Mechanobiological modeling of endochondral ossification: an experimental and computational analysis.

Long bone formation starts early during embryonic development through a process known as endochondral ossification. This is a highly regulated mechanism that involves several mechanical and biochemical factors. Because long bone development is an extremely complex process, it is unclear how biochemical regulation is affected when dynamic loads are applied, and also how the combination of mechanical and biochemical factors affect the shape acquired by the bone during early development. In this study, we develop a mechanobiological model combining: (1) a reaction-diffusion system to describe the biochemical process and (2) a poroelastic model to determine the stresses and fluid flow due to loading. We simulate endochondral ossification and the change in long bone shapes during embryonic stages. The mathematical model is based on a multiscale framework, which consisted in computing the evolution of the negative feedback loop between Ihh/PTHrP and the diffusion of VEGF molecule (on the order of days) and dynamic loading (on the order of seconds). We compare our morphological predictions with the femurs of embryonic mice. The results obtained from the model demonstrate that pattern formation of Ihh, PTHrP and VEGF predict the development of the main structures within long bones such as the primary ossification center, the bone collar, the growth fronts and the cartilaginous epiphysis. Additionally, our results suggest high load pressures and frequencies alter biochemical diffusion and cartilage formation. Our model incorporates the biochemical and mechanical stimuli and their interaction that influence endochondral ossification during embryonic growth. The mechanobiochemical framework allows us to probe the effects of molecular events and mechanical loading on development of bone.

Genetic variation of vascular endothelial growth factor pathway does not correlate with the severity of retinopathy of prematurity.

OBJECTIVE: The aim of this study was to assess the genetic effects of the vascular endothelial growth factor (VEGF) pathway on retinopathy of prematurity (ROP). STUDY DESIGN: A prospective study from a tertiary center that enrolled 204 Japanese infants (<35 weeks of gestational age (GA)) having no anomalies. ROP developed in 127, but not in 77 infants. The relative severity was defined as non-severe, moderate and severe ROP for GA, based on the staging criteria. VEGF (g.-634G>C, g.+13553C>T) and VEGF-receptor (KDR g.+4422(AC)11 to 14, Flt-1 c.+6724(TG)13 to 23) gene polymorphisms and clinical variables were assessed by uni/multivariate analyses. RESULT: The frequency of polymorphisms did not differ between ROP and non-ROP patients. The TT genotype of g.+13553 showed a higher odds ratio for non-severe ROP than CC genotype (P=0.006). Multivariate analyses indicated that low birth weight, blood transfusion and respiratory distress syndrome, but not polymorphisms, were the risk factors of advanced ROP (≥ stage 3). CONCLUSION: A genotype of the VEGF pathway weakly affects the severity of ROP compared with other clinical factors.

Activation of p38 mitogen-activated protein kinase is required for in vivo brain-derived neurotrophic factor production in the rat hippocampus.

Several lines of evidence strongly suggest that brain-derived neurotrophic factor (BDNF) is associated with the formation, storage and recall of memory in the hippocampus and that it is important to maintain a considerable level of hippocampal BDNF in order to keep normal functions. BDNF can be synthesized in an activity-dependent manner. In fact, kainic acid or AMPA enhances BDNF levels in hippocampal granule neurons. However, the mechanisms of BDNF production are largely unclear. Recently, we have found that riluzole, which blocks voltage-gated sodium channels and thereby reduces glutamate release, actually strengthens immunoreactivity of BDNF in hippocampal granule neurons of rats. Therefore, we examined the riluzole-activated signaling pathways for BDNF production. Riluzole increased levels of phospho-p38 mitogen-activated protein kinase (p38 MAPK), as well as BDNF levels. Inhibition of p38 MAPK by SB203580 reduced riluzole effects, while activation of p38 MAPK by anisomycin increased levels of BDNF, suggesting that p38 MAPK can mediate BDNF production. Riluzole-induced elevation of phospho-activating transcription factor-2, a transcription factor downstream of p38 MAPK, was also observed. A blocker of N-type voltage-gated calcium channels reduced the effects of riluzole on BDNF production and p38 MAPK activation. We also examined a possible involvement of the adenosine A1 receptor in BDNF production because riluzole can influence ecto-nucleotide levels. An A1 receptor agonist inhibited riluzole-induced elevation of BDNF levels, whereas an antagonist not only increased levels of BDNF and active p38 MAPK but also augmented riluzole effects. These results indicate that, in the rat hippocampus, there is an in vivo signaling pathway for BDNF synthesis mediated by p38 MAPK, and that N-type voltage-gated calcium channels and/or adenosine A1 receptors contribute to p38 MAPK activation.

