Wholly vascularized millimeter-sized engineered tissues by cell-sized microscaffolds.

The in vitro fabrication of wholly vascularized millimeter-sized engineered tissues is still a key challenge in the tissue engineering field. Recently we reported a unique approach 'sedimentary culture' using a collagen microfiber (CMF) to fabricate large-scale engineered tissues. The millimeter-sized tissues with high extracellular matrix (ECM) density were easily obtained by centrifugation of cells and CMFs and subsequent cultivation because the CMFs acted as a micrometer-sized scaffold. However, cell distribution in the obtained tissues was not homogeneous because of the different sedimentation velocity of the cells and CMFs because of their size difference. Here we report the fabrication of wholly vascularized millimeter-sized engineered tissues using cell-sized CMFs. To avoid dissolving, vacuum drying was performed at 200 °C for 24 h for thermal crosslinking of primary amine groups of type I collagen. The 200- and 20-µm-sized CMFs (CMF-200 and CMF-20) were obtained by homogenization and subsequent sonication of the crosslinked collagen. Interestingly, the CMF-20 indicated a similar sedimentation velocity with cells because of their same size range, thus uniform millimeter-sized tissue with homogeneous cell distribution was fabricated by the sedimentary culture method. To form a whole blood capillary structure in the tissues, fibronectin (FN) was adsorbed on the surface of CMF-20 to stimulate endothelial cell migration. The distribution of the blood capillary network in 1.6-mm-sized tissues was markedly improved by FN-adsorbed CMF-20 (FN-CMF-20). Sedimentary culture using FN-CMF-20 will create new opportunities in tissue engineering for the in vitro fabrication of wholly vascularized millimeter-sized engineered tissues.

Repair of neglected Achilles tendon rupture using gastrocnemius fascial flaps.

BACKGROUND: Neglected Achilles tendon rupture is a rare disorder. In this article, we discuss the results of 10 patients with neglected Achilles tendon ruptures who were treated surgically. METHODS: Between 1980 and 1997, we treated 10 patients (6 men and 4 women) using gastrocnemius fascial flaps according to the method described by Lindholm. The mean age of the patients at the time of the operation was 51 years (range 38-57 years). They were followed-up for 26-192 months. RESULTS: There were significant differences between the American Orthopaedic Foot and Ankle Society (AOFAS) scale score before the operation (72.6+/-5.3) and the score at the most recent follow-up (98.1+/-2.5) (p<0.0001). On Cybex isokinetic strength testing, the peak torque deficiencies in plantar flexion ranged from 8% to 68% at the low setting and from 19% to 33% at the high setting preoperatively, and ranged from -9% to 17% at the low setting and from -13% to 23% at the high setting postoperatively. There were no re-ruptures. CONCLUSION: Our data indicate that the reconstructive technique using gastrocnemius fascial flaps can result in an excellent clinical and functional outcome.

Hydroxyapatite and bFGF Coating of Detachable Coils for Endovascular Occlusion of Experimental Aneurysm.

SUMMARY: The purpose of this study was to evaluate the effect of hydroxyapatite (HAp) and fibroblast growth factor-basic (bFGF) coating on Guglielmi detachable coils (GDCs) in an experimental aneurysm model. A total of 18 aneurysms were experimentally made in the common carotid arteries of swine. Embolization was done on these aneurysms using standard GDCs and coated GDCs with HAp (GDC-HAp) and with bFGF (GDC-HAp-bFGF). The animals were then killed 14 days after embolization. The development of tissue scarring and coverage the aneurysm's orifice were evaluated macroscopically. No significant difference of volume ratio of the coils exited in each groups. Macroscopically, covering ratio of fibrous membrane at the neck of aneurysms were 88.3 +/- 14.7% in a group with GDC-HAp-bFGF, while it were 26.7 +/- 15.3% in a group with standard GDC and it was 41.7 +/- 31.7% in a group with GDC-HAp. These results indicated that coating by hydroxyapatite and bFGF might facilitate a wound healing in an experimental aneurysm model.

