Isolates, antimicrobial susceptibility profiles and multidrug resistance of bacteria cultured from samples collected from sheep in New Zealand (2003-2016).

Aim: To describe the common species, antimicrobial susceptibility and multidrug resistance (MDR) of bacteria cultured from samples submitted to veterinary diagnostic laboratories from sheep in New Zealand between 2003 and 2016. Methods: Bacterial culture and antimicrobial susceptibility test data from June 2003 to March 2016 for animals identified as sheep were obtained from two commercial veterinary diagnostic laboratories in New Zealand. Submission information included animal signalment, geographic origin, specimen description, the organisms cultured, and where available, antimicrobial susceptibilities of the isolates. MDR was defined as any isolate with resistance to ≥3 antimicrobial classes. Results: There were 1,971 unique laboratory submissions, yielding 2,188 isolates. Of the 1,971 submissions, the most commonly represented breeds were Romney (933; 47.3%), Romney cross (264; 13.4%), and Coopworth (197; 10.0%), and there were more submissions from females (1,006; 51.0%) than males (184; 9.3%). Most submissions were from Canterbury (549; 27.9%), Southland (471; 23.9%), and Manawatu-Wanganui (272; 13.8%) regions. Other signalment data were inconsistently described. Submitted samples most commonly originated from the gastrointestinal tract (852; 43.2%), faeces (378; 12.1%), or liver (146; 7.4%). Of the 2,188 isolates, 1,771 (80.9%) were identified by species and 247 (11.4%) by genus, with the most common isolates being Salmonella spp. (880; 40.2%), Campylobacter spp. (408; 18.6%), Listeria spp. (140; 6.4%) and Yersinia spp. (113; 5.2%). Susceptibility results were available for 117/2,188 (5.3%) isolates from 51/1,971 (2.6%) submissions. No antimicrobial susceptibility data were available for Salmonella spp., Campylobacter spp., Listeria spp. or Yersinia spp. Overall for the isolates tested, susceptibility to the fluoroquinolones and tetracyclines was greatest, and MDR was found in 24/117 (20.5%) isolates. MDR was a more frequent finding for Enterococcus spp., Bacillus spp., and Proteus mirabilis, but was infrequent in isolates of Staphylococcus aureus, alpha-haemolytic streptococci, Escherichia coli or Enterobacter spp. Conclusions and clinical relevance: This is the first report on antimicrobial susceptibility and MDR for isolates from laboratory submissions from sheep in New Zealand. The low numbers of isolates submitted for antimicrobial susceptibility testing during the period studied mean that these findings provide limited insights into antimicrobial resistance in this population, and highlight the need to address significant gaps in our understanding of why veterinarians do not more frequently submit samples from sheep for bacterial culture and susceptibility testing. Abbreviation: AMR: Antimicrobial resistance; MDR: Multidrug resistance.

Bacterial isolates, antimicrobial susceptibility and multidrug resistance in cultures from samples collected from beef and pre-production dairy cattle in New Zealand (2003-2016).