Possible contribution of hyalocytes to idiopathic epiretinal membrane formation and its contraction.

AIM: To address the cellular components and the contractile mechanisms of the idiopathic epiretinal membrane (ERM). METHODS: Ten surgically removed ERMs were fixed in 4% paraformaldehyde and analysed by whole-mount immunohistochemistry with anti-glial fibrillar acidic protein (GFAP) and alpha smooth-muscle actin (alphaSMA) antibodies. Type I collagen gel contraction assay, an established wound-healing assay in vitro, was performed using cultured bovine hyalocytes or normal human astrocytes (NHA) to evaluate the contractile property of the cells in the presence of tissue growth factor (TGF)-beta2. The expression of alphaSMA was also analysed by western blot analysis to examine myofibroblastic transdifferentiation of the cells. Vitreous-induced collagen gel contraction was also evaluated. RESULTS: All membranes were composed of alphaSMA immunopositive cells in contracted foci and GFAP immunopositive cells in the periphery. No apparent double positive cells were observed in any membranes examined. Cultured hyalocytes showed overexpression of alphaSMA and hypercontraction of collagen gels in response to TGF-beta2, while glial cells showed marginal change. The vitreous from ERM patients also caused overexpression of alphaSMA and hypercontraction of the gels embedding hyalocytes, which were almost completely inhibited in the presence of anti-TGF-beta2 neutralising antibody. CONCLUSIONS: Hyalocytes might be one of the critical components of ERM mediating its contractile property through the effect of TGF-beta2 in the vitreous fluid.

Residual internal limiting membrane in epiretinal membrane surgery.

BACKGROUND/AIM: To examine the degree of the residual internal limiting membrane (ILM) after epiretinal membrane (ERM) peeling. METHODS: Sixty-one eyes of 59 patients with ERM were enrolled. After ERM peeling, residual ILM was visualised with Brilliant Blue G (BBG). The residual ILM pattern was divided into three groups: (1) residual type (ILM mostly remained), (2) half type (approximately half of ILM remained), (3) no residual type (ILM mostly removed with ERM). If ILM remained, residual ILM was removed in all cases and histologically examined using the flat mount method in 10 cases. The correlation between the degree of ERM evaluated by preoperative best-corrected visual acuity (BCVA) and residual ILM pattern was also examined. RESULTS: Twenty-eight eyes (45.9%) were of the residual type. Three eyes (4.9%) were of the half type, and 30 eyes (49.2%) were of no residual type. The mean preoperative BCVA showed no significant correlation with the residual ILM pattern. Flat mount immunohistochemistry revealed many remnant cells, both glial fibrillar acidic protein positive and negative, on residual ILMs in all specimens examined. No recurrence that needed surgical treatment was observed. CONCLUSION: Residual ILM with remnant cells seems to be frequent after ERM removal. Intraoperative staining with BBG may be helpful in determining the extent of ILM removal.

Vascular endothelial growth factor expression by hyalocytes and its regulation by glucocorticoid.

