Basic research and clinical application of beta-tricalcium phosphate (ß-TCP).

The mechanism of bone substitute resorption involves two processes: solution-mediated and cell-mediated disintegration. In our previous animal studies, the main resorption process of beta-tricalcium phosphate (ß-TCP) was considered to be cell-mediated disintegration by TRAP-positive cells. Thus, osteoclast-mediated resorption of ß-TCP is important for enabling bone formation. We also report the results of treatment with ß-TCP graft in patients since 1989. Two to three weeks after implantation, resorption of ß-TCP occurred from the periphery, and then continued toward the center over time. Complete or nearly complete bone healing was achieved in most cases within a few years and was dependent upon the amount of implanted material, the patient's age, and the type of bone (cortical or cancellous). We have previously reported that an injectable complex of ß-TCP granules and collagen supplemented with rhFGF-2 enabled cortical bone regeneration of rabbit tibiae. Based on the experimental results, we applied this technique to the patients with femoral and humeral fractures in elderly patients, and obtained bone union.

A novel evaluation system to monitor bone formation and ß-tricalcium phosphate resorption in opening wedge high tibial osteotomy.

PURPOSE: The aim of this study was to establish an evaluation system to monitor bone formation and beta-tricalcium phosphate (TCP) resorption in opening high tibial osteotomy (HTO). METHODS: From 2003 to 2005, opening HTO was performed in 36 patients using a Puddu plate and ß-TCP blocks with 60 and 75 % porosity. Thirty-one patients were used for evaluation. All patients underwent CT examination at 2 weeks and 6 years. The CT image data were divided into three areas, and CT values of each area were analysed using the imaging software, Osirix. RESULTS: CT image analysis at 2 weeks showed that the mean CT-attenuation values (in Hounsfield units) of the implanted area with ß-TCP of 60 % porosity, the implanted area with ß-TCP of 75 % porosity, and cancellous bone were, 1,694.0 ± 94.2, 1,010.9 ± 81.1, and 178.0 ± 45.1, respectively. Six years after surgery, these values were 574.1 ± 273.5, 168.8 ± 75.1, and 174.9 ± 69.3, respectively. CONCLUSION: ß-TCP with 75 % porosity was completely resorbed and replaced by bone. ß-TCP with 60 % porosity was resorbed, but approximately 1/3 still remained even 6 years after surgery. The imaging software, Osirix, enabled scanning of the whole area to measure CT values. This system is the first to quantitatively evaluate ß-TCP resorption and bone formation in opening HTO. LEVEL OF EVIDENCE: Laboratory studies.

An Injectable Complex of ß-tricalcium Phosphate Granules, Hyaluronate, and rhFGF-2 on Repair of Long-bone Fractures with Large Fragments.

We evaluated the effects of an injectable complex of ß-tricalcium phosphate (ß-TCP) granules, hyaluronate, and recombinant human fibroblast growth factor-2 (rhFGF-2) on repair of unstable intertrochanteric fractures in elderly patients. Twenty-five patients (range, 76-91 years) having 31.A2 fractures (AO classification) were treated with injection of the complex followed by intramedullary nails. Bone regeneration and ß-TCP resorption, unions of intertrochanteric fractures and displaced lesser trochanters to the shaft, and varus deformity of the femoral neck were assessed by X-ray and CT scans. Fracture union occurred in all cases and union of the displaced lesser trochanter to the shaft was obtained in 24 cases by 12 weeks. It is of interest that ß-TCP granules were completely resorbed and marked new bone formation around the lesser trochanter was observed in all cases compared to cases not treated with the complex. Based on the results of intertrochanteric fractures, we applied this technique to two patients with subtrochanteric or humeral fractures in elderly patients, and obtained bone union. This complex is a paste-like material that is easy to handle, and it may be of considerable use in treatment of both unstable intertrochanteric fractures and other cortical bone defects with minimal surgical invasion.

Use of an Injectable Complex of ß-Tricalcium Phosphate Granules, Hyaluronate, and Fibroblast Growth Factor-2 on Repair of Unstable Intertrochanteric Fractures.

We evaluated effects of an injectable complex of ß-tricalcium phosphate (ß-TCP) granules, hyaluronate, and recombinant human fibroblast growth factor-2 (rhFGF-2) on repair of unstable intertrochanteric fractures in elderly patients. Twenty-five patients (range, 76-91 years) having 31.A2 fractures (AO classification) were treated with injection of the complex followed by intramedullary nails. Bone regeneration and ß-TCP resorption, unions of intertrochanteric fractures and displaced lesser trochanters to the shaft, and varus deformity of the femoral neck were assessed by X-ray and CT scans. Fracture union occurred in all cases and union of the displaced lesser trochanter to the shaft was obtained in 24 cases by 12 weeks. It is of interest that ß-TCP granules were completely replaced by bone and marked new bone formation around the lesser trochanter was observed in all cases compared to cases not treated with the complex. This complex is a paste-like material that is easy to handle, and it may be of considerable use in treatment of both unstable intertrochanteric fractures and other cortical bone defects with minimal surgical invasion.