Aims: To describe the common species and the antimicrobial susceptibility of bacteria cultured from samples submitted to commercial veterinary diagnostic laboratories from beef and pre-production dairy cattle between 2003-2016, and to describe the proportion of isolates with multidrug resistance (MDR). Methods: Bacterial culture and antimicrobial susceptibility data from July 2003 to March 2016 were obtained from commercial veterinary diagnostic laboratories in New Zealand. Results were included from samples from beef cattle, irrespective of age or sex, dairy-breed females aged <2 years and dairy-breed males of any age. Submission information provided included the specimen description, the organisms cultured, and the antimicrobial susceptibilities of isolates, if tested. Antimicrobial resistance (AMR) was defined as any isolate not showing susceptibility to an antimicrobial compound and MDR as any isolate showing AMR to ≥3 antimicrobial classes. Results: There were 1,858 unique laboratory submissions, yielding 2,739 isolates. Of these submissions, most were from the Canterbury (389; 21.9%), Manawatu (388; 21.9%) Waikato (231; 12.4%) and Hawke's Bay (136; 7.3%) regions. There were 163 unique species identifications for the 2,739 isolates; the most common were Yersinia pseudotuberculosis (452; 16.5%), Campylobacter jejuni (249; 9.1%), Escherichia coli (230; 8.4%) and Salmonella enterica serovar Typhimurium (143; 5.2%). Only 251/2,739 (9.2%) isolates from 122/1,858 (6.6%) submissions had antimicrobial susceptibility results. There were no sensitivity results for Yersinia spp., and only one each for Salmonella spp., and Campylobacter spp. Amongst the isolates tested, susceptibility to ampicillin was lowest (33/56; 58.9%). Overall, 57/251 (20.7%) isolates tested for antimicrobial susceptibility had MDR, and MDR was most common for Enterococcus spp. (12/17; 71%) and E. coli (13/30; 43%). Conclusions and Clinical Relevance: This is the first report on antimicrobial susceptibility and MDR in New Zealand beef and pre-production dairy cattle. Findings highlight the limited use of bacterial culture and sensitivity testing by veterinarians and deficits in the information accompanying submissions. A national antimicrobial resistance surveillance strategy that specifically includes this population is recommended.

Antimicrobial resistance patterns of bacteria isolated from canine urinary samples submitted to a New Zealand veterinary diagnostic laboratory between 2005-2012.

AIMS: To identify and describe culture and antimicrobial resistance (AMR) patterns in bacteria isolated from canine urinary samples submitted to a New Zealand veterinary diagnostic laboratory. METHODS: Records from a veterinary diagnostic laboratory were examined for bacterial isolates cultured from canine urine samples between January 2005 and December 2012. Culture and susceptibility results were compiled with information on the age, sex and breed of dog. Repeat submissions were removed. Susceptibility was assessed using results of the Kirby-Bauer disk diffusion method, for a standard panel including amoxicillin-clavulanic acid (AMC), cefovecin (from 2010-2012), cephalothin, clindamycin, enrofloxacin and trimethoprim-sulphonamide (TMS). RESULTS: A total of 5,786 urine samples were submitted for analysis, and 3,135 bacterial isolates were cultured from 2,184 samples. Of these 3,135 isolates, 1,104 (35.2%) were Escherichia coli, 442 (14.1%) were Staphylococcus spp., 357 (11.4%) Proteus mirabilis and 276 (8.8%) were Enterococcus spp. The frequency of culture-positive samples increased with increasing age in both female and male dogs (p<0.001). The percentage of E. coli isolates resistant to AMC and cephalothin increased between 2005 and 2012 (p<0.001), as did resistance to enrofloxacin (p=0.022), but there was no change in resistance to TMS (p=0.696). Enrofloxacin was the antimicrobial with the least resistance shown by the four most common bacteria isolated during the course of the study. CONCLUSIONS AND CLINICAL RELEVANCE: The results of this study provide important regional information regarding the prevalence of bacterial uropathogens and their susceptibility patterns. There was an increase in resistance to some commonly used antimicrobials in the treatment of urinary tract infections. Having access to regional antimicrobial susceptibility results is crucial when forming guidelines for the use of antimicrobials for the treatment of urinary tract infections. Given changes in practising habits and antimicrobial usage over time, ongoing monitoring and surveillance of resistance in pathogens is needed.

Furthering the understanding of silicate-substitution in α-tricalcium phosphate: an X-ray diffraction, X-ray fluorescence and solid-state nuclear magnetic resonance study.