AIM: Tumour necrosis factor-alpha (TNF-alpha) is one of the major inflammatory cytokines involved in the pathogenesis of various vitreoretinal diseases. The authors investigated the effect of hypoxia, TNF-alpha and dexamethasone on vascular endothelial growth factor (VEGF) expression by cultured hyalocytes. METHODS: Hyalocytes were isolated from bovine vitreous. Hypoxic and TNF-alpha-dependent effects on cultured hyalocytes were investigated using several assays to determine VEGF protein expression, hypoxia-inducible factor (HIF)-1alpha protein levels, HIF-1alpha-DNA-binding ability and VEGF mRNA stability. The effects of dexamethasone on VEGF expression and its intracellular signalling under hypoxic or TNF-alpha stimulated conditions were also examined. RESULTS: Hypoxic conditions and TNF-alpha stimulation induce VEGF expression in hyalocytes. These stimuli also stabilise HIF-1alpha protein and increase its DNA-binding ability. Dexamethasone significantly inhibits both HIF-1alpha protein levels and HIF-1alpha-DNA-binding activity, and also decreases the hypoxic- and TNF-alpha -dependent induction of VEGF expression in hyalocyte. However, dexamethasone has no significant effect on the stability of VEGF mRNA. CONCLUSIONS: Hyalocytes may be involved in various vitreoretinal diseases by increasing HIF-1alpha protein stability and HIF-1alpha-DNA binding, and thus increasing VEGF production under pathological conditions. Dexamethasone seems to be capable of inhibiting hypoxic and TNF-alpha dependent VEGF production, presumably via its inhibitory effects on HIF-1alpha protein levels and its DNA-binding activity.

Extensor tendon rupture associated with osteoarthritis of the distal radioulnar joint.

Non-rheumatoid osteoarthritis of the distal radioulnar joint can cause extensor tendon rupture. We analysed the radiographic morphology of the distal radioulnar joint to identify the risk factors for this complication. Forty-one wrist X-rays of 37 patients with extensor tendon rupture caused by distal radioulnar joint osteoarthritis were evaluated retrospectively for the severity of osteoarthritis by the Kellgren/Lawrence scoring system. Measurements were obtained from posteroanterior views. All but one wrist had severe osteoarthritic changes exceeding grade 3. The radiographic features that were different from those of the contralateral wrists included deepening and widening of the sigmoid notch, radial shift of the ulnar head and dorsal inclination of the sigmoid notch. There was no significant association between tendon rupture and the morphology of the ulnar head or ulnar variance. The scallop sign, dorsal inclination of the sigmoid notch and radial shift of the ulnar head are radiological risk factors for extensor tendon ruptures.

Threonine 74 of MOB1 is a putative key phosphorylation site by MST2 to form the scaffold to activate nuclear Dbf2-related kinase 1.

Mammalian nuclear Dbf2-related (NDR) kinases (LATS1 and 2, NDR1 and 2) play a role in cell proliferation, apoptosis and morphological changes. These kinases are regulated by mammalian sterile 20-like kinases (MSTs) and Mps one binder (MOB) 1. Okadaic acid (OA), which activates MST2, facilitates the complex formation of MOB1, MST2 and NDR1 in HEK293FT cells. The in vitro biochemical study demonstrates the phosphorylation of MOB1 by MST2. The phosphorylated MOB1 alone is capable to partially activate NDR1 in vitro, but MST2 is also required for the full activation. The knockdown of MOB1 or MST2 abolishes the OA-induced NDR1 activation in HEK293FT cells. Among MOB1 mutants, in which each serine or threonine residue is replaced with alanine, MOB1 T74A and T181A mutants fail to activate NDR1. Thr74, but not Thr181, is phosphorylated by MST2 in vitro, although MOB1 is also phosphorylated by MST2 at other site(s). The interaction of MOB1 T74A with NDR1 is barely enhanced by OA treatment. These findings indicate that the phosphorylation of MOB1 at Thr74 by MST2 is essential to make a complex of MOB1, MST2 and NDR1, and to fully activate NDR1.

Closed rupture of the flexor tendons caused by carpal bone and joint disorders.

We analysed 21 patients with closed rupture of the flexor tendons caused by carpal bone and joint disorders. The tendon that ruptured depended on the location of the bone perforation into the carpal tunnel. Radiocarpal arthrography was performed in 13 patients and capsular perforation was demonstrated by contrast medium leakage into the carpal canal in 11 patients. This proved a useful diagnostic test. The flexor tendon(s) were reconstructed with free tendon graft in 17 patients, cross-over transfer of flexor tendons from adjacent digits in two and buddying to an adjacent flexor tendon in one patient. Postoperative total active range of motion in the fingers after 13 free tendon graft reconstructions averaged 213 degrees (range 170-265 degrees ). The active range of motion of the thumb-interphalangeal joint after free tendon graft reconstruction in three cases improved from 0 degrees to 33 degrees on average (range 10 degrees -40 degrees ).

