Transosseous application of ultrasound on healing at tendon graft-bone interface and fracture healing of long bones. Experimental and clinical study

The process of ligamentization includes histological and structural remodelling of the tendon graft during healing at the tendon-bone interface and thereafter. A number of essential genes are involved in this process and their expression can be regulated through complex biochemical pathways. However, there is little information documenting the mechanism of healing at the tendon graft-bone interface at molecular and cellular level. Fracture healing is also a complex biological process that involves the spatial and temporal action of many different cell types, proteins and the expression of hundreds of genes all working towards restoring the bone’s functional and mechanical properties. About 10 % of fractures require further surgical procedures due to impaired healing, such as delayed union or pseudarthrosis. The purpose of this study is: a) to investigate the effect of transosseous application of low intensity ultrasound (LiUS) during ligamentization process on the healing at tendon graft-bone interface: experimental and clinical study and b) to investigate the clinical application of transosseous LiUS on the enhancement and monitoring of fracture healing process in patients with fracture of long bones. We investigated the healing at tendon graft-bone interface in rabbits after transosseous application of LiUS by analyzing the expression levels of TGFb1, biglycan, collagen I and tRNAGly using semiquantitative RT-PCR, and through subsequent histological analysis. We also investigated the clinical application of LiUS during the ligamentization process in patients who underwent arthroscopically assisted anterior cruciate ligament (ACL) reconstruction by analyzing the serum levels of TGFb1, IGF, OPG, sRANKL, NTx and procollagen I using ELISA, and through subsequent Multiple Direction Computer Tomography. We observed statistically significant alterations in gene expression and protein level of all factors after application of LiUS in both experimental and clinical study, respectively. Histological and imaging examination supported our molecular findings, indicating a faster healing rate and a more efficient ligamentization process after ultrasound treatment. These results suggest that application of LiUS enhances the healing rate of the tendon graft-bone interface in ACL reconstruction, possibly by affecting the expression levels of genes significant for the tendon to bone healing through important biochemical pathways. The clinical efficacy of the transosseous application of LiUS for both enhancement and monitoring of the bone healing process was investigated in patients with fracture of long bones. The effect of LiUS on fracture healing was evaluated using radiograph and computer tomography. Both the LiUS application and the ultrasonic measurements are supported by an integrated telemedicine system which also incorporates an ultrasound wearable device and a patient management system. Transosseous application of LiUS was well tolerated by all patients. No complications or adverse effects were observed and we showed that the propagation velocity of ultrasound increased post-operatively which has been found to be a most sensitive parameter to reflect structural changes into the newly formed callus. Taken together our findings suggest that transosseous application of LiUS, with the ultrasound transducers placed directly on the periosteum at the fracture site, can enhance the mechanical properties of the fracture callus and reduce the time to fracture healing. Therefore, transosseous application of LiUS can be used for both enhancement and monitoring of the bone healing process in patients with fracture of long bones.