A case of superficial peroneal nerve injury during ankle arthroscopy.

We report a case of superficial peroneal nerve (SPN) injury caused by ankle arthroscopy. A 20-year-old woman underwent arthroscopy on her right ankle because of chronic ankle pain after a sprain. After arthroscopy, the patient complained of pain on the dorsum of her right foot and felt a radiating pain from the anterolateral portal to the dorsomedial aspect of her foot. Eight months after arthroscopy, we found that a neuroma had developed on the intermediate dorsal cutaneous nerve, and performed neurolysis of the SPN. Her symptoms gradually decreased after surgery, and had disappeared by 45 months. To avoid such an injury of the SPN, the safest placement of the anterolateral portal is necessary and is, according to our previous anatomic study, 2 mm lateral to the peroneus tertius tendon.

Hydroxyapatite coating of detachable coils for endovascular occlusion of experimental aneurysm.

SUMMARY: The purpose of this study was to evaluate the effect of hydroxyapatite (HAp) coating on Guglielmi detachable coils (GDCs) in an experimental aneurysm model. A total of 12 aneurysms were experimentally made in the common carotid arteries of swine using a microsurgical technique. Embolization was done on these aneurysms using standard GDCs and GDCs coated with HAp (GDC-HAp). The animals were then killed 14 days after embolization. The physical properties of coated coils and the development of tissue scarring and coverage the aneurysm's orifice were evaluated macroscopically and microscopically. Macroscopically, a scar formation and coverage at the neck of aneurysms were observed in a group with GDC-HAp, while such findings were not seen in a group with GDC. With light microscope, fibroblasts were seen in the neck of the aneurysms in a group using GDC-HAp, whereas only a fibrin- like net was seen in a group using GDC. In a group with GDC-HAp, inflammatory response was more intense in the dome of the aneurysm with faster re-endothelial coverage of the neck of the aneurysm than the ones in a group with GDC. These results indicated that GDC-HAp might create a clinically beneficial biological surface in an experimental aneurysm model.

Repair of articular cartilage defects with cultured chondrocytes in Atelocollagen gel. Comparison with cultured chondrocytes in suspension.

We attempted to repair full-thickness articular cartilage defects in rabbit knee joints with allogeneic cultured chondrocytes embedded in Atelocollagen gel. An articular cartilage defect was created on the patellar groove of the femur. The defect was filled with chondrocytes cultured in the collagen gel and covered with periosteal flap (G group). In three other experimental groups, the same defects were transplanted with chondrocytes in monolayer culture with periosteal flap (M group), periosteal graft only (P group), or left empty (E group). At 4, 12, and 24 weeks after operation, the reparative tissue was analyzed macroscopically and histologically. At 4 weeks after operation, the surfaces of the reparative tissue were smooth, and the defects were filled with reparative tissues that resembled hyaline cartilage in all four groups. However, the reparative tissues degenerated gradually with time in the M, P, and E groups. In contrast, in the G group, the reparative tissue retained its thickness, and there was a steady integration of the grafted tissue into the adjacent normal cartilage at 24 weeks after operation. The results suggest that transplantation of allogeneic chondrocytes cultured in Atelocollagen gel is effective in repairing an articular cartilage defect.

Human chondrocyte proliferation and matrix synthesis cultured in Atelocollagen gel.

To evaluate the potential of Atelocollagen gel as a carrier for chondrocyte transplantation, histological and biochemical characteristics of the chondrocytes in gel culture were compared with those in conventional monolayer cultures. Articular chondrocytes from 20 patients were isolated by enzyme digestion, embedded in Atelocollagen gel, and cultured for up to 4 weeks. The effects on proliferation, morphological changes, and synthesis of proteoglycans were analyzed by cell counts, light and electron microscopy, and measurement of isomers of chondroitin sulfates. Chondrocytes embedded in the Atelocollagen gel gradually proliferated and produced chondroitin 6-sulfate, maintaining the chondrocyte phenotype for up to 4 weeks. In contrast, although monolayer chondrocytes increased in number, most could be characterized as being fibroblast-like cells with a reduced capability of producing chondroitin 6-sulfate. The results suggest that Atelocollagen gel permitted a gradual proliferation and matrix synthesis of chondrocytes and maintaining its phenotype. Atelocollagen gel represents an important carrier for the clinical application of cultured chondrocyte transplantation for repair of cartilage defects.