Aqueous and vitreous penetration of levofloxacin after topical and/or oral administration.

PURPOSE: To investigate the aqueous and vitreous penetration of levofloxacin, the drug was administered topically and/or orally to patients undergoing vitrectomy. METHODS: Thirty-six patients undergoing initial vitrectomy with phacoemulsification and aspiration (PEA) were enrolled, and were divided randomly into three groups. Group 1 was treated with topical application of levofloxacin (three times on the day before surgery and seven times on the day of surgery), Group 2 received oral administration of levofloxacin (200 mg twice on the day before surgery and 200 mg at 3 hours before surgery), and Group 3 received both topical and oral levofloxacin according to the above schedules. The concentration of levofloxacin was measured in aqueous humor and vitreous fluid samples obtained during surgery. RESULTS: In Groups 1, 2, and 3, the mean levofloxacin concentration in aqueous humor was 0.765+/-0.624 micro g/mL, 1.279+/-0.440 micro g/mL, and 1.823+/-0.490 micro g/mL, respectively, while the mean levofloxacin concentration in vitreous fluid was <0.02 micro g/mL, 1.455+/-0.445 micro g/mL, and 1.369+/-0.530 micro g/mL, respectively. CONCLUSIONS: Oral administration of levofloxacin at a dose of 400 mg/day was sufficient for the prophylaxis of ocular infections, because the drug concentrations in both aqueous humor and vitreous fluid were higher than the MIC90 values for major ocular pathogens. Topical application of levofloxacin achieved adequate drug levels in aqueous humor, but not in vitreous fluid, while combined topical and oral administration had an additive effect on the drug concentration in aqueous humor.

Long-term clinical outcomes and therapeutic benefits of triamcinolone-assisted pars plana vitrectomy for proliferative vitreoretinopathy: a case study.

PURPOSE: To investigate intraoperative visibility and long-term clinical outcome following triamcinolone acetonide (TA)-assisted pars plana vitrectomy (PPV) for proliferative vitreoretinopathy (PVR). METHODS: A retrospective interventional noncomparative clinical study was carried out on 21 eyes from 21 patients with more than grade C2 PVR, all of whom underwent TA-assisted PPV. Two of the specimens were observed with an electron microscope. After treatment, outcome measures, including changes in best-corrected visual acuity (BCVA), intraocular pressure (IOP) elevation, corneal pathology, and occurrence of endophthalmitis, were recorded. Patient follow-up time was >36 months (mean +/-standard deviation = 47.3 +/- 6.7 months). RESULTS: TA improved the intraoperative visualization of the epiretinal membrane (ERM), allowing it to be easily removed together with the partially internal limiting membrane (ILM) using micro forceps. The excised tissue consisted of proliferative cells and an extracellular matrix underlying the ILM. After the operation, 71.4% of the eyes had improved BCVA. Three of the eyes showed sustained IOP elevation (14.3%); two of these cases were controlled by the administration of eyedrops, while the third required filtering surgery. In two cases, an absorption delay of the TA granule on the retinal surface was observed. One eye developed corneal stromal opacity. No other severe complications occurred during the observation period. CONCLUSIONS: TA-assisted PPV offers improved visualization during the surgical management of PVR, and allows surgeons to excise the ERM safely and effectively without the risk of serious complications.

The two locations of ganglions causing radial nerve palsy.

Ganglions associated with radial nerve palsy at two different locations were identified at the elbow in 14 patients. The first type, found in 13 patients, arose from the anterior capsule of the proximal radioulnar joint and was located proximal to the proximal edge of the supinator muscle. It compressed the main radial nerve anteriorly. The second type, which has not been reported before in patients without abnormalities in the elbow joint, was found in the remaining patient. It was located in the supinator muscle, distal to the proximal edge of the supinator muscle, and compressed the posterior interosseous nerve against the proximal radius. Magnetic resonance imaging makes it possible to identify ganglions in a wide area around the elbow. This examination should be carried out in view of the possible presence of both types of ganglion. Magnetic resonance imaging also provides more accurate information than computed tomography or ultrasonography about the location and characteristics of the mass.

Ligand-of-Numb protein X is an endocytic scaffold for junctional adhesion molecule 4.

