Biocompatibility testing of NiTi screws using immunohistochemistry on sections containing metallic implants.

NiTi is one of the most innovative concepts to have appeared in the field of metallic biomaterials in recent years but its biocompatibility remains controversial. We evaluated the biocompatibility of Nitinol screws using immunohistochemistry to observe the distribution of bone proteins during bone remodeling process around NiTi implant. Results were compared with screws made of Vitallium, c.p. titanium, Duplex austenitic-ferritic stainless steel (SAF), and Stainless Steel 316L. Screws were implanted in rabbit tibia for 3, 6, and 12 weeks. Embedding was performed in the hard resin Technovit, and for the immunohistochemical procedure undecalcified sections with bone-anchored implants could thus be used. The immunostaining method developed seemed to be a reliable technique to stain proteins in undecalcified sections. Biocompatibility results of the NiTi screws compared with the other screws showed a slower osteogenesis process characterized by no close contact between implant and bone, disorganized migration of osteoblasts around the implant, and a lower activity of osteonectin synthesis.

Long-term growth hormone-releasing factor administration on growth hormone, insulin-like growth factor-I concentrations, and bone healing in the Beagle.

Twelve 11 month old male Beagles were assigned to two treatment groups: a control group (saline) and a group receiving human growth hormone (GH)-releasing factor (hGRF) [1-29]NH2 (25 micrograms/kg, SC, TID). Treatment was started 6 days prior to surgery (day 1) and continued until necropsy (3 dogs per group/day) on d 29 or 58. Two porous polyethylene rods were surgically implanted on the lateral diaphysis of the femoral shaft and a 3 mm hole was drilled through the cortex between the two implants of each dog on day 1. Blood and urine were collected on d -6, 27 and 56. Human GRF injections produced a significant (P < 0.05) increase in GH release following each injection. An increase in GH response was also observed (P < 0.05) over time. The concentration of insulin-like growth factor-1 (IGF-1) increased for 5 weeks and then reached a plateau. None of the hematologic or urine measured parameters was affected by the treatment (P > 0.05). Albumin, calcium, and protein concentrations were higher (P < 0.05) on d 27 and 56 in GRF-treated animals. Histological sections of the onlay sites showed that bony ingrowth tended to be greater into the porous polyethylene material in GRF-treated animals than the controls at d 28 and 57, while no difference was observed in the degree of periosteal bone formation around the implants at either time period (P > 0.05). Bone formation into the cortical defect was greater in the GRF-treated dogs when compared to controls at day 57 only. In conclusion, chronic hGRF [1-29]NH2 treatment in Beagle dogs produced an increased GH response over time and increased IGF-1 concentrations. It also appeared to promote bony ingrowth into a porous polyethylene onlay and into a bony deficit.

Excretion of a radiolabelled anticancer biodegradable polymeric implant from the rabbit brain.

The elimination of a clinically used anticancer biodegradable polymer implant (Gliadel) in the rabbit brain was studied. The implant is composed of N,N-bis(2-chloroethyl)-N-nitrosourea (BCNU) (1.6 wt%) dispersed in a copolyanhydride matrix of 1,3-bis(p-carboxyphenoxypropane) (CPP) and sebacic acid (SA) in a 20:80 molar ratio. Four groups of rabbits were implanted with wafers loaded with BCNU, one in a 14C-SA-labelled polymer, another in a 14C-CPP-labelled polymer and two groups with 14C-BCNU in a non-labelled polymer, one for BCNU disposition study and one for residual drug study. In the rabbits implanted with the 14C-SA-labelled polymer, approximately 10% of the radioactivity was found in the urine and 2% in the faeces, and about 10% remained in the device 7 d after implantation. In contrast, only 4% of the radioactivity of the 14C-CPP-labelled polymer was found in urine and faeces during this period. However, a drastic increase in the CPP excretion was found after 9 d, and at 21 d, 64% of the implanted 14C-CPP was found in the urine and faeces, and 29% was still in the recovered wafers. Approximately 50% of the BCNU in the wafers was released in 3 d, and over 95% was released after 6 d in the rabbit brain. This study demonstrates that BCNU-loaded polyanhydride is biodegradable and is excreted from the body primarily through the renal system. The water-soluble components SA and BCNU were rapidly excreted, while the insoluble CPP was gradually eliminated after a lag time of 9 d.