High-purity (SupT) and reagent-grade (ST), stoichiometric and silicate-containing α-tricalcium phosphate (α-TCP: ST0/SupT0 and Si-TCP x=0.10: ST10/SupT10) were prepared by solid-state reaction based on the substitution mechanism Ca3(PO4)(2-x)(SiO4)x. Samples were determined to be phase pure by X-ray diffraction (XRD), and Rietveld analysis performed on the XRD data confirmed inclusion of Si in the α-TCP structure as determined by increases in unit cell parameters; particularly marked increases in the b-axis and ß-angle were observed. X-ray fluorescence (XRF) confirmed the presence of expected levels of Si in Si-TCP compositions as well as significant levels of impurities (Mg, Al and Fe) present in all ST samples; SupT samples showed both expected levels of Si and a high degree of purity. Phosphorus ((31)P) magic-angle-spinning solid-state nuclear magnetic resonance (MAS NMR) measurements revealed that the high-purity reagents used in the synthesis of SupT0 can resolve the 12 expected peaks in the (31)P spectrum of α-TCP compared to the low-purity ST0 that showed significant spectral line broadening; line broadening was also observed with the inclusion of Si which is indicative of induced structural disorder. Silicon ((29)Si) MAS NMR was also performed on both Si-TCP samples which revealed Q(0) species of Si with additional Si Q(1)/Q(2) species that may indicate a potential charge-balancing mechanism involving the inclusion of disilicate groups; additional Q(4) Si species were also observed, but only for ST10. Heating and cooling rates were briefly investigated by (31)P MAS NMR which showed no significant line broadening other than that associated with the emergence of ß-TCP which was only realised with the reagent-grade sample ST0. This study provides an insight into the structural effects of Si-substitution in α-TCP and could provide a basis for understanding how substitution affects the physicochemical properties of the material.

Three cases of a presumptive atypical myopathy in New Zealand horses.

CASE HISTORY: Three weanling Thoroughbred fillies were presented during autumn with depression, muscle rigidity and, in one case, colic symptoms and cardiovascular shock. CLINICAL FINDINGS: All fillies had abnormal physical examinations that included elevated heart rates and respiratory rates coupled with muscle rigidity through the back and rump. Biochemistry revealed markedly elevated creatinine kinase and aspartate aminotransferase which indicated a myopathy. DIAGNOSIS AND TREATMENT: All three horses were diagnosed with presumptive equine atypical myopathy. The horses received supportive therapy as per the literature available at the time regarding this condition; two responded to supportive therapy and survived, and one was euthanased due to a rapid deterioration in clinical status. PATHOLOGICAL FINDINGS: Following post mortem of one case, histology of the trapezius muscle demonstrated an acute, severe myofibre degeneration. CLINICAL RELEVANCE: Atypical myopathy and a very similar disorder termed seasonal pasture myopathy in North America are potentially fatal, pasture-related syndromes that have been described in Europe and America but have not been previously described in New Zealand. This report describes three presumptive cases of this unique syndrome in New Zealand for the first time; it outlines the characteristics of the condition; and includes recently published information regarding diagnosis and treatment.

Curettage and diathermy: a treatment for feline nasal planum actinic dysplasia and superficial squamous cell carcinoma.

OBJECTIVE: To evaluate curettage and diathermy as a treatment for actinic dysplasia and superficial squamous cell carcinoma of the feline nasal planum. METHODS: Thirty-four cats clinically assessed to have actinic dysplasia and superficial squamous cell carcinoma involving less than 50% of the nasal planum were treated with a three-cycle curettage and diathermy procedure. Degree of dysplasia, response to treatment, adverse effects, owner perceptions, time to recurrence and proportion disease free at 1 year were evaluated. RESULTS: Lesions ranged from actinic keratoses to invasive squamous cell carcinoma. A complete response to treatment was obtained in all cats. The median follow-up time was 18 · 2 (IQR: 12 · 0-22 · 8) months. Two cats had a clinical recurrence of lesions at 161 and 192 days after treatment. The probability of remaining disease free after 12 months was 0 · 94 (95% CI: 0 · 85-1 · 0). Median time to recurrence was not reached. The procedure was well tolerated with a good cosmetic outcome and no significant post-operative complications. CLINICAL SIGNIFICANCE: This study suggests that curettage and diathermy is an effective treatment for feline actinic dysplasia and for superficial squamous cell carcinoma involving less than 50% of the nasal planum. Curettage and diathermy is an easily mastered technique, requiring minimal equipment.

The presence of p16 CDKN2A protein immunostaining within feline nasal planum squamous cell carcinomas is associated with an increased survival time and the presence of papillomaviral DNA.