Η επουλωτική εξεργασία στη διεπιφάνεια οστού-τενοντίου μοσχεύματος αποτελεί μία πολύπλοκη διαδικασία η οποία φαίνεται ότι επηρεάζεται από διάφορους βιολογικούς παράγοντες αλλά και παρά! γοντες μηχανικού στρες. ?στόσο, ο ακριβής μηχανισμός της δεν έχει αποσαφηνιστεί πλήρως. Η πώρωση των καταγμάτων αποτελεί ένα εξίσου πολύπλοκο βιολογικό και μηχανικό φαινόμενο κατά το οποίο στα u948 ιακά αποκα_! 2 ίστανται οι δομικές, λειτουργικές και μηχανικές ιδιότητες του οστού. Δεν είναι σπάνιες οι περιπτώσεις εμφάνισης επιπλοκών (καθυστερημένη πώρωση, ψευδάρθρωση) αυξάνοντας το κόστος θεραπείας κα ! 52 ώς απαιτούνται περαιτέρω χειρουργικές επεμβάσεις. Σκοπός της συγκεκριμένης διατριβής είναι: α) η διερεύνηση και αξιολόγηση της επίδρασης των διοστικών υπερήχων χαμηλής έντασης (LiUS) στην ευόδωση και επιτάχυνση της επουλωτικής εξεργασίας στη δι u949 πιφάνεια οστού- τενοντίου μοσχεύματος μετά από πλαστική αποκατάσταση προσθίου χιαστού συνδέσμου (ΠΧΣ): πειραματική και κλινική μελέτη, και β) η διερεύνηση και αξιολόγηση της κλινικής εφαρμογή! ς των διοστι! κ u974 ν LiUS στην επιτάχυνση της πώρωσης καταγμάτων μακρών οστών καθώς και αξιολόγηση δυνατότητας παρακολούθησης της εξέλιξης της πώρωσης. Κατά την πειραματική μελέτη διερευνήθηκε η διαδικασία συνδεσμοποίησης κατά την ανακατασκευή του ΠΧΣ σε κουνέλια με έλεγχο και αξιολόγηση της έκφρασης των γονιδίων TGF-β1, biglycan, κολλαγόνου τύπο! υ Ι, και tRNAGly με ημιποσοτική RT-PCR και ιστολογική ανάλυση. Η κλινική εφαρμογή LiUS στην ευόδωση και επιτάχυνση της επουλωτικής εξεργασίας στη διεπιφάνεια οστού- τενοντίου μοσχεύματος διερευνήθηκε σ! ε ασθενείς μ! εά από αρθροσκοπική αποκατάσταση ΠΧΣ με έλεγχο των επιπέδων αυξητικών παραγόντων (TGF-β1, IGF) και πρωτεϊνών (OPG, sRANKL, τελοπεπτίδιο κολλαγόνου τύπου Ι - NTx, προκολλαγόνο τύπου Ι) με μέθοδο ELISA καθώς και με απεικονι! στικό έλεγχο. Παρατηρήθηκαν στατιστικά σημαντικές μεταβολές των επιπέδων έκφρασης των γονιδίων και των πρωτεϊνών μετά την εφαρμογή LiUS τόσο στην πειραματική όσο και την κλινική μελέτη αντίστοιχα. Οι μεταβλές αυτές σχετίζονται με ταχύτερη ωρίμανση και ενσωμάτωση του τενοντίου μοσχεύματος στο οστικό τούνελ κατά την ανακατασκευή του ΠΧΣ με τενόντιο μόσχευμα όπως αποδείχθηκε από την ιστολογι_! 4 ή και την απε! ι u954 ονιστική ανάλυση. Φαίνεται λοιπόν ότι η εφαρμογή των LiUS ευοδώνει και επιταχύνει την επουλωτική εξεργασία στη διεπιφάνεια οστού-τενοντίου μοσχεύματος μέσω μοριακών μονοπατιών και μηχανισμών! που επηρεάζουν την έκφραση σημαντικών γονιδίων και πρωτεϊνικών προϊόντων τους. Η κλινική εφαρμογή διοστικών LiUS για την ευόδωση και επιτάχυνση της πώρωσης καθώς και για την παρακολούθηση της εξέλιξης της πώρωσης των καταγμάτων πραγματοποιήθηκε σε ασθενείς με κάταγμα μακρ! ών οστών. Η αξιολόγηση της επίδρασης των υπερήχων πραγματοποιήθηκε με ακτινολογικό έλεγχο και υπολογιστική τομογραφία. Η εφαρμογή διοστικών υπερήχων τόσο για τη θεραπεία όσο και για την παρακλούθηση τη ! 62 πώρωσης, υποστηρίζεται από ένα ολόκληρο σύστημα τηλεϊατρικής, το οποίο ενσωματώνει μια φορητή συσκευή και μια κεντρική μονάδα διαχείρισης ασθενών. Η εφαρμογή απέδειξε άριστη ανοχή από τους ασ_! 2 ενείς χωρίς επιπλοκές και ανεπιθύμητες ενέργειες και παρατηρήθηκε ότι η ταχύτητα διάδοσης των υπερήχων αυξάνονταν μετεγχειρητικά γεγονός που υποδηλώνει τον σχηματισμό και ωρίμανση πώρου. Η διοστική εφαρμογή LiUS, με την εμφύτευση υπερηχητικών μετατροπέων απευθείας πάνω στο οστό στην περιοχή του κατάγματος, συμβάλλει στην ευόδωση των μηχανικών ιδιοτήτων του πώρου μειώνοντας το χ`! 1 όνο επούλωσης. Φαίνεται λοιπόν ότι το σύστημα διοστικών LiUS μπορεί να χρησιμοποιηθεί για ταυτόχρονη ευόδωση και παρακολούθηση της εξέλιξης της πώρωσης σε ασθενείς με κατάγματα μακρών οστών