Acute and subchronic neurotoxicity studies with tri-N-butyl phosphate in adult Sprague-Dawley rats.

Male and female adult Sprague-Dawley rats received tributyl phosphate (TBP) in corn oil by gavage in acute (single-dose) and subchronic (three-month) studies. Dosage levels in the acute study were 100, 325, and 1000 mg/kg; they were 32.5, 100, and 325 mg/kg/day in the subchronic study. Behavioral evaluations were performed in both studies, and neuropathological evaluations were performed in the three-month study only. The methods used in the studies are as described in the U.S. Environmental Protection Agency neurotoxicity guidelines. Mean body weight decreases were statistically significant compared to controls for male rats at the high-dosage level only in the acute study. Transient changes only, attributable to the general toxicity of the material, were noted in forelimb grip strength and mean activity level during the first 24 hours after dosing for 1000 mg/kg rats. In the subchronic study high-dose males and females had statistically significant body weight decreases; some mortality also was observed at this dosage level. The motor activity levels and qualitative and quantitative functional observational battery measurements were comparable between treatment and control groups, and there were no gross or neurohistopathological findings in the rats indicative of treatment-related effects. Based on these study results, TBP was not neurotoxic to rats following either acute or subchronic exposures.

Metabolic disposition and elimination studies of a radiolabelled biodegradable polymeric implant in the rat brain.

The metabolic disposition and elimination process of the anhydride co-polymer poly[1,3-bis(p-carboxy-phenoxypropane):sebacic acid] 20:80 [P(CPP:Sa)20:80] implanted in the rat brain was studied. Two polymers were prepared, one with [14C]SA and unlabelled CPP, and the other co-polymer with [14C]CPP and unlabelled SA. With these two polymers we were able to study the metabolic disposition of each monomer after polymer degradation. Polymer wafers loaded with N,N-bis(2-chloroethyl)-N-nitrosourea or without the drug were implanted in the rat brain. For the rats implanted with the [14C]SA-labelled polymer, approximately 40% of the radioactivity was found in the expired CO2, 10% in the urine, about 2% in the faeces and about 10% remained in the device 7 d after implantation. On the other hand, only 4% of the [14C]CPP monomer was eliminated by urine and faeces during this period. The drug-loaded polymer degraded faster than the blank polymer. This study supports the theory that the polymer is a biodegradable material that can be used for the direct and specific delivery of drugs into a targeted organ and can provide continued release of drugs over a period of time.

Efficacy of diazepam and avizafone against soman-induced neuropathology in brain of rats.

The purpose of this investigation was to compare the efficacy of diazepam and a water soluble pro-diazepam drug, avizafone (lysyl, peptido-aminobenzophenone diazepam pro-drug) in preventing or reducing the severity of soman-induced neuropathology in rats and to determine the temporal relationship between seizure initiation, anticonvulsant administration and the incidence and severity of soman-induced neuropathology. Brains from rats, treated with a convulsant dose of soman (pinacolyl methylphosphonofluoridate) and anticonvulsants such as diazepam and avizafone, were evaluated by light microscopy for evidence of neuropathology. All rats received atropine methyl nitrate (20 mg/kg, ip)+the bispyridinium acetylcholinesterase reactivator HI-6 (125 mg/kg, ip; 1-(((4-(aminocarbonyl)pyridinio) methoxy)methyl)-2-((hydroxyimino)methyl)-pyridinium dichloride) in the same solution 10 min before soman (130 micrograms/kg,sc). Three days later the rats were perfused and the tissue fixed for histological evaluation. Necrosis and/or malacia (degenerative changes) and hemorrhage were observed in some groups. The sites where pathology was most frequently observed and with greater severity were the piriform cortex, amygdala and (dorsal) thalamus. Less severe changes were observed in the cerebral cortex and hippocampus. There were no changes in the hypothalamus. Diazepam given 10 minutes before soman prevented the occurrence of soman-induced convulsions and neuropathology (i.e. degenerative changes were not then seen). Diazepam given at the start of the soman-induced convulsions reduced considerably the convulsions and the degree of neuropathology. Avizafone given 10 minutes before soman reduced slightly the effect of soman. Other treatments (diazepam given 30, 60 and 120 minutes after the start of the convulsions and avizafone given at the start of convulsions) showed little or no effect on the neuropathology associated with soman administration. The results of this study have demonstrated that the use of an anticonvulsant, such as diazepam, must be initiated shortly after soman exposure in order for any therapeutic benefit to be realized.