Pertussis immunization with acellular vaccines in Ghanaian children.

In the present study, the persistence of antibodies to pertussis antigens was assessed in 51 Ghanaian children immunized with one of two acellular vaccines and one whole cell vaccine in early infancy. The effect of a booster dose 1 year after primary immunization was also examined. Antibody titres to pertussis toxin (PT) and filamentous haemagglutinin (FHA) were measured 1 month and 1 year after primary immunization and 1 month after the booster dose. Although geometric titres (GMTs) to FHA were significantly higher in the two types of acellular vaccinees in the whole cell vaccinees 1 month after primary immunization, GMTs to FHA and PT after 1 year were not significantly different in the three groups. Geometric mean titres to PT and FHA following the booster dose were significantly higher in the acellular vaccinees than in the whole cell vaccinees. Seropositivity rates to PT and FHA in the acellular vaccinees, which were more than 93.3% 1 month after primary immunization, ranged from 50.0 to 77.8% after 1 year. In conclusion, the acellular vaccines did not produce higher antibody levels than the whole cell vaccine 1 year after primary immunization. The booster dose was essential to maintaining sufficient seropositivity to pertussis antigens.

A randomized controlled trial of two acellular pertussis-diphtheria-tetanus vaccines in primary immunization in Ghana: antibody responses and adverse reactions.

Two acellular pertussis vaccines combined with diphtheria and tetanus toxoids (APDT vaccines) were compared with a whole cell PDT (WCPDT) vaccine in primary immunization in Ghana. One is a liquid vaccine which is used for general immunization in Japan and the other is a freeze-dried vaccine newly developed as a heat-stable vaccine. Eighty-nine infants were recruited in the study. Sixty-eight who completed three doses of the immunization were assessed for immunological responses. Twenty-one dropped out because of sickness or moving from the study area. A total of 242 vaccinations in 89 infants were followed up for adverse reactions. Geometric mean titres (GMTs) to filamentous haemagglutinin in the two APDT vaccinees were significantly higher than in the WCPDT recipients. GMTs to pertussis toxin, diphtheria and tetanus toxoids were not significantly different among the three groups. Seropositive rates to pertussis antigens, tetanus and diphtheria toxoids were 94.4 to 100% in the two APDT vaccines. Systemic reactions within 7 days of inoculation were similarly low in the three groups, but significantly fewer infants had local reactions after either of the two APDT vaccines than after the WCPDT vaccine.

Effect of serum treatment on pertussis antibody determination by ELISA.

Determination of anti-PT and anti-FHA antibodies by ELISA and neutralization titer by CHO-cell method were performed with inactivated and uninactivated human sera. The findings were as follows: in inactivated sera, higher anti-PT ELISA antibody titers were shown compared with the titers determined with corresponding inactivated sera, especially in sera having low ELISA antibody titers; however, anti-FHA ELISA antibody titers remained identical, with the uninactivated or inactivated sera. CHO-cell neutralization titer remained identical and stable between the uninactivated and inactivated sea. CHO-cell neutralization titers correlated with anti-PT ELISA antibody titers in sera without inactivation. Anti-PT ELISA antibody titers in inactivated sera, however, did not correlate with the corresponding CHO-cell neutralization titers. These results suggest the possible existence of (a) substance(s) with affinity to PT which enhance(s) the ELISA reaction when sera are inactivated. Therefore, the inactivation of sera is not favorable for anti-PT determination by ELISA.

Clinical use of Biken acellular DPT in two-month old infants.

We have examined the antibody titer and side effects in two-month-old infants (1.5-2.5) who received vaccinations of acellular DPT Combined Vaccine Adsorbed "Biken" Lots 22 and 23. We observed high-antibody titers far in excess of prevention levels. No particular increase in side effects was observed in the two-month-old infants, except a normal increase in once-only side effects. The results suggest that this vaccine can protect infants under one year of age, who are especially liable to be affected by this illness.

Safety follow-up in a cohort of Biken acellular DPT vaccine recipients in Japan.

In October 1984 in Sweden, a phase II trial of Biken acellular Pertussis vaccine was started and in 1986, a phase III trial of the same vaccine was begun. During the phase III trial, there were three cases of deaths out of 1,385 of study children at two, four and ten weeks after the second dose of the vaccine, due to severe invasive bacterial infections such as H. influenzae, Pneumococcus, or Meningococcus infection. A number of arguments arose about the results of the Phase III trial. No one can either prove or disprove the association between invasive bacterial infection and administration of acellular pertussis vaccine. The purpose of this paper is to discuss the side effects of Biken acellular DPT vaccine. The pediatricians inquired about the physical status of the children who received Biken acellular DPT vaccine. During the observation period, three out of 940 infants suffered from infectious diseases. One suffered from measles, the other from varicella and the last from mumps. Our retrospective study did not reveal any severe invasive bacterial infection cases cases such as the ones experienced in Sweden.

Characterization of mutant strains producing pertussis toxin cross reacting materials.