Bandage distraction technique for ankle arthroscopy.

In ankle arthroscopy, the joint space of the talocrural joint is often too narrow for insertion of the scope and instruments. Various distraction devices for this procedure have been used to widen the joint space. Bandage distraction is effective and noninvasive, but it is difficult to extend the posterior joint space sufficiently for insertion of the scope. Here we describe a new bandage distraction method that can extend the posterior joint space adequately. Using our method, the anterior and posterior joint spaces on direct lateral radiographs were measured after adding the distraction force in nine healthy volunteers (18 ankles; three men and 6 women). This was compared to a previously reported method. The posterior joint space was widened a greater amount when our new bandage distraction technique was used.

Hyaluronic acid enhances proliferation and chondroitin sulfate synthesis in cultured chondrocytes embedded in collagen gels.

The effects of hyaluronic acid (HA) on the proliferation and chondroitin sulfate (CS) synthesis of chondrocytes embedded in collagen gels were examined. Articular cartilage was isolated from the humerus, femur, and tibia of 21 10-week-old Japanese white rabbits. Chondrocytes isolated by collagenase digestion were embedded in type I collagen gels and cultured in Dulbecco's modified Eagle's medium (DMEM) with various doses of HA for 4 weeks. Histological and biochemical evaluations were performed at postculture weeks 1, 2, 3, and 4. For biochemical evaluations, isomers such as chondroitin 6-sulfate (delta(di)-6S) and chondroitin 4-sulfate (delta(di)-4S) synthesized by cultured chondrocytes were determined by high performance liquid chromatography (HPLC) combined with fluorometry. Morphological and histological studies demonstrated that HA-treated chondrocytes in collagen gel proliferated profusely while maintaining their phenotype. At postculture week 4, 0.1 mg/ml of HA induced an eightfold increase in cell counts compared with HA pretreatment values, or 1.5-fold more than control group. Synthesis of delta(di)-6S (delta(di)-6S content/cell) in groups treated with 0.01 and 0.1 mg/ml of HA significantly increased, while gel accumulation rates in groups treated with 0.1 and 1.0 mg/ml of HA scored significantly higher values than other groups. In collagen gel culture, HA enhanced the proliferation and delta(di)-6S synthesis of chondrocytes while maintaining their phenotype. In clinical application, since the supply of autologous chondrocytes for transplantation is not unlimited, the HA-treated culture method may be useful for increasing the number of chondrocytes and thus improving the quality of implants.

Effects of basic fibroblast growth factor on proliferation and phenotype expression of chondrocytes embedded in collagen gel.

1. Whether basic fibroblast growth factor (bFGF) can stimulate proliferation and synthesis of chondroitin sulfate of chondrocytes in gel culture while maintaining the phenotype was studied. 2. At 3 weeks in culture, the cell number in 1.0 ng/ml (18.5+/-2.1 x 10(5)) and 10.0 ng/ml of the bFGF group (15.3+/-1.9 x 10(5)) was significantly larger than that in 0 ng/ml (12.3+/-2.1 x 10(5)), 0.1 ng/ ml (11.7+/-2.2 x 10(5)) and 100.0 ng/ml of the bFGF group (9.8+/-2.3 x 10(5)). 3. All doses of bFGF used in this study suppressed synthesis of chondroitin 6-sulfate. 4. Chondrocyte phenotype in gel culture was maintained for 4 weeks even with stimulation of bFGF.

Thermoregulatory response in female patients during lower abdominal surgery in the head-down tilt position.