In humans, oral SCCs are either caused by papillomavirus (PV) infection or by other carcinogens such as tobacco. As these 2 groups of SCCs have different causes they also have different clinical behaviors. Immunostaining using anti-p16(CDKN2A) protein (p16) antibodies is used to indicate a PV etiology in human oral SCCs and p16-positive SCCs have a more favorable prognosis. The present study investigated whether p16 immunostaining within feline nasal planum SCCs was similarly associated with the presence of PV DNA and with a longer survival time. Intense p16 immunostaining was visible in 32 of 51 (63%) SCCs. In 30 cats with nonexcised SCCs, cats with p16-positive neoplasms had a longer estimated mean survival time (643 days) than cats with p16-negative SCCs (217 days, P = .013). Papillomavirus DNA was amplified more frequently from p16-positive nasal planum SCCs (28 of 32) than p16-negative SCCs (5 of 19, P < .001). The different survival times in cats with p16-positive and p16-negative SCCs suggests that p16 could be a useful prognostic indicator in these common feline cancers. As the clinical behavior of the SCCs can be subdivided using p16 immunostaining, the 2 groups of SCCs may be caused by different factors, supporting a PV etiology in a proportion of feline nasal planum SCCs.

Widespread mismatch repair expression in feline small intestinal lymphomas.

Small intestinal lymphoma is a common feline tumour that most often develops in older cats, but also occurs in younger animals. In man, germline defects in the mismatch repair (MMR) genes most commonly cause hereditary non-polyposis colorectal cancer (HNPCC), or Lynch syndrome, while MMR defects have also been implicated in the development of lymphoid tumours in mice and in people. It was hypothesized that inherited MMR defects predispose a proportion of younger cats to the development of small intestinal lymphoma. MMR expression in 10 small intestinal lymphomas from younger cats (group 1, mean age 4.5 years) was compared with MMR expression in 30 small intestinal lymphomas from older cats (group 2, mean age 12.6 years). The cross-reactivity of the antibodies specific for the human MMR proteins MLH1, MSH2 and MSH6 with the corresponding proteins in feline tissues was first confirmed by western blotting. MMR expression was then investigated immunohistochemically in feline lymphomas. MLH1, MSH2 and MSH6 were detected immunohistochemically within neoplastic lymphocytes from all tumours examined. There were no significant differences between the two groups in either the intensity of immunolabelling or the percentage of neoplastic cells within which MMR proteins were detected. These results confirm the cross-reactivity of the human MMR antibodies with the corresponding proteins in feline tissues, but do not support the hypothesis that inherited germline MMR defects are a significant cause of feline small intestinal lymphomas.

A study of neonatal cryptosporidiosis of foals in New Zealand.

AIM: To assess the occurrence of Cryptosporidium oocysts in faecal specimens from foals, and investigate an outbreak of neonatal cryptosporidiosis in foals revealed in the course of the study. METHODS: Faecal specimens from foals received by a diagnostic veterinary laboratory in New Zealand between 2006 and 2007 were submitted to Massey University and tested microscopically for the presence of Cryptosporidium oocysts. The Cryptosporidium isolates in the oocyst-positive specimens were genetically identified to species level. In addition, specimen submission data from the participating laboratory for 2005-2007 were examined. In the course of the study, the identification of one Cryptosporidium-positive specimen triggered an on-farm investigation. RESULTS: Twelve faecal specimens submitted by the participating laboratory between 2006 and 2007 were tested further, and three were positive for C. parvum. Specimen submission records indicated a total of 67 faecal specimens were tested for Cryptosporidium by the participating laboratory between 2005 and 2007; 12 (18%) were positive. The on-farm investigation on a broodmare farm revealed a high incidence of neonatal diarrhoea in foals; C. parvum was the only enteropathogen found in the faeces of 4/4 affected foals examined. The diarrhoea in all those foals was self-limiting, manifesting during the second week of life, resembling foal heat diarrhoea, and accompanied by a short but intense period of shedding oocysts. CONCLUSIONS AND CLINICAL RELEVANCE: The fact that Cryptosporidium parasites were identified in 18% of faecal specimens from foals analysed for this agent in 2005-2007 by the participating laboratory indicated that infection with this agent in foals is not uncommon. Collectively, the results of this and previous studies performed in New Zealand indicate C. parvum is a cause of diarrhoea in newborn foals, potentially accounting for a proportion of cases empirically diagnosed as foal heat diarrhoea. It is therefore advisable to take precautions when handling diarrhoeic foals, until this potentially zoonotic agent is ruled out in the laboratory.

