Increased antibiotic release from a bone cement containing bacterial cellulose.

BACKGROUND: Major disadvantages of antibiotic bone cements include limited drug release and reduced strength resulting from the addition of high doses of antibiotics. Bacterial cellulose, a three-dimensional hydrophilic mesh, may retain antibiotics and release them gradually. We hypothesized that the addition of cellulose to antibiotic bone cement would improve mechanical strength and antibiotic release. QUESTIONS/PURPOSES: We therefore examined the mechanical strength and antibiotic release of cellulose antibiotic cement. METHODS: A high dose of antibiotics (5 g per 40 g cement powder) was incorporated into bacterial cellulose and then mixed with bone cement. We compared the compression strength, fracture toughness, fatigue life, and elution kinetics of this formulation with those of plain cement and a traditional antibiotic cement. RESULTS: The average values for compression strength, fracture toughness, and fatigue life of the cellulose antibiotic cement were 97%, 97%, and 78% of the values obtained for plain cement, respectively. The corresponding values for the traditional antibiotic cement were 79%, 82%, and 17%, respectively. The cumulative elution over 35 days was 129% greater from the cellulose antibiotic cement than from the traditional antibiotic cement. CONCLUSIONS: With a high dose of antibiotics, incorporating cellulose into the bone cement prevented compression and fracture fragility, improved fatigue life, and increased antibiotic elution. CLINICAL RELEVANCE: Antibiotic cements containing cellulose may have applications in clinical situations that require high levels of antibiotic release and preservation of the mechanical properties of the cement.

Optimum predrilled hole size for bone screws used in osteochondral fixation: in vitro biomechanical study and clinical case.

BACKGROUND: Bone screws have a mechanical advantage over bone pegs for fixation of osteochondral fractures. The purpose of this study was to investigate predrilling methods to facilitate bone screw insertion. METHODS: The 162 bone screws (major diameter 3.0 mm; minor diameter 2.5 mm; length 10 mm) used were made from porcine tibial cortical bone. Metal screws of the same design were used as a control. The screws were inserted, without tapping, into predrilled holes of six different sizes (2.4-2.9 mm diameter; 80%-97% of the screw) made in cancellous bone harvested from porcine femoral condyle, and pullout strength was determined. The volumetric bone mineral densities of porcine bones were compared to those of human bones. In a clinical case, an osteochondral fracture of the patella was repaired using three autologous bone screws. RESULTS: Bone screws occasionally broke due to high insertion torque (108-158 N.mm); the incidences were 30% and 19% with the 80% and 83% sized holes, respectively. None of the screws inserted into holes of size >/= 87% broke, and their insertion torques never exceeded 100 N.mm. Metal screws were inserted without breakage regardless of hole size. Bone screws inserted into 80%-93% holes had the greatest pullout strengths (range 154-165 N), whereas 97% holes had a mean strength of 84 N. Metal screws had large pullout strengths when inserted into 80% and 83% holes (164 and 169 N, respectively). The mineral density of porcine bone was lower than that of human bone (1167 and 1357 mg/cm(3) for cortical bone and 193 and 269 mg/cm(3) for cancellous bone, respectively). In the clinical case, bone screws were inserted smoothly into 90% holes. CONCLUSIONS: The optimal size of predrilled holes for bone screws (87%-93%) differs from that for metal screws.

Proximal half angle of the screw thread is a critical design variable affecting the pull-out strength of cancellous bone screws.

BACKGROUND: Screws with strong pull-out strength have been sought for the treatment of cancellous bone. We hypothesized that an obliquely angled screw thread has advantages over conventional vertical thread with a minimal proximal half angle. METHODS: Metal and bone screws were made of stainless steel and porcine cortical bone. Their proximal half angle was set at 0 degrees , 30 degrees , or 60 degrees . The screws were inserted into porcine cancellous bone. At 0 degrees , the thread faced the recipient bone vertically. Pullout tests at a rate of 30 mm/min (n=40, each screw type) and microcomputed tomography (n=6) were conducted. FINDINGS: The pull-out strength of the screws was maximal at 30 degrees ; 348.8 (SD, 44.1)N with metal and 326.6 (39.4)N with bone. It was intermediate at 0 degrees ; 301.9 (35.9)N with metal and 278.2 (30.6)N with bone. It was minimal at 60 degrees; 126.5 (39.0)N with metal and 174.8 (29.7)N with bone. Cancellous bone was damaged between the threads at 30 degrees , while intact cancellous bone was preserved between the threads at 0 degrees. INTERPRETATION: A proximal half angle of around 30 degrees is appropriate because the pullout force is applied to the recipient bone evenly. Commercial cancellous screws can be improved by changing the thread shape to minimize the damage to recipient bone.

