Reconstruction of Vertebral Bone Defects using an Expandable Replacement Device and Bioactive Glass S53P4 in the Treatment of Vertebral Osteomyelitis: Three Patients and Three Pathogens.

BACKGROUND AND AIMS: Bioactive glass S53P4 is an antibacterial bone substitute with bone-bonding and osteostimulative properties. The bone substitute has been successfully used clinically in spine; trauma; orthopedic; ear, nose, and throat; and cranio-maxillofacial surgeries. Bioactive glass S53P4 significantly reduces the amount of bacteria in vitro and possesses the capacity to kill both planktonic bacteria and bacteria in biofilm. Three patients with severe spondylodiscitis caused by Mycobacterium tuberculosis, Candida tropicalis, or Staphylococcus aureus were operatively treated due to failed conservative treatment. The vertebral defects were reconstructed using bioactive glass S53P4 and an expandable replacement device. MATERIAL AND METHODS: Decompression and a posterolateral spondylodesis, using transpedicular fixation, were performed posteriorly in combination with an anterior decompression and reconstruction using an expandable vertebral body replacement device. For patients 1 and 2, the expander was covered with bioactive glass S53P4 only, and for patient 3, the glass was mixed with autograft bone. RESULTS: The patients healed well with complete neurological recovery. Fusion was observed for all patients. The total follow-up was 4 years for patient 1, 1 year and 8 months for patient 2, and 2 years and 2 months for patient 3. No relapses or complications were observed. CONCLUSION: The antibacterial properties of bioactive glass S53P4 also make it a suitable bone substitute in the treatment of severe spondylodiscitis.

Bioactive glass S53P4 as bone graft substitute in treatment of osteomyelitis.

Bioactive glass (BAG)-S53P4 is an osteoconductive bone substitute with proven antibacterial and bone bonding properties. In a multicentre study 11 patients with verified chronic osteomyelitis in the lower extremity and the spine were treated with BAG-S53P4 as a bone substitute. The cavitary bone defect and the surrounding of a spinal implant were filled with BAG-S53P4. The most common pathogen causing the infection was Staphylococcus aureus. The mean follow-up was 24 months (range 10-38). BAG-S53P4 was well tolerated. Nine patients healed without complications. One patient who achieved good bone formation sustained a superficial wound infection due to vascular problems in the muscle flap, and one patient had an infection due to a deep haematoma. This study shows that BAG-S53P4 is a good and well-tolerated bone substitute, and can be used in treatment of osteomyelitis with good primary results.

Lateral approach for fixation of the fractures of the distal tibia. Outcome of 20 patients. Technical note.

INTRODUCTION: The treatment of fractures of the distal third of the tibia remains still controversial. It is often difficult to get and retain good reduction by non-operative or nailing methods. Open reduction and plate fixation offers good reduction and retention of the achieved position. However, increased soft tissue damage and high complication rate has led to search less invasive surgical methods such as minimal invasive plating technique. MATERIAL AND METHODS: Lateral approach for the distal tibia allows to reduce and to plate both the fibula and the tibia using only one skin incision. We have used lateral approach for 20 consecutive tibia fracture patients and report clinical and radiological results after an average follow-up of 31 months. RESULTS: All fractures united, but two malunions were developed after good primary reduction. Seventeen patients achieved excellent or good subjective result while one had moderate and two poor results. Four superficial wound infections were noticed and they were treated conservatively. CONCLUSION: We conclude that lateral approach for the distal tibia is a demanding, but useful surgical method for treatment of the distal tibia fractures especially in cases where no medial comminution of the tibia is present and when the fibula has to be fixed, too.

Crystal structure of a recombinant anti-estradiol Fab fragment in complex with 17beta -estradiol.