Widespread mismatch repair protein expression in canine cutaneous mast cell tumors.

Cutaneous mast cell tumors (MCTs) are common canine neoplasms. Some dog breeds more frequently develop MCTs, suggesting a genetically mediated predisposition. In humans, the most common inherited cancer predisposition is caused by germline defects in the mismatch repair (MMR) genes. To investigate whether inherited defects in the MMR genes predispose some dogs to MCT development, MMR expression in 22 MCTs from young and predisposed breed dogs was compared with MMR expression in 22 MCTs from old dogs of non-MCT-predisposed breeds. MMR expression was investigated immunohistochemically using antibodies against MLH1, MSH2, and MSH6. Mast cells within all MCTs expressed MLH1, MSH2, and MSH6. There were no significant differences in the intensity of immunoreactivity or the percentage of cells expressing MMR proteins between MCTs from the 2 groups of dogs. There were no significant differences in MMR protein expression between grade II and grade III MCTs. These results do not support the hypothesis that inherited MMR defects predispose some dogs to MCT development.

Clinically overt infections with methicillin-resistant Staphylococcus aureus in animals in New Zealand: a pilot study.

AIM: To describe clinically overt infections with methicillin resistant Staphylococcus aureus (MRSA) in animals in New Zealand, characterise clinical isolates, and track their sources. METHODS: MRSA isolates identified in 2005 and 2006 by a veterinary diagnostic laboratory were referred to Massey University for confirmation and characterisation. Clinical information was extracted from the laboratory records or obtained from referring clinicians. RESULTS: Seven MRSA isolates from animals and contact persons were characterised. All the isolates belonged to the British epidemic MRSA 15 strain (EMRSA-15). Three EMRSA-15 were isolated from post-operative infections in two dogs. An EMRSA-15 indistinguishable from the isolate recovered from one dog was isolated from the anterior nares of a healthy hospital staff member involved in the care of the animal, suggesting nosocomial transmission. Other EMRSA-15 isolates of uncertain clinical significance were isolated from the femoral head of a cat, and from a sample of cow's milk. AlleMRSA-15 isolates were resistant to ciprofloxacin, and four were resistant to erythromycin; the latter four isolates also exhibited inducible resistance to clindamycin. CONCLUSIONS: MRSA can cause clinically overt and difficult-to-treat infections in animals in New Zealand. The rapid emergence of EMRSA-15 as the dominant MRSA strain in humans has resulted in infection spill over to animals. CLINICAL RELEVANCE: Little is known about the incidence of clinically overt infections with MRSA in animals. The cases described here illustrate the complexities involved in the pharmacological management of EMRSA-15 infections, which is compounded by the universal resistance to beta-lactams, and by the strain's fluoroquinolone resistance and frequent inducible resistance to clindamycin. Such complexities indicate there is a need to develop specific empirical antimicrobial treatment strategies and antibiotic susceptibility testing protocols in countries where EMRSA-15 is dominant.

Comparison of the histology and immunohistochemistry of vaccination-site and non-vaccination-site sarcomas from cats in New Zealand.