Junctional adhesion molecule 4 (JAM4) is a cell adhesion molecule that interacts with a tight junction protein, membrane-associated guanylate kinase inverted 1 (MAGI-1). Our previous studies suggest that JAM4 is implicated in the regulation of paracellular permeability and the signalings of hepatocyte growth factor. In this study, we performed yeast two-hybrid screening to search for an unidentified JAM4-binding protein and obtained one isoform of Ligand-of-Numb protein X1 (LNX1), LNXp70, that is an interactor of Numb. Ligand-of-Numb protein X1 is expressed in kidney glomeruli and intestinal epithelial cells, where JAM4 is also detected. Immunoprecipitation from kidney lysates supports the in vivo interaction of proteins. Biochemical studies reveal that JAM4 directly binds the second PDZ domain of LNX1 through its carboxyl terminus. Junctional adhesion molecule 4, LNX1 and Numb form a tripartite complex in vitro and are partially colocalized in heterologous cells. Ligand-of-Numb protein X1 facilitates endocytosis of JAM4 and is involved in transforming growth factor beta -induced redistribution of JAM4 in mammary epithelial cells. Experiments using dominant-negative constructs and RNA interference insure that Numb is necessary for the LNX1-mediated endocytosis of JAM4. All these findings indicate that LNX1 provides an endocytic scaffold for JAM4 that is implicated in the reorganization of cell junctions.

Surgical results of combined pars plana vitrectomy, phacoemulsification, and intraocular lens implantation.

PURPOSE: To evaluate the results and complications of combined pars plana vitrectomy (PPV), phacoemulsification and aspiration (PEA), and intraocular lens (IOL) implantation. METHODS: A total of 117 eyes from 114 patients who had undergone PPV combined with PEA and IOL implantation were retrospectively analyzed. Combined surgery was performed for a wide variety of vitreoretinal diseases. Intraoperative and postoperative complications were also reviewed. RESULTS: The postoperative BCVA improved by 2 lines or more in 85 eyes (72.6%). Intraoperative complications consisted of retinal tears in 14 eyes (12.0%) and posterior capsular rupture in 2 eyes (1.7%). Iatrogenic retinal tears occurred more frequently in eyes with a macular hole than in eyes with any other disease (p=0.005, chi-square test). Postoperative complications consisted of posterior capsule opacification (PCO) (21 eyes), transient IOP elevation (29 eyes), vitreous hemorrhage (6 eyes), anterior chamber fibrin exudation (11 eyes), posterior iris synechia (8 eyes), neovascular glaucoma (1 eye), and recurrent retinal detachment (RD) (2 eyes). Fibrin exudation occurred more frequently in eyes with proliferative diabetic retinopathy (PDR) and RD than in eyes with any other disease (p=0.03, chi-square test). PCO occurred more frequently in eyes with PDR than in eyes with any other disease (p=0.03, chi-square test). CONCLUSIONS: The present study suggests that a high success rate can be achieved when recently improved PPV techniques are combined wi th PEA and IOL implantation. The complications that were observed following this combined treatment varied with respect to the vitreoretinal disease present prior to surgery.

Closed rupture of the flexor tendons of the little finger secondary to non-union of fractures of the hook of the hamate.

We report six patients with closed flexor tendon rupture affecting the little finger, occurring secondarily to non-union of the hook of the hamate bone. The ununited fragments were separated from the basal part of the hook by more than 1mm. The fragments were also rounded and showed marginal sclerosis. Non-union was located in the middle part of the hook in three patients, the tip in two, and the base in one. At operation, the fragments were removed in all patients. Five patients were treated by free tendon grafts using three palmaris and two plantaris grafts and one underwent tendon transfer. Postoperative total range of active motion of the little finger averaged 218 degrees (range 185-265 degrees ). All patients returned to their original employment. This series would suggest that flexor tendon rupture can occur after fracture of the hook of the hamate bone, even when the ununited fragment is small and/or rounded.

Rehabilitation of a bilateral maxillectomy patient with a free fibula osteocutaneous flap.

