratory failure. RESULTS: Sequence analysis of the patient's TNSALP gene revealed two heterozygous mutations [c.653T>C (I201T), c.1171C>T (R374C)]. Transfection studies of the unique I201T variant in COS-7 cells yielded a mutant TNSALP protein with only a residual enzyme activity (3.7%) compared with wild-type, whereas the R374C variant was previously shown to reduce normal activity to 10.3%. 3D-modeling of the mutated enzyme showed that I201T resides in a region that does not belong to any known functional site. CONCLUSION: We note that I201, which has been conserved during evolution, is buried in a hydrophobic pocket and, therefore, the I>T-change should affect its functional properties. Residue R374C is located in the interface between monomers and it has been previously suggested that this mutation affects dimerization. These findings explain the patient's clinical picture and severe course.

Outcome and patients' satisfaction after functional treatment of acute lateral ankle injuries at emergency departments versus family doctor offices

BACKGROUND: In some Western countries, more and more patients seek initial treatment even for minor injuries at emergency units of hospitals. The initial evaluation and treatment as well as aftercare of these patients require large amounts of personnel and logistical resources, which are limited and costly, especially if compared to treatment by a general practitioner. In this study, we investigated whether outsourcing from our level 1 trauma center to a general practitioner has an influence on patient satisfaction and compliance. METHODS: This prospective, randomized study, included n = 100 patients who suffered from a lateral ankle ligament injury grade I-II (16, 17). After radiological exclusion of osseous lesions, the patients received early functional treatment and were shown physical therapy exercises to be done at home, without immobilization or the use of stabilizing ortheses. The patients were randomly assigned into two groups of 50 patients each: Group A (ER): Follow-up and final examination in the hospital's emergency unit. Group B (GP): Follow-up by general practitioner, final examination at hospital's emergency unit. The patients were surveyed regarding their satisfaction with the treatment and outcome of the treatment. RESULTS: Female and male patients were equally represented in both groups. The age of the patients ranged from 16 - 64 years, with a mean age of 34 years (ER) and 35 years (GP). 98% (n = 98) of all patients were satisfied with their treatment, and 93% (n = 93) were satisfied with the outcome. For these parameters no significant difference between the two groups could be noted (p = 0.7406 and 0.7631 respectively). 39% of all patients acquired stabilizing ortheses like ankle braces (Aircast, Malleoloc etc.) on their own initiative. There was a not significant tendency for more self-acquired ortheses in the group treated by general practicioners (p = 0,2669). CONCLUSION: Patients who first present at the ER with a lateral ankle ligament injury grade I-II can be referred to a general practitioner for follow-up treatment without affecting patient satisfaction regarding treatment and treatment outcome.

The clinical and structural long-term results of open repair of massive tears of the rotator cuff

BACKGROUND: At a mean follow-up of 3.1 years, twenty-seven consecutive repairs of massive rotator cuff tears yielded good and excellent clinical results despite a retear rate of 37%. Patients with a retear had improvement over the preoperative state, but those with a structurally intact repair had a substantially better result. The purpose of this study was to reassess the same patients to determine the long-term functional and structural results. METHODS: At a mean follow-up interval of 9.9 years, twenty-three of the twenty-seven patients returned for a review and were examined clinically, radiographically, and with magnetic resonance imaging with use of a methodology identical to that used at 3.1 years. RESULTS: Twenty-two of the twenty-three patients remained very satisfied or satisfied with the result. The mean subjective shoulder value was 82% (compared with 80% at 3.1 years). The mean relative Constant score was 85% (compared with 83% at 3.1 years). The retear rate was 57% at 9.9 years (compared with 37% at 3.1 years; p = 0.168). Patients with an intact repair had a better result than those with a failed reconstruction with respect to the mean absolute Constant score (81 compared with 64 points, respectively; p = 0.015), mean relative Constant score (95% and 77%; p = 0.002), and mean strength of abduction (5.5 and 2.6 kg; p = 0.007). The mean retear size had increased from 882 to 1164 mm(2) (p = 0.016). Supraspinatus and infraspinatus muscle fatty infiltration had increased (p = 0.004 and 0.008, respectively). Muscles with torn tendons preoperatively showed more fatty infiltration than muscles with intact tendons preoperatively, regardless of repair integrity. Shoulders with a retear had a significantly higher mean acromion index than those without retear (0.75 and 0.65, respectively; p = 0.004). CONCLUSIONS: Open repair of massive rotator cuff tears yielded clinically durable, excellent results with high patient satisfaction at a mean of almost ten years postoperatively. Conversely, fatty muscle infiltration of the supraspinatus and infraspinatus progressed, and the retear size increased over time. The preoperative integrity of the tendon appeared to be protective against muscle deterioration. A wide lateral extension of the acromion was identified as a previously unknown risk factor for retearing.