Median nerve collagen wrapping in revision surgery: Clinical outcome of 10 patients

 Nerve wrap protectors are bioabsorbable synthetic materials made of collagen or extracellular matrix that provide a non-constricting encasement for injured peripheral nerves. They are designed to be used as an interface between the nerve and the surrounding tissue. After hydrated, they transform into a soft, pliable, nonfriable, easy to handle porous conduit. The wall of the nerve wrap has a longitudinal slit that allows to be placed around the injured nerve. Τhis article presents the surgical technique for median nerve neurolysis and nerve coverage using a collagen or an extracellular matrix nerve wrap protector in 10 patients with recurrent or persistent carpal tunnel syndrome. All patients had a mean of three previous open carpal tunnel operations, which were not successful. The mean follow-up was 3 years. Under axillary nerve block anaesthesia with the use of pneumatic tourniquet, a standard open carpal tunnel approach was done incorporating the previous incision. Scar tissue was excised in a healthy bed and the median nerve was thoroughly released with external neurolysis. An appropriate length of nerve wrap protector was cut longitudinally according to the length of nerve release. The nerve wrap was loosely sutured with separate polypropylene sutures No. 7-0. A dorsal splint was applied for a mean of 2 weeks followed by progressive passive and active range of motion rehabilitation exercises of the wrist and fingers. At the last follow-up, all patients showed improvement of clinical symptoms, static two-point discrimination test and median nerve conduction studies, and absence of Tinel sign. Differences in outcome and complications with respect to the nerve wrap materials used were not observed.

An association study between hypoxia inducible factor-1alpha (HIF-1α) polymorphisms and osteonecrosis.

Bone hypoxia resulting from impaired blood flow is the final pathway for the development of osteonecrosis (ON). The aim of this study was to evaluate if HIF-1α, the major transcription factor triggered by hypoxia, is genetically implicated in susceptibility to ON. For this we analyzed frequencies of three known HIF-1α polymorphisms: one in exon 2 (C111A) and two in exon 12 (C1772T and G1790A) and their association with ON in a Greek population. Genotype analysis was performed using PCR-RFLP and rare alleles were further confirmed with sequencing. We found that genotype and allele frequency of C1772T and G1790A SNP of HIF-1α (SNPs found in our cohort) were not significantly different in ON patients compared to control patients. Furthermore these SNPs could not be associated with the different subgroups of ON. At the protein level we observed that the corresponding mutations (P582S and A588T, respectively) are not significant for protein function since the activity, expression and localization of the mutant proteins is practically indistinguishable from wt in HEK293 and Saos-2 cells. These results suggest that these missense mutations in the HIF-1α gene are not important for the risk of developing ON

HIF-1α SNP genotype and allele frequencies between subcategories of ON patients and control subjects.

*<p>Fisher chi-square analysis, P values <0.05 are considered as statistically significant.</p>**<p>OR, Odds ratio; CI, confidence interval.</p><p>Calculations were performed for genotypes: CC vs CT+TT.</p

HIF-1α SNP genotype and allele frequencies between ON patients and control subjects.