We have isolated 120 mutant strains producing pertussis toxin (PT) cross reacting materials (CRMs) from B. pertussis, strain Tohama, phase I by nitrosoguanidine treatment. Strains producing higher PT tend to show higher virulence in mice. No direct correlation between the virulence and other factors, such as filamentous hemagglutinin, adenylate cyclase or dermonecrotic heat labile toxin, was found. Most CRMs were less reactive to the anti-S1 monoclonal antibody, 1B7. When the PT CRMs produced by strains 69D, 74E or 79G, which were less or non-toxic, were mixed with A protomer purified from native PT, the PT activity assayed by clustering of CHO-cells increased significantly, but not when they were mixed with B oligomer. These CRMs may be composed of defective S1 and intact S2, S3, S4 and S5. Molecular sizes of PT CRMs outside and inside the cells were analysed by sucrose density gradient centrifugation. The sizes of the CRMs were in the range of 10K to 210K, but the biological activity of PT was detected at only the same molecular size, 106 K, as native PT. The majority of the CRM was released into culture medium if all five subunits were assembled; otherwise they accumulated inside the cell without completion of assembly to form the hexamer in the PT-form. One of the non-toxic mutants named 79G showed one point mutation from G to A at the 730th base from the Eco R1 site of the PT gene. Replacement of Cys-41 with Tyr-41 in S1 must have resulted from this mutation. 79G PT composed of S234 (5) was accumulated both inside and outside the cells because the mutant S1 could not form the disulfide bond in the molecule to form the hexamer with the B oligomer, and also S1 must be degraded because of its instability in the cells. Nevertheless 79 GPT showed high immunoprotectivity in mice by active or passive immunization against ic or aerosol challenge with B. pertussis, strain 18323, respectively. It may have a proper conformational structure for protective immunogenicity and could become a good candidate strain for production of a safer and effective pertussis vaccine in the future.

The pyrogenicity of pertussis vaccine in mice and the factors in the vaccine responsible for this effect.

The injection of whole cell pertussis vaccine into mice produced a biphasic fever reaction with two peaks appearing after about one and four hours, respectively. A method for the quantitative determination of each peak fever activity was developed and the factor responsible for each activity was investigated. The first and the second peak fever activities did not parallel each other in individual vaccines. The earlier fever activity appeared to correlate with endotoxin activity in individual vaccines while the later appeared to correlate with histamine-sensitizing factor (HSF) activity. The later peak fever activity was greatly reduced by heating the vaccine at 100 degrees C for 30 min while the first was little affected by such treatment. It was concluded that the fever activity of pertussis vaccine in mice may be ascribed to the combined actions of endotoxin and a heat-labile substance, possibly HSF.

The purification and characterization of an acellular pertussis vaccine.

An acellular pertussis vaccine manufactured by Biken was investigated for purity, potency and toxicity. The vaccine was composed of almost equal proportions of pertussis toxin (PT) and filamentous hemagglutinin (FHA). The purity of the vaccine was 97-99%. The protective effects of component vaccines containing various ratios of PT and FHA were tested and it was found that the ratio of 1:1 provided the most effective vaccine.

Further studies on complementation between mutants of Clostridium perfringens.

1. Mutants devoid of lambda- and kappa-toxin and hemagglutinin (HA), respectively, were isolated from Cl. perfringens PB6K. The lambda- and HA- mutants could be classified into a and b groups by complementation but the kappa- mutants were all of the a group. 2. All b group mutants isolated, irrespective of the marker used for isolation, were pleiotropically negative or leaky with respect to theta-, lambda- and kappa-toxin and HA production. 3. Lambda-toxin produced by complementation was proved to be a rennet-like protease. 4. The activities of 12 extracellular enzymes, including sialidase, of several b group strains and the parent PB6K were compared, but no definite differences were observed. From this finding, the productions of these enzymes were concluded not to be regulated by the same mechanism as theta-, lambda- and kappa-toxin and HA. 5. Cl. perfringens CN3870 was also studied. Findings were similar to those on PB6K except for very low activity of HA.

Vascular leakage through intraendothelial channels induced by cholera toxin in the skin of guinea pigs.

Guinea pigs were injected intravenously with Evans blue solution (1/2), 1, 5 and 22 hours after intradermal injection of Vibrio cholerae 569B toxin. Then they were fixed by perfusion with diluted Karnovsky's aldehyde mixture 1(1/2), 5, 10, 15 and 90 minutes later. In some cases, horseradish peroxidase was mixed in the dye solution, and chopped tissue slices were incubated histochemically. Dermal blood vessels from these samples were examined under the electron microscope. The fine structure of the endothelial junctions remained unchanged as compared with controls. However, extended rough surfaced endoplasmic reticulum was observed in the endothelial cells at 30 minutes after the toxin injection, when cutaneous bluing was not marked yet. At 22 hours after toxin injection, endothelial perikarya were filled with prominent Golgi complexes, multivesicular bodies and systems of caveolae and vesicles which coalesced, forming intraendothelial channels after diaphragms disappeared. Endothelial fenestrae were numerous in the attenuated portions.