AIMS: To compare the histology and immunohistochemistry of vaccination-site sarcomas (VSSs) with non-vaccination-site sarcomas (NVSSs) in cats in New Zealand. To determine whether VSSs in cats in New Zealand have similar histological and immunohistochemical features to those previously described in feline vaccine-associated sarcomas (VASs) in North American studies. METHODS: A retrospective survey of skin biopsies submitted between 2004 and 2006 was performed to identify cutaneous sarcomas from both vaccination and non-vaccination sites in cats. Vaccination sites included the interscapular, shoulder, or dorsal or lateral cervical and thoracic regions. All sarcomas were examined histologically, and smooth muscle actin and desmin were assessed immunohistochemically. Features previously described in VASs were assessed and compared. RESULTS: Sarcomas from 34 cats were identified, 10 of which occurred at vaccination sites. Compared with NVSSs, VSSs were more likely to be located in the hypodermis and have greater cellular pleomorphism, higher mitotic rates, more frequent peripheral lymphocytic aggregates and multinucleated giant cells. VSSs were also more likely than NVSSs to show partial myofibroblastic differentiation, demonstrable using immunohistochemistry. The histological and immunohistochemical features of VSSs in cats in New Zealand are consistent with those previously described in VASs in cats in North America. CONCLUSIONS: The results of this study suggest that VASs occur in cats in New Zealand. CLINICAL RELEVANCE: The occurrence of VASs in cats in New Zealand would provide further support for restriction of the vaccination of cats to the minimum necessary to protect health, and adoption of the New Zealand Veterinary Association guidelines on vaccination.

Physicochemical degradation studies of calcium phosphate glass ceramic in the CaO-P2O5-MgO-TiO2 system.

The aim of this work was to evaluate the in vitro degradation behaviour of a 45CaO-37P(2)O(5)-5MgO-13TiO(2) (mol.%) glass ceramic, under two different simulated physiological conditions: normal physiological pH 7.4, and pH 3.0, which was designed to simulate the acidic conditions produced by osteoclast cells. The in vitro testing was carried out at 37 degrees C for up to 42 days for the pH 7.4 solution and for up to 1 day for the pH 3.0 solution. The incorporation of TiO(2) into the glass structure leads to the precipitation of specific crystalline phases in the glass matrix, namely alpha- and beta-Ca(2)P(2)O(7), TiP(2)O(7) and CaTi(4)(PO(4))(6). The degradation testing at pH 3.0 showed a higher weight loss compared with degradation testing at pH 7.4; the weight loss under the acidic condition after 1 day (24 h) was about 10 times higher than the weight loss after 42 days of immersion at pH 7.4. The ionic release profile of Ca(2+), PO(4)(3-), Mg(2+) and Ti(4+) showed a continuous increase in concentration over all immersion times for both testing solutions. After 1 day of immersion at pH 3.0, the concentration levels of Mg(2+), Ca(2+), PO(4)(3-) were about six times higher than the levels achieved after 42 days of immersion at pH 7.4. The glass ceramic showed similar degradation to hydroxyapatite, and therefore has potential to be used in certain clinical applications where relatively slow resorption of the implant and replacement by bone is required, e.g. cranioplasty.

In vivo assessment of hydroxyapatite and silicate-substituted hydroxyapatite granules using an ovine defect model.

Phase pure hydroxyapatite (HA) and two silicate-substituted hydroxyapatites (0.8 and 1.5 wt% Si, or 2.6 and 4.9 wt% SiO4) were prepared by aqueous precipitation methods. The filter-cakes of HA and silicate-substituted hydroxyapatite (SiHA) compositions were processed into granules 1.0-2.0 mm in diameter and sintered at 1200 degrees C for 2 h. The sintered granules underwent full structural characterisation, prior to assessment in an ovine defect model by implantation for a period of 6 and 12 weeks. The results indicate that HA and SiHA implants were well accepted by the host tissue, with no evidence of inflammation. New bone formation was observed directly on the surfaces and in the spaces between the granular implants. Quantitative histomorphometry as determined by the percentage of bone ingrowth and bone coverage for both SiHA implant compositions was significantly greater than that for phase pure HA. These findings indicate that the in vivo bioactivity of hydroxyapatite was significantly improved by the incorporation of silicate ions into the HA structure, making SiHA ceramics attractive alternatives to conventional HA materials for use as bone graft substitute ceramics.