Rehabilitation of patients who have undergone bilateral maxillectomy is difficult because of extensive loss of bone and soft tissue. In this clinical report, prosthodontic rehabilitation of oral function in a bilateral maxillecitomy patient combined with a new fibular osteocutaneous flap, which was designed to have two oronasal slits for the retention of an obturator prosthesis, was described. A 58-year-old man with a maxillary alveolar carcinoma underwent bilateral maxillectomy. The defect was reconstructed using a vascularized fibular bone wrapped circumferentially with a peroneal flap, which was fixed with miniplates between the right malar prominence and cut edge of the left zygoma remaining two slits anterior and posterior to the graft. Two and half weeks after the surgery, a delayed surgical obturator was delivered and an obturator prosthesis was delivered 6 weeks after the surgery. This obturator prosthesis could be extended into the slits to engage the tissue undercuts, and was stable during use. Mastication, deglutition, articulation and the mid-facial profile of the patient were rehabilitated. After installation of the obturator prosthesis, relining of the prosthesis base was carried out alongside the healing process of the graft, and adjustment of occlusions and high-pressure spots was carried out. No clinical disorders were observed either in the grafted tissue or the obturator prosthesis with a 3-year prognosis. Newly designing a fibular osteocutaneous flap combined with tissue-borne obturator prosthesis is one successful approach to the restoration of oral function, and increases the patient's quality of life after bilateral maxillectomy.

Glycoconjugates with NeuAc-NeuAc-Gal-Glc are more effective at preventing adhesion of Helicobacter pylori to gastric epithelial cells than glycoconjugates with NeuAc-Gal-Glc.

Helicobacter pylori (H. pylori) adheres to human gastric epithelial cells, eliciting various gastroduodenal diseases. Gangliosides play a critical role in bacterial adhesion to cell surfaces. The present study examined how residues of gangliosides are important for inhibition of adhesion of H. pylori to MKN-45 cells. We measured adhesion or detachment effects of gangliosides on the interaction between MKN-45 cells and H. pylori, as well as interleukin-8 production. Among the gangliosides, O-Ac-GD3, GT(1b), GD(1a), GD(1b), GT(1a), and GD3 had potent dose dependent inhibitory effects on adhesion of H. pylori to MKN-45 cells, interleukin-8 production, and vacuole formation induced by H. pylori toxin binding to Vero cells. GD3 also accelerated bacterial detachment of MKN-45 cells with adherent H. pylori in a dose dependent manner. Such results strongly suggest that the mechanism involved in the inhibition of H. pylori adhesion is mediated by the variations of the residues of the NeuAc-NeuAc-Gal-Glc chain of gangliosides. NeuAc-NeuAc-Gal-Glc exhibits a more inhibitory effect on adhesion than the NeuAc-Gal-Glc chain. Such gangioside and oligosaccrharide sequences appear to have therapeutic importance for prevention of H. pylori adhesion, as well as reduction of both inflammation and gastric mucosal injuries.

Preoperative assessment of anterolateral thigh flap cutaneous perforators by colour Doppler flowmetry.

An anterolateral thigh flap is very useful in head and neck reconstruction because of its long and large-caliber vascular pedicle, large skin territory and elevation simultaneous with tumour resection. However, the number and locations of cutaneous perforators vary individually, and thus, it is not widely used because flap elevation is often complicated and time-consuming owing to unexpected anatomical variations. To overcome this disadvantage, we assessed the number and locations of cutaneous perforators preoperatively by colour Doppler flowmetry. These data were compared with the intraoperative anatomical findings and their reliability evaluated. A total of 48 cutaneous perforators were found by preoperative colour Doppler flowmetry scanning of 17 anterolateral thigh flaps. All the perforators except two were found intraoperatively. Doppler scanning failed to detect four perforators. Colour Doppler flowmetry assessment therefore has a 92% true-positive rate and a 95.8% positive predictive value. All the flaps except one included multiple perforators, and sufficient blood circulation was observed in all cases. No flaps were unexpectedly changed to anteromedial thigh flaps or contralateral anterolateral thigh flaps because of inappropriate cutaneous perforators or the absence of perforators. Though this investigation is relatively time-consuming (30-40 min) and requires skill, it is very useful for preoperative flap planning and increases the reliability and safety of elevating an anterolateral thigh flap.