Subacute inhalation toxicity of benzaldehyde in the Sprague-Dawley rat.

Benzaldehyde was administered by inhalation to female and male Sprague-Dawley rats for 14 consecutive days (low level: 500 ppm; medium level: 750 ppm; high level: 1000 ppm). Effects of this chemical were investigated during and at the end of the exposure period. Throughout the experiment, significant hypothermia and a reduction of motor activity were observed in all rats exposed to benzaldehyde and were accompanied in high-level rats by a severe impairment of the central nervous system, as evidenced by abnormal gait, tremors, and a positive Straub sign. Histopathologic examination of tissues from exposed rats showed a goblet cell metaplasia that was largely confined to the respiratory epithelium lining the nasal septum in male rats. No other abnormal microscopic changes were observed. A no effect level was not observed in these studies.

Quantitative techniques in neuropathology.

In the future, quantitative techniques will probably be used in industry as part of Tier II studies for the evaluation of chemicals and drugs for their neurotoxic potential. Movement towards quantifying some structures or neuropathological changes will be made possible by advances in tissue preparation and computer technology. Emphasis will need to be placed on standardized techniques, good quality samples and sampling techniques in order to produce good quantitative data in a reasonable time. In this paper, different sampling techniques are evaluated using a cross section of rat sural nerve as the tissue for quantitative investigation.

Distribution of the bispyridinium oxime [14C] HI-6 in male and female rats.

Female rats poisoned with multiple LD50s of soman or tabun have been shown previously to respond to the protective effects of HI-6 more positively than male rats. This present study was designed first to determine the distribution pattern and concentration of [14C] HI-6 in rats, and secondly, to determine the possibility that HI-6 might be located in high concentrations in critical tissues in the female as opposed to the male. To these ends, [14C] HI-6 was administered to groups of male and female rats and its radiolabelled distribution determined by whole body autoradiography and/or by measurement of its actual concentration, by scintillation spectrometry. The experiments were repeated in the presence of 2 x LD50 soman and supporting therapy with atropine. In both sexes, HI-6 levels were highest in the kidney, followed in order by cartilage greater than plasma greater than liver greater than heart greater than or equal to lung greater than or equal to diaphragm greater than brain and spinal cord. The relative distribution in the two sexes was confirmed by both methods and was not significantly altered in the presence of soman and atropine. The lack of a measurable difference in tissue distribution of [14C] HI-6 derived radioactivity between males and females suggested that the hormone-dependent difference in the protective effects previously observed was not due to selective accumulation of [14C] HI-6 in organs believed to be important in its therapeutic activity, such as brain or diaphragm.

Correlation between behavioral and pathological changes in the evaluation of neurotoxicity.