Highly active mutants of carbonyl reductase S1 with inverted coenzyme specificity and production of optically active alcohols.

A wild type NADPH-dependent carbonyl reductase from Candida magnoliae (reductase S1) has been found not to utilize NADH as a coenzyme. A mutation to exchange the coenzyme specificity in reductase S1 has been designed by computer-aided methods, including three-dimensional structure modeling and in silico screening of enzyme mutants. Site-directed mutagenesis has been used to introduce systematic substitutions of seven or eight amino acid residues onto the adenosine-binding pocket of the enzyme according to rational computational design. The resulting S1 mutants show NADH-dependency and have lost their ability to utilize NADPH as a coenzyme, but retain those catalytic activities. Kinetic parameter V(max) and K(m) values of those mutants for NADH are 1/3- to 1/10-fold those of the wild type enzyme for NADPH. As a model system for industrial production of optically active alcohols, the S1 mutants can be applied to an asymmetric reduction of ketones, cooperating with a coenzyme-regeneration system that uses an NAD-dependent formate dehydrogenase.

Temporal water deficit and wood formation in Cryptomeria japonica.

Cell behavior in the cambium and developing xylem of 3-year-old Japanese cedar (Cryptomeria japonica D. Don.) trees, during and after an 11-day suspension of irrigation, was analyzed. Leaf xylem pressure potential and tangential strain of the stem surface were monitored throughout the experiment. Anatomical features and numbers of developing tracheids and cambial cells were observed in four trees, sampled on Days 0, 4, 8 and 11 after irrigation was suspended. Daytime xylem pressure potential decreased to -1.9 MPa on Day 7 and remained the same until irrigation was resumed on Day 11. The transverse dimensions of the tracheids, which began to form secondary walls, began to decrease on Day 4. The number of cells in the cambial zone and cell expansion zone decreased abruptly on Day 8. Tangentially aligned developing tracheids with collapsed cell walls were observed in samples harvested on Days 8 and 11. Secondary wall formation was recognized in these tracheids. After the resumption of irrigation, xylem pressure potential recovered rapidly to the same value as before the suspension of irrigation. Tangential strain increased within 30 min after the resumption of irrigation, and continued to increase until the onset of light the next day. Eighteen days after the resumption of irrigation, anatomical features of cells in the cambium and cell-expansion zone were similar to those observed before suspension of irrigation.

A new type of aminoacyltransferase from Saccharothrix sp. AS-2 favorable for the synthesis of D-amino acid-containing peptides.

A unique enzyme with some properties favorable for the synthesis of D-amino acid-containing peptides has been purified from the culture broth of Saccharothrix sp. AS-2. The purification steps included ammonium sulfate fractionation, chromatographies on CM-Toyopearl 650M and ProtEx Butyl, and sucrose density-gradient isoelectric focusing. The enzyme, consisting of four subunits of 56 kDa, showed its maximum transfer activity at around pH 8.2 and 35 degrees C, and had an isoelectric point of 5.8. The enzyme yielded homooligomers from methyl esters of D-Asp(OMe), D-Met, D-Phe, D-Trp, D-Tyr, and L-Glu(OMe), but showed no hydrolytic activity toward any of the D- or L-amino acid methyl esters tested. The homooligomers were not formed from the corresponding free amino acids. The reaction of Ac-D-Phe-OMe with DL-Ala-NH(2), DL-Leu-NH(2), DL-Phe-NH(2), or DL-Trp-NH(2) was effectively catalyzed by the enzyme, both the DD- and DL-stereoisomers of the expected N-acetyldipeptide being yielded. The resulting dipeptides remained unhydrolyzed even after 48 h incubation. Also, it showed no detectable hydrolytic activity toward casein, diastereomers of diAla, diMet, and diPhe, D-/L-amino acid amides, or D-/L-amino acid p-nitroanilides, indicating that the enzyme had no peptidase activity leading to secondary hydrolysis of the growing peptide. The enzyme activity was strongly depressed by phenylmethanesulfonyl fluoride, but not by penicillin G or ampicillin, suggesting that the protein is a serine enzyme lacking penicillin-binding ability. These observations lead us to the conclusion that the enzyme from Saccharothrix sp. AS-2 characterized in this study is a new type of aminoacyltransferase with an amino acid ester as the acyl donor, and has potential utility as a catalyst for the synthesis of D-amino acid-containing peptides.