Effect of the proportion of organic material in bone on thermal decomposition of bone mineral: an investigation of a variety of bones from different species using thermogravimetric analysis coupled to mass spectrometry, high-temperature X-ray diffraction, and Fourier transform infrared spectroscopy.

Thermogravimetric analysis linked to mass spectrometry (TGA-MS) shows changes in mass and identifies gases evolved when a material is heated. Heating to 600 degrees C enabled samples of bone to be classified as having a high (cod clythrum, deer antler, and whale periotic fin bone) or a low (porpoise ear bone, whale tympanic bulla, and whale ear bone) proportion of organic material. At higher temperatures, the mineral phase of the bone decomposed. High temperature X-ray diffraction (HTXRD) showed that the main solids produced by decomposition of mineral (in air or argon at 800 degrees C to 1000 degrees C) were beta-tricalcium phosphate (TCP) and hydroxyapatite (HAP), in deer antler, and CaO and HAP, in whale tympanic bulla. In carbon dioxide, the decomposition was retarded, indicating that the changes observed in air and argon were a result of the loss of carbonate ions from the mineral. Fourier transform infrared (FTIR) spectroscopy of bones heated to different temperatures, showed that loss of carbon dioxide (as a result of decomposition of carbonate ions) was accompanied by the appearance of hydroxide ions. These results can be explained if the structure of bone mineral is represented by [Formula: see text] where V(Ca) and V(OH) correspond to vacancies on the calcium and hydroxide sites, respectively, and 2-x-y = 0.4. This general formula is consistent in describing both mature bone mineral (i.e., whale bone), with a high Ca/P molar ratio, lower HPO4(2-) content, and higher CO3(2-) content, and immature bone mineral (i.e., deer antler), with a low Ca/P ratio, higher HPO4(2-), and lower CO3(2-) content.

Preparation of macroporous calcium phosphate cement tissue engineering scaffold.

Unlike sintered hydroxyapatite there is evidence to suggest that calcium phosphate cement (CPC) is actively remodelled in vivo and because CPC is formed by a low-temperature process, thermally unstable compounds such as proteins may be incorporated into the matrix of the cement which can then be released after implantation. The efficacy of a macroporous CPC as a bone tissue engineering scaffold has been reported; however, there have been few previous studies on the effect of macroporosity on the mechanical properties of the CPC. This study reports a novel method for the formation of macroporous CPC scaffolds, which has two main advantages over the previously reported manufacturing route: the cement matrix is considerably denser than CPC formed from slurry systems and the scaffold is formed at temperatures below room temperature. A mixture of frozen sodium phosphate solution particles and CPC powder were compacted at 106 MPa and the sodium phosphate was allowed to melt and simultaneously set the cement. The effect of the amount of porogen used during processing on the porosity, pore size distribution and compressive strength of the scaffold was investigated. It was found that macroporous CPC could reliably be fabricated using cement:ice ratios as low as 5:2.

Effect of porosity reduction by compaction on compressive strength and microstructure of calcium phosphate cement.

Hydroxyapatite (HA) calcium phosphate cements (CPCs) are attractive materials for orthopedic applications because they can be molded into shape during implantation. However their low strength and brittle nature limits their potential applications to principally non-load-bearing applications. Little if any use has been made of the HA cement systems as manufacturing routes for preset HA bone grafts, which although not moldable pastes, are resorbable, unlike HA sintered ceramic. It is known that the strength of cements can be increased beyond that attainable from slurry systems by compaction, and this study investigates whether compaction significantly alters the specific surface area and pore-size distribution of CPC prepared according to the method of Brown and Chow. Compaction pressures of between 18 and 106 MPa were used to decrease the porosity from 50 to 31%, which resulted in an increase in the wet compressive strength from 4 to 37 MPa. The Weibull modulus was found to increase as porosity decreased; in addition the amount of porosity larger than the reactant particle size increased as porosity decreased. It is proposed that this was caused by a combination of voids created by the aqueous solvent used in fabrication and shrinkage that occurs on reaction. The specific surface area was unchanged by compaction.