*<p>Fisher chi-square analysis, P values <0.05 are considered as statistically significant.</p>**<p>OR, Odds ratio; CI, confidence interval.</p><p>Calculations were performed for genotypes: CC vs CT+TT, GG vs GA+AA.</p><p>nc: not calculated.</p

Frequencies of HIF-1α polymorphisms between ON patients and controls.

*<p>Frequencies of rare alleles.</p>**<p>P values of deviation from HWE, in patients and controls.</p

P582S and A588T mutations do not affect HIF-1α transcriptional activity, localization and expression.

<p>HIF-1α transcriptional activity was determined 24 h after co-transfection of Saos-2 (A) and HEK293 cells (B) with plasmids expressing the indicated proteins together with the pGL3–5HRE-VEGF reporter plasmid. Values (as relative luminescence units) were determined as a ratio of firefly luciferase activity over Renilla luciferase activity and represent the mean of four different experiments performed in triplicate (+/- S.E.). Statistical differences were assessed using unpaired-t-test. P values>0.05 were considered as statistically non-significant (ns). Saos-2 (C) and HEK293 cells (D) where transfected with plasmids expressing the indicated proteins and their localization was observed after 24 h, with immunofluoresence, using an anti-FLAG antibody. Protein levels of FLAG-HIF-1α wt and mutants were determined in Saos-2 (E) and HEK293 (F) cells after 24-48 h post-transfection, in Western blot using a monoclonal anti-HIF-1α antibody and anti-tubulin as loading control. Densitometric analysis of the bands was performed with the public domain software for image analysis ‘ImageJ’. FLAG-HIF-1α quantities were normalized against corresponding tubulin and expressed as fold increase against wt FLAG-HIF-1α (100%).</p

Analysis of HIF-1α polymorhisms using RFLP.

<p>(A) Analysis of HIF-1α gene C1772T polymorphism with HphI enzyme, shown on 2% agarose electrophoresis. CT heterozygous genotype yielded three bands 467, 251 and 216 bp; C allele wt yielded two bands (251 and 216 bp). T allele remained uncut and yielded one fragment at 467 bp. Molecular weight standards are shown on the right. (B) Chromatograms of DNA sequence analysis of HIF-1α exon12 fragment showing the corresponding C, CT, T allelic variations at position 1772. (C) Analysis of HIF-1α gene G1790A polymorphism with AciI enzyme, shown on 2% agarose electrophoresis. G allele wt yielded two bands (269 and 198 bp). A allele would remained uncut and yielded one fragment at 467 bp, as represented. Molecular weight standards are shown on the right. (D) Analysis of HIF-1α gene C111A polymorphism with BglII enzyme, shown on 2% agarose electrophoresis. C allele wt yielded two bands (143 and 44 bp). A allele would remained uncut and yielded one fragment at 187 bp. Molecular weight standards are shown on the right.</p

Survivorship of Monoblock Trabecular Metal Cups in Primary THA: Midterm Results

Monoblock trabecular metal cups are made of a novel porous material intended to enhance ingrowth and improve fixation. We prospectively followed 223 consecutive patients with 245 trabecular metal acetabular cups implanted during primary total hip arthroplasties to determine the overall survivorship of the implant, and any association of survivorship to primary diagnosis and age, and to determine the fate of polar gaps and cysts. Minimum followup was 36 months (mean, 60 months; range, 36–112 months). Patients were assessed with the Harris Hip score and the Oxford questionnaire and radiographically with standardized serial radiographs. At last followup, all cups were radiographically stable with no evidence of migration or progressive radiolucencies. The survivorship with reoperation as the end point was estimated at 98.75% with a 95% confidence interval. Three reoperations occurred during the first 36 months. The Harris hip score increased from 48 to 94 and the Oxford score was 16.4 at the last examination. We observed no difference in terms of survivorship among patients with osteoarthritis, osteonecrosis, or hip dysplasia. Seven of 14 (50%) osteoarthritis cysts and 10 of 33 (33.3%) polar gaps detected on postoperative radiographs decreased or filled, whereas none of the remainder deteriorated with time. Our midterm results suggest this implant may enhance fixation, but long-term followup is needed to confirm our findings