BACKGROUND: The head-down tilt (HDT) position suppresses sympathetic nervous activity. We investigated the effect on thermoregulation of the HDT position during lower abdominal surgery under general anaesthesia. METHODS: Tympanic membrane temperature was measured to assess core temperature, and forearm-fingertip and calf-toe temperature gradients were used to assess peripheral vasoconstriction in 30 female patients less than 60 years of age. RESULTS: During surgery in the HDT position (n = 10), the tympanic membrane temperature was similar to that of patients in the supine position (n = 10) (36.2 degrees C and 36.2 degrees C), but in patients operated in the HDT position and premedicated with an angiotensin converting enzyme inhibitor (5 mg of enalapril, n = 10) it was reduced (35.7 degrees C, P < 0.05). In both groups of patients operated in the HDT position, forearm and calf skin-surface temperature gradients were lower by 2.3 degrees C than those in patients operated in the supine position (P < 0.05). CONCLUSIONS: During lower abdominal surgery the head-down tilt position does not augment core hypothermia in spite of suppressed peripheral vasoconstriction, and the renin-angiotensin system may be of importance for thermoregulation.

Premedication with famotidine augments core hypothermia during general anesthesia.

BACKGROUND: Animal studies have provided considerable evidence to support a role of histamine in the central nervous system in thermoregulation, and premedication with a histamine H2 receptor antagonist before general anesthesia is used to reduce the risk of acid aspiration. The authors investigated whether premedication with famotidine had an effect on thermoregulation during general anesthesia. METHODS: In a randomized, placebo-controlled study, 30 ASA physical status 1 or 2 patients, scheduled for open abdominal surgery, were given either placebo or 40 mg oral famotidine 3 h before induction of anesthesia. Epidural buprenorphine (4 micrograms/kg) was injected, and anesthesia was maintained with 0.4-0.6% isoflurane and 66% nitrous oxide in oxygen. The tympanic membrane temperature was measured to assess core temperature, and forearm-fingertip and calf-toe skin-surface temperature gradients were used to assess peripheral vasoconstriction. Tympanic membrane temperature triggering initial vasoconstriction (a skin temperature gradient of 0 degree C) identified the vasoconstriction threshold. RESULTS: Tympanic membrane temperature during surgery in the patients premedicated with famotidine was significantly less than those in the patients without famotidine. Famotidine significantly reduced the thermoregulatory threshold for vasoconstriction in the leg (35.0 +/- 0.5 degree C, P < 0.05), compared to that in the placebo group (36.4 +/- 0.6 degree C) Once triggered, thermoregulatory vasoconstriction produced a core-temperature plateau and no further hypothermia was observed for the duration of the study. Neither mean arterial pressure nor heart rate were significantly different between the two groups. CONCLUSIONS: Premedication with famotidine augments intraoperative hypothermia. The mechanism appears to be inhibition of centrally mediated thermoregulatory control.

Influence of workload on the antihypertensive effect of exercise.

1. The relation between workload and the antihypertensive effect of exercise therapy in hypertensive patients, and the mechanism of that effect, were investigated. 2. Twenty-six patients participated in the study and were randomly assigned to 10 weeks of either low or high workload exercise. In the low workload group, 16 mild hypertensive patients were treated with bicycle ergometer exercise at approximately 50% of their maximum oxygen consumption (VO2max) for 60 min three times a week for 10 weeks. In the high workload group, 10 mild hypertensive patients exercised on the same schedule, but at approximately 75% of VO2max. 3. After 10 weeks of exercise, the low workload group had significantly lower systolic (9 mmHg), mean (6 mmHg) and diastolic (6 mmHg) blood pressures. In the high workload group, decreases in systolic (3 mmHg), mean (4 mmHg) and diastolic (5 mmHg) blood pressure were not statistically significant. 4. In the low workload group, changes in haemodynamic and humoral variables were not significant, except for a reduction in plasma norepinephrine at week 7. Cardiac index and plasma norepinephrine tended to decrease. In the high workload group, plasma norepinephrine and the renin-angiotensin system were transiently stimulated after 4 weeks of exercise. Stroke volume significantly increased (+26.4%) after 10 weeks of high workload exercise. 5. Based on these results and better patient compliance with the exercise programme in the low workload group than in the high workload group, low workload exercise therapy was recommended to mild hypertensive patients.