Recently the EPA has implemented new guidelines under Section 4 of TSCA to undertake neurotoxicity testing. It is expected that other agencies, both national and international, will follow suit. The evaluation of neurotoxicity will be based primarily on behavioral and morphological observations and particularly on the correlation between them. Initially, considerable information will have to be gathered from various laboratories to form the background data on which decisions of toxic effects can be made. This paper presents our experience to date with behavioral and neuropathological procedures for the evaluation of chemical compounds or dietary regimens in rats for potential neurotoxicity. Reference is made to acrylamide, 2,5-hexanedione, 3,3'-iminodipropionitrile, amphetamine, physostigmine, ethanol, triethyltin, food and water deprivation, and carbon tetrachloride. The question of whether every change induced by xenobiotics can be considered as a sign of toxicity is discussed.

Morphometric evaluation of hepatocellular proliferative lesions in the rat liver.

Classification of rat hepatocellular proliferative lesions can vary between pathologists as the many qualitative histologic criteria have not been satisfactorily evaluated and ranked for prognostic value. Computer-assisted morphometry offers an objective method to evaluate certain cellular features. The Solt-Farber resistant hepatocyte model was used in this study to produce populations of rats with a full range of hepatocellular proliferative lesions. Cellular features within the lesions were then measured morphometrically and the data were analyzed by animal age and by subjective lesion diagnosis. The nuclear/cytoplasmic ratio followed by the cell area and nuclear area were found to be the most important parameters for separating microscopic foci and islands of cellular alteration, an early hyperplastic lesion, from other hepatocellular proliferative lesions. The coefficient of variation, as a relative measure of heterogeneity, increased in a linear manner for cell, nuclear and nucleolar areas as the animals aged and was significantly higher for cell and nuclear area in hepatocellular carcinoma compared to other hepatocellular proliferative lesions. Hepatocyte nodules (representing primarily late hyperplastic lesions) and persistent hepatocyte nodules (lesions with similarities to hepatocellular adenoma) could not be satisfactorily separated within the limits of this study. As these borderline lesions show a continuum of cytologic change, other features, such as architectural change, are necessary to satisfactorily classify them on a subjective basis. An alternative approach is to use discriminant functions derived from morphometric studies.

Induction of urinary bladder hyperplasia in Sprague-Dawley rats orally administered tri-n-butyl phosphate.

The effects of tri-n-butyl phosphate (TBP) were investigated in the Sprague-Dawley rat over an 18-wk period. Groups of randomized female (average weight [AW] = 206 +/- 10 g) and male (AW = 294 +/- 13 g) rats were divided into low-dose, high-dose, and control groups (12 rats/sex X group). Tri-n-butyl phosphate was administered by gavage once a day for 5 days/wk over an 18-wk period. Low-dose animals received 0.20 g/kg X day throughout the experiment and high-dose animals received 0.30 g/kg X day for the first 6 wk. For the remaining 12 wk, the high-dose level was increased to 0.35 g/kg X day. Histopathological examination of tissues revealed that all test rats examined developed diffuse hyperplasia of the urinary bladder epithelium. Similar changes were not found in the control animals.

Subchronic oral toxicity of tributoxyethyl phosphate in the Sprague-Dawley rat.

The effects of the widely used alkyl phosphate, tributoxyethyl phosphate (TBOP), were investigated in male and female Sprague-Dawley rats. This chemical was administered (low dose: 0.25 ml/kg X day; high dose: 0.50 ml/kg X day) by gavage once a day for 5 days/wk, over a period of 18 wk. Histopathological examination of tissues in treated male rats showed the presence of cardiac lesions, including myocardial necrosis with inflammatory cell infiltration. This study indicated that oral administration of TBOP may have caused or contributed to the early onset of a common background finding in this rat strain.

Subacute oral toxicity of tri-n-butyl phosphate in the Sprague-Dawley rat.

Tri-n-butyl phosphate was administered by gavage to male and female Sprague-Dawley rats for 14 consecutive days (low dose: 0.14 ml kg-1; high dose: 0.42 ml kg-1). Effects of this chemical were investigated at the end of the feeding period. Histopathological examination of the testes (high-dose group) showed the presence of microscopic degenerative changes in the seminiferous tubules. No other abnormal microscopic changes were observed.