Preparation and characterization of magnesium/carbonate co-substituted hydroxyapatites.

A new synthesis/processing method has been devised to produce magnesium/carbonate co-substituted hydroxyapatite ceramics that do not decompose to tricalcium phosphate (TCP) on sintering. Using this method, a series of magnesium/carbonate co-substituted hydroxyapatite (Mg/CO(3)-HA) compositions, containing between 0 and 0.35 wt % Mg and approximately 0.9 wt % CO(3) were prepared. Sintering the Mg/CO(3)-HA compositions in a CO(2)/H(2)O atmosphere yields a single crystalline phase that appears to be identical to stoichiometric HA. In contrast, when the compositions were prepared in the absence of carbonate and were sintered in air, the phase composition was a biphasic mixture of HA and TCP e.g. for 0.25 wt % Mg substitution the phase composition was approximately 60%HA/40% TCP. Clearly, both the synthesis route and the processing (i.e. sintering) route are of importance in the production of a single-phase Mg/CO(3)-HA ceramic. Fourier transform infrared (FTIR) spectroscopy has indicated that the Mg/CO(3)-HA ceramics still contained carbonate groups after sintering at 1200 degrees C. Chemical analysis by X-ray fluorescence spectroscopy (XRF) and C-H-N analysis has shown that the cation/anion molar ratio (i.e. [Ca+Mg]/[P+C/2]) of the different compositions were 1.68(+/-0.01), which is equivalent to the Ca/P molar ratio of stoichiometric HA. Although the magnesium/carbonate co-substitution had a positive effect in preventing phase decomposition during sintering, it appeared to have a negative effect on the densification of the MgCO(3)-HA ceramics, compared to stoichiometric HA.

Structural analysis of Si-substituted hydroxyapatite: zeta potential and X-ray photoelectron spectroscopy.

The aim of this study was to determine the effect of the incorporation of silicon on the surface charge of hydroxyapatite (HA) and to assess surface structural changes of HA and Si-HA induced by dissolution in both static and dynamic systems. X-ray photoelectron spectroscopy (XPS) analysis showed that SiO(4)(4-) groups were substituted for PO(4)(3-) groups in the silicon-hydroxyapatite (Si-HA) lattice according to a previously proposed substitution mechanism without the formation of other crystalline phases, such as tricalcium phosphate or calcium oxide. The substituted silicon induced a decrease in the net surface charge and the isoelectric point of HA as determined by zeta potential (ZP) measurements. At physiological pH=7.4 the surface charge of Si-HA was significantly lowered compared to unmodified HA, i.e. -50+/-5 to -71+/-5 eV, caused by the presence of silicate groups in the HA lattice, which may account for a faster in vitro apatite formation using SBF testing. XPS results indicated that silicon seems to be preferentially leached out from Si-HA surface compared to other ionic species after dissolution studies in tris-buffer using a dynamic system.

A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules.

Phase pure hydroxyapatite (HA) and a 0.8 wt % silicon substituted hydroxyapatite (SiHA) were prepared by aqueous precipitation methods. Both HA and SiHA were processed into granules 0.5-1.0 mm in diameter and sintered at 1200 degrees C for 2 h. The sintered granules underwent full structural characterization, prior to implantation into the femoral condyle of New Zealand White rabbits for a period of 23 days. The results show that both the HA and SiHA granules were well accepted by the host tissue, with no presence of any inflammatory cells. New bone formation was observed directly on the surfaces and in the spaces between both HA and SiHA granular implants. The quantitative histomorphometry results indicate that the percentage of bone ingrowth for SiHA (37.5%+/-5.9) was significantly greater than that for phase pure HA (22.0%+/-6.5), in addition the percentage of bone/implant coverage was significantly greater for SiHA (59.8%+/-7.3) compared to HA (47.1%+/-3.6). These findings indicate that the early in vivo bioactivity of hydroxyapatite was significantly improved with the incorporation of silicate ions into the HA structure, making SiHA an attractive alternative to conventional HA materials for use as bone substitute ceramics.