Mild exercise decreases plasma endogenous digitalislike substance in hypertensive individuals.

Changes in a plasma endogenous digitalislike substance were investigated in relation to the antihypertensive mechanism of mild exercise. Fifteen women with mild essential hypertension and seven normotensive female volunteers were divided into exercised hypertensive (n = 10), nonexercised hypertensive (n = 5), and nonexercised normotensive (n = 7) groups. A 4-week general clinical observation period preceded the study period of 10 weeks. The exercised hypertensive individuals were treated with a lactate threshold exercise that corresponded to approximately 50% of the maximum oxygen consumption three times a week, whereas the nonexercised groups were observed at the outpatient clinic as control groups. In the exercised group, systolic blood pressure fell by 7 mm Hg (p = 0.05), diastolic by 6 mm Hg (p less than 0.01), and mean blood pressure by 7 mm Hg (p less than 0.01) after 10 weeks. The reduction in the plasma endogenous digitalislike substance was significant after 7 (-1.02 ng/ml, p less than 0.05) and 10 (-1.04 ng/ml, p less than 0.05) weeks in this group. It positively correlated with the reduction in diastolic (r = 0.70, p less than 0.05) or mean (r = 0.66, p less than 0.05) blood pressure and with changes in plasma norepinephrine (r = 0.76, p less than 0.05). The mean corpuscular volume of erythrocytes decreased (-1.7 fl, p less than 0.01) after 10 weeks of exercise, and the plasma volume index tended to decrease (-108 ml/m2, p = 0.28). In the control groups, significant changes in blood pressure and plasma endogenous digitalislike substance were not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Active and inactive renin after exercise.

The effects of graded exercise on plasma concentrations of active and inactive renin were studied in seven healthy men. Exercise was performed on a cycle ergometer at four different exercise intensities (corresponding to 30%, 50%, 80% and 87% of VO2max) for 10 min each. Concentrations of active renin and total renin after activation by trypsin were measured by direct immunoradiometric assay. Non-trypsin-activated renin concentration (inactive) was obtained by subtraction. Active renin concentrations at 30%, 50%, 80% and 87% of VO2max were 1.2, 1.9, 3.1 and 4.6 times higher than the control concentration, respectively. Similar increases in plasma renin concentration, determined by conventional enzymatic assay, were observed at every stage. In contrast, changes in inactive renin concentration were not significant at any stage. Significant increases in noradrenaline concentration were found at every exercise stage, but adrenaline, aldosterone and lactate concentrations were significantly elevated only after exercise at 50%, 80% and 87% of VO2max. The similarity between the changes in concentration of active renin and noradrenaline would suggest that sympathetic nerve activity may have been responsible either for the release of active renin or for the conversion of inactive renin to its active form in the kidney.

Changes of dopamine and atrial natriuretic factor by mild exercise for hypertensives.

Changes of humoral factors related to the regulation of fluid volume were investigated in exercise training for hypertensives. Twelve patients with essential hypertension were treated with an aerobic exercise for 10 weeks. A significant reduction in blood pressure from 161 +/- 3/100 +/- 2 mmHg at week 0 to 142 +/- 5/94 +/- 3 mmHg at week 4 was observed which continued until week 10. Urine dopamine was increased significantly at the 4th week from 386 +/- 29.4 micrograms/day at week 0 to 524 +/- 46.3 micrograms/day and plasma atrial natriuretic factor (ANF) was significantly reduced at the 4th week, from 41.5 +/- 2.7 pg/ml at week 0 to 32.6 +/- 3.7 pg/ml. Plasma volume was found reduced significantly from 2,531 +/- 166 ml/m2 at week 0 to 2,221 +/- 165 ml/m2 at week 10. These results suggest that the increase of dopamine and reduction of plasma ANF which took place at the early stage might be related to, at least in part, the depletion of plasma volume and the reduction of blood pressure in mild exercise for hypertensives.