The crystal structure of a Fab fragment of an anti-17beta-estradiol antibody 57-2 was determined in the absence and presence of the steroid ligand, 17beta-estradiol (E2), at 2.5 and 2.15-A resolutions, respectively. The antibody binds the steroid in a deep hydrophobic pocket formed at the interface between the variable domains. No major structural rearrangements take place upon ligand binding; however, a large part of the heavy chain variable domain near the binding pocket is unusually flexible and is partly stabilized when the steroid is bound. The nonpolar steroid skeleton of E2 is recognized by a number of hydrophobic interactions, whereas the two hydroxyl groups of E2 are hydrogen-bonded to the protein. Especially, the 17-hydroxyl group of E2 is recognized by an intricate hydrogen bonding network in which the 17-hydroxyl itself forms a rare four-center hydrogen bond with three polar amino acids; this hydrogen bonding arrangement accounts for the low cross-reactivity of the antibody with other estrogens such as estrone. The CDRH3 loop plays a prominent role in ligand binding. All the complementarity-determining regions of the light chain make direct contacts with the steroid, even CDRL2, which is rarely directly involved in the binding of haptens.

Polyelectrolyte multilayers prepared from water-soluble poly(alkoxythiophene) derivatives.

Electronically conducting polyanion and polycation based on poly(alkoxythiophene) derivatives, poly-3-(3'-thienyloxy)propanesulfonate (P3TOPS) and poly-3-(3'-thienyloxy)propyltriethylammonium (P3TOPA) have been synthesized. Both polymers are water-soluble and exhibit high conjugation length in solution and in the solid state. These polyelectrolytes were used to prepare conducting and electroactive polyelectrolyte multilayers by the sequential layer-by-layer adsorption technique. In aqueous solutions multilayers of P3TOPS with inactive polyelectrolytes (e.g., poly(diallyldimethylammonium chloride), PDADMA) displayed electrochemical and optical behavior similar to polythiophene films prepared in organic media. Their in-plane conductivity was low (ca. 1.6 x 10(-)(5) S cm(-)(1)). The conductivity could, however, be increased by a factor of ca. 40 in "all-thiophene" films, in which P3TOPA was substituted for the inactive polycation (PDADMA). The interpenetration of layers is of prime importance in films containing conducting components. The interpenetration of P3TOPS was studied by measuring the charge-transfer rate across an insulating polyelectrolyte multilayer between the substrate and the P3TOPS layer with modulated electroreflectance. The extent of interpenetration was 8-9 polyelectrolyte layers, the length scale (7-15 nm) depending on the nature of the insulating layer and, especially, on the ionic strength of the solution used for the adsorption of P3TOPS.

A time-resolved study of the mechanism of the energy transfer from a ligand to the lanthanide(III) ion in solutions and solid films.

The photochemical properties of some lanthanide chelates developed for immunohistochemistry have been studied in water and in solid Langmuir-Blodgett films. The fluorescence and triplet-state lifetimes of 4-(phenylethynyl)pyridine (PET), di[(phenylethynyl)pyridine] (D-PET), phenylterpyridine (PTP) and their tetra- or penta-acid derivatives (-TA or -PA) were measured in the presence and absence of Gd(III)-, Tb(III)- and Eu(III)-ions. The mechanism for the total process and the rate constants and quantum yields for the individual reaction steps and for the total process were determined in water solution. Time-resolved absorption and luminescence methods were also used to study the energy transfer between an amphiphilic 4-[4-[(C(10)H(12))(2)NCO]phenylethynyl]-pyridine tetra acid (A-PET-TA) and the Tb(III)- and Eu(III)-ions in solid Langmuir-Blodgett films. Luminescence and transient absorption rate constants were determined.

Crystallization and preliminary X-ray analysis of a recombinant Fab fragment in complex with 17beta-oestradiol.

The recombinant Fab fragment of the anti-17beta-oestradiol antibody 57-2 has been a target for several protein-engineering experiments. A method for production, purification and crystallization of the Fab fragment alone (apo form) and in complex with the major female sex hormone 17beta-oestradiol is reported here. Diffracting apo-form crystals were only obtained with microseeding; crystals of the Fab-steroid complex were produced by co-crystallization in the presence of oestradiol and cross-seeding with the apo-form crystals. The crystals were grown using vapour-diffusion methods with reservoir solutions containing 10-14% PEG 4000 or 8-12% PEG 8000 and Tris-HCl buffer at high pH (9.0-9.5). Both the apo and complex crystals belong to space group P2(1)2(1)2(1) and diffract to 2.0 A resolution. High-resolution X-ray data sets suitable for structure determination were collected from flash-cooled crystals using 25% glycerol as the cryoprotectant.