Mechano-regulated tenascin-C orchestrates muscle repair

Tenascin-C (TNC) is a mechano-regulated, morphogenic, extracellular matrix protein that is associated with tissue remodeling. The physiological role of TNC remains unclear because transgenic mice engineered for a TNC deficiency, via a defect in TNC secretion, show no major pathologies. We hypothesized that TNC-deficient mice would demonstrate defects in the repair of damaged leg muscles, which would be of functional significance because this tissue is subjected to frequent cycles of mechanical damage and regeneration. TNC-deficient mice demonstrated a blunted expression of the large TNC isoform and a selective atrophy of fast-muscle fibers associated with a defective, fast myogenic expression response to a damaging mechanical challenge. Transcript profiling mapped a set of de-adhesion, angiogenesis, and wound healing regulators as TNC expression targets in striated muscle. Expression of these regulators correlated with the residual expression of a damage-related 200-kDa protein, which resembled the small TNC isoform. Somatic knockin of TNC in fast-muscle fibers confirmed the activation of a complex expression program of interstitial and slow myofiber repair by myofiber-derived TNC. The results presented here show that a TNC-orchestrated molecular pathway integrates muscle repair into the load-dependent control of the striated muscle phenotype.

Fat implant is superior to muscle implant in vestibular schwannoma surgery for the prevention of cerebrospinal fluid fistulae

OBJECTIVE: Meticulous sealing of opened air cells in the petrous bone is necessary for the prevention of cerebrospinal fluid (CSF) fistulae after vestibular schwannoma surgery. We performed a retrospective analysis to determine whether muscle or fat tissue is superior for this purpose. METHODS: Between January 2001 and December 2006, 420 patients underwent retrosigmoidal microsurgical removal by a standardized procedure. The opened air cells at the inner auditory canal and the mastoid bone were sealed with muscle in 283 patients and with fat tissue in 137 patients. Analysis was performed regarding the incidence of postoperative CSF fistulae and correlation with the patient's sex and tumor grade. RESULTS: The rate of postoperative CSF leak after application of fat tissue was lower (2.2%) than after use of muscle (5.7%). Women had less postoperative CSF leakage (3.4%) than men (5.6%). There was an inverse correlation with tumor grade. Patients with smaller tumors seemed to have a higher rate of CSF leakage than those with large tumors without hydrocephalus. Only large tumors with severe dislocation of the brainstem causing hydrocephalus showed a higher incidence of CSF leaks. CONCLUSION: Fat implantation is superior to muscle implantation for the prevention of CSF leakage after vestibular schwannoma surgery and should, therefore, be used for the sealing of opened air cells in cranial base surgery.

Silencing of microRNAs in vivo with 'antagomirs'

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.

Neer Award 2007: Reversion of structural muscle changes caused by chronic rotator cuff tears using continuous musculotendinous traction. An experimental study in sheep

HYPOTHESIS: Chronic rotator cuff tears are associated with irreversible architectural muscle changes and a high rate of repair failure. The changes observed in man and their irreversibility with a single stage repair can be reproduced in sheep. It was the purpose of this experiment to test the hypothesis that slow, continuous elongation of a retracted musculotendinous unit allows reversal of the currently irreversible structural muscle changes. MATERIALS AND METHODS: The infraspinatus tendon of 12 sheep was released using a greater tuberosity osteotomy and allowed to retract for 4 months. Then, a new device was mounted on the scapular spine and used to extend the infraspinatus muscuculotendinous unit transcutaneously by 1 mm per day. Thereafter, the tendon was repaired back to the greater tuberosity. We assessed the muscular architecture using magnetic resonance imaging, macroscopic dissection, histology, and electron microscopy. Fatty infiltration (in Hounsfield units 1/4 HU) and muscular cross-sectional area (in % of the control side) were monitored with computed tomography at tendon release, initiation of elongation, repair, and at sacrifice. RESULTS: Sixteen weeks after tendon release, the mean tendon retraction was 29 +/- 6 mm (14% of original length, P = .008). In 8 sheep, elongation was achieved as planned (group I), but in 4, the elongation failed technically (group II). The mean traction time was 24 +/- 6 days with a mean traction distance of 19 +/- 4 mm. At sacrifice, the mean pennation angle in the infraspinatus of group I was not different from the control side (29.8 degrees +/-7.5 degrees vs. 30 degrees +/-6 degrees , P = .575). In group II, the pennation angle had increased from 30 degrees +/-6 degrees to 55 degrees +/-14 degrees (P = .035). There was no fatty infiltration at the time of tendon release. After retraction, there was a significant increase in fatty infiltration of the infraspinatus muscle and a decrease of its cross-sectional area to 57% of the contralateral side (P = .0001). During traction, the degree of fatty infiltration remained unchanged (36 HU to 38 HU, P = .381), and atrophy improved to a muscle square area of 78% of the contralateral side (P = .0001) in group I. In group II, an increase of fatty infiltration was measured from 36 HU to 28 HU; however, this increase was not significant (P = .144). Atrophy did not change in group II (57-55%, P = .946). At sacrifice, the remaining muscle mass was 64% in group I and 46% in group II (P = .019). DISCUSSION: Our preliminary results document, that continuous elongation of a retracted, fatty infiltrated and atrophied musculotendinous unit is technically feasible. CONCLUSION: In the sheep, continuous elongation can lead to restoration of normal muscle architecture, to partial reversal of muscle atrophy, and to arrest of the progression of fatty infiltration. LEVEL OF EVIDENCE: Basic science level 2; Prospective comparative therapeutic study.