Production, crystallization and preliminary X-ray analysis of the human integrin alpha1 I domain.

Integrin alpha1beta1 is one of the main collagen receptors in many cell types. A fast large-scale production, purification and crystallization method for the integrin alpha1 I domain is reported here. The alpha1 I domain was crystallized using the vapour-diffusion method with a reservoir solution containing a mixture of PEG 4000, sodium acetate, glycerol and Tris-HCl buffer. The crystals belong to the C2 space group, with unit-cell parameters a = 74.5, b = 81.9, c = 37.3 A, alpha = gamma = 90.0, beta = 90.8 degrees. The crystals diffract to 2.0 A and a 94.2% complete data set to 2.2 A has been collected from a single crystal with an Rmerge of 5.8%.

Operative treatment of displaced intra-articular fractures of the calcaneus using absorbable internal fixation: a prospective study of twenty-five fractures.

OBJECTIVE: To document the use of absorbable implants in the operative treatment of displaced intra-articular fractures of the calcaneus. DESIGN: Prospective clinical study. SETTING: Level one trauma center, university hospital. PATIENTS: Twenty-five displaced intra-articular calcaneal fractures in twenty-one patients. INTERVENTION: The fractures were reduced operatively and fixed with absorbable self-reinforced polyglycolide rods. MAIN OUTCOME MEASUREMENTS: Reduction, healing, and stability of fixation were assessed on radiographs. The functional result was scored using the method of Crosby and Fitzgibbons. Patient satisfaction was recorded. RESULTS: The mean Böhler angle was -12 degrees (standard deviation 22 degrees) preoperatively and 20 degrees (standard deviation 13 degrees) postoperatively. After a two-year follow-up, seven patients had excellent, five good, three fair, and six poor functional results. Subjectively, fifteen patients were satisfied and six were dissatisfied. Twelve patients returned to work within one year, three were unemployed, two were retired prior to the accident, and four were unable to work because of the calcaneal fracture. The postoperative Böhler angle and later the appearance or absence of posttraumatic subtalar arthritis were significant prognostic factors. CONCLUSIONS: Results with absorbable implants are similar to those with other operative methods. Advantages include the absence of secondary procedures for implant removal and undisturbed computed tomography scans postoperatively.

Dislocated fractures of the talus treated with biodegradable internal fixation.

Six patients with a displaced fracture of the neck or body of the talus were treated using biodegradable screws and rods. During an average follow-up time of 24 (range, 18-31) months, there were no redisplacements nor collapses due to avascular necrosis. All the fractures united. The functional result was mainly dependent on the presence or absence of other injuries, being excellent in 4, good in 1 and poor in 1 patient who also had bilateral highly comminuted calcaneal fractures. Thus, biodegradable implants seem to be suitable for the fixation of displaced fractures of the talus.

Tibial condylar fractures fixed with totally absorbable, self-reinforced polyglycolide screws. A preliminary report.

Six patients with displaced split-depression-type tibial condylar fractures were treated with absorbable, self-reinforced polyglycolide screws. One patient underwent reoperation because of an unacceptable primary fracture reduction unrelated to the implant material. The others healed well, with good functional and anatomical results despite one slight redisplacement in a medial condylar fracture treated without plaster cast immobilisation. Absorbable polyglycolide screws seem to be suitable for the fixation of selected tibial condylar fractures. The implant removal operation is thus avoided.

Potentiometric response of conducting polymer electrodes for oxygen in neutral aqueous solutions.

The effect of oxygen on the potential of electrodes coated with a thin layer of various conducting polymers and immersed in aqueous neutral buffer solution has been studied. A theoretical equation describing the dependence of the potential on the concentration of oxygen has been derived and shown to be in conformity with the experimental results for polyaniline, polypyrrole, and poly(3-methylthiophene). The theory is based on the formation of charge-transfer complexes between oxygen molecule and the reduced (undoped) form of conducting polymer.

An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

Structure of Escherichia coli inorganic pyrophosphatase at 2.2 A resolution.

The refined crystal structures of hexameric soluble inorganic pyrophosphatase from E. coli (E-PPase) are reported to R factors of 18.7 and 18.3% at 2.15 and 2.2 A, respectively. The first contains one independent monomer; the other, two independent monomers, in an R32 unit cell. Because the E-PPase monomer is small with a large open active site, there are relatively few hydrophobic interactions that connect the active-site loops to the five-stranded twisted beta-barrel that is the hydrophobic core of the molecule. The active-site loops are, however, held in place by interactions between monomers around the threefold and twofold symmetry axes of the D(3) hexamer. Consequently, mutations of active-site residues (such as Glu20 and Lysl04) often affect protein stability and oligomeric structure. Conversely, mutations of residues in the interface between monomers (such as His136 and Hisl40) not only affect oligomeric structure but also affect active-site function. The effects of the H136Q and H140Q variants can be explained by the extended ionic interaction between H140, D143 and H136' of the neighbouring monomer. This interaction is further buttressed by an extensive hydrogen-bonding network that appears to explain why the E-PPase hexamer is so stable and also why the H136Q and H140Q variant proteins are less stable as hexamers.

Crystallographic identification of metal-binding sites in Escherichia coli inorganic pyrophosphatase.

We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) to R-factors of 18.3% and 17.1% at 2.2 and 2.3 angstroms, respectively. Both structures contain two independent monomers in the asymmetric unit of an R32 cell. The difference between the structures is that the latter contains 1.5 Mg2+ ions per monomer. One metal ion binds to the "tight" metal-binding site identified by equilibrium dialysis studies, and is coordinated to Asp65, Asp70, and Asp102. The other metal ion, shared between two monomers at a hitherto unidentified metal-binding site in the dyad interface between trimers, is coordinated through water molecules to Asp26s and Asn24s from two monomers. The hexamers with metal bound to them are more tightly associated than the ones without metal bound to them. Combined with our other mechanistic and structural data, the results suggest that, at high metal concentrations, E-PPase may bind at least 4.5 metals per monomer: two in the active site before binding substrate, two with substrate, and 0.5 in the dyad interface. Glu20 interacts via a water molecule with Asp70 and appears in the related yeast PPase structure (Heikinheimo, manuscript in preparation) to be involved in binding the second metal ion. Magnesium ion therefore stabilizes the hexamer form through both direct and indirect effects. The direct effect is by tighter association at the subunit interface; the indirect effect occurs because magnesium stabilizes the correct conformation of the loop between Glu20 and Ile32, a loop involved a trimer-trimmer interactions. Our results thus provide a structural explanation for the solution studies that show that the E20D variant is partially hexameric and that the hexamer form can be stabilized by binding magnesium ion.

Biodegradable self-reinforced polyglycolide screws and rods in the fixation of displaced malleolar fractures in the elderly. A comparison with metallic implants.

Biodegradable self-reinforced polyglycolide screws and rods were constructed for internal fixation of fractures in cancellous bone. The self-reinforced texture was achieved by embedding polyglycolide fibres in a polyglycolide matrix. In a prospective clinical study, a total of 37 patients at least 65 years of age with displaced malleolar fractures were managed by open reduction and internal fixation using either biodegradable screws and rods or metallic implants in a randomly allocated series. The results were assessed approximately one year after the fracture. One wound infection occurred after metallic fixation. Reoperation because of displacement of the fracture was needed in one patient after biodegradable fixation. The functional results were satisfactory in most patients. There were no major difference in the end results between both operative methods used.

Malleolar fractures in alcoholics treated with biodegradable internal fixation. 6/16 reoperations in a randomized study.

29 alcohol abusers with displaced malleolar fractures were randomized to treatment with biodegradable self-reinforced polyglycolide screws or metallic implants. During an average follow-up time of 7 (0-15) months, 8 patients out of 16 treated with biodegradable fixation had postoperative redisplacement of the fracture and 6 were reoperated. 1 fracture in 13 patients with metallic fixation had a slight displacement postoperatively which did not require reoperation.

Structure and function analysis of Escherichia coli inorganic pyrophosphatase: is a hydroxide ion the key to catalysis?

Using site-directed mutagenesis, we have completed replacing all 17 putative active site residues of Escherichia coli inorganic pyrophosphatase (PPase). We report here the production of 11 new variant proteins and their initial characterization, including thermostability, hydrophobicity, oligomeric structure, and specific activity at pH 8. Studies of the pH-rate profiles of 12 variants containing substitutions for potentially essential residues showed that the effect of the mutation was always to increase the pKa of a basic group essential for both substrate binding and catalysis by 1-3 pH units. The D70E variant had the lowest activity at all pHs; the K29R, R43K, and K142R variants also had low kcat/Km values. The principal effect seen in the other variant proteins was higher and sharper pH optima; their pH-independent kcat and kcat/Km values changed at most by a factor of 8. Our results suggest that the most likely candidate for the essential basic group affected by all mutations in the active site is a hydroxide ion stabilized by coordination to the essential Mg2+ ions. Analyzing our results using the structure recently obtained for E. coli PPase [Kankare et al. (1994) Protein Eng. 7, 823-830] led us to identify a group of residues, centered around Asp70 and including Tyr55, Asp65, Asp67, Asp102, and Lys104, that we believe binds the magnesium ions that are critical for the activity, possibly by stabilizing the essential hydroxide. Others, including Lys29, Arg43, and Lys142, are more spread out and more positively charged. They appear to be involved in binding substrate and product. Tyr55 is also a key part of the hydrophobic core of E. coli PPase; when it or residues that interact with it are conservatively mutated, there are changes in the overall structure of the enzyme as assayed by thermostability, hydrophobicity, or oligomeric structure.

A controlled immunohistochemical study of inflammatory cells in disc herniation tissue.

STUDY DESIGN: The presence and abundance of inflammatory cells was studied immunocytochemically in lumbar disc herniations (DH) and macroscopically normal discs for comparison. OBJECTIVES: The objective of the study was to characterize inflammatory cells that appear in herniated disc tissue and to study the relative abundance of various types of inflammatory cells. SUMMARY OF BACKGROUND DATA: Only few macrophages were observed in control discs, whereas abundant macrophages were present in half of the DH. Other types of inflammatory cells were less often abundant in the present material. In about a third of the DH interleukin-1 beta-expressing cells were also observed. METHODS: Twenty-four DH and control tissue from five discs were studied immunocytochemically, using specific monoclonal antibodies to various types of inflammatory cells and interleukin-1 beta. The results were compared with corresponding clinical data. Macrophages were studied with an antibody to CD68 antigen and Ber-MAC3 antibody separately. RESULTS: The obtained results suggest a variable inflammatory cell response in DH, which seems to be often dominated by macrophages at the time of operation. Thus previous suggestions of sometimes very active inflammation in DH tissue are supported. CONCLUSIONS: Inflammation may be important in disc tissue pathophysiology, possibly also in discogenic pain mechanisms.

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution.

The structure of E.coli soluble inorganic pyrophosphatase has been refined at 2.7 A resolution to an R-factor of 20.9%. The overall fold of the molecule is essentially the same as yeast pyrophosphatase, except that yeast pyrophosphatase is longer at both the N- and C-termini. Escherichia coli pyrophosphatase is a mixed alpha + beta protein with a complicated topology. The active site cavity, which is also very similar to the yeast enzyme, is formed by seven beta-strands and an alpha-helix and has a rather asymmetric distribution of charged residues. Our structure-based alignment extends and improves upon earlier sequence alignment studies; it shows that probably no more than 14, not 15-17 charged and polar residues are part of the conserved enzyme mechanism of pyrophosphatases. Six of these conserved residues, at the bottom of the active site cavity, form a tight group centred on Asp70 and probably bind the two essential Mg2+ ions. The others, more spreadout and more positively charged, presumably bind substrate. Escherichia coli pyrophosphatase has an extra aspartate residue in the active site cavity, which may explain why the two enzymes bind divalent cation differently. Based on the structure, we have identified a sequence motif that seems to occur only in soluble inorganic pyrophosphatases.