The processing and characterization of animal-derived bone to yield materials with biomedical applications: part 1: modifiable porous implants from bovine condyle cancellous bone and characterization of bone materials as a function of processing.

A study on the development of a process to form materials suitable for biomedical xenograft implants from bovine cancellous bone is presented. Bone cubes cut from the condyle portion of bovine femurs sourced from abattoir waste were subjected to a defatting and subsequent deproteination procedure to produce shape-modifiable materials in which the biocompatible mineral calcium hydroxycarbonate apatite component was preserved in the original osseous architecture of the bovine bone. Optimum defatting was achieved by (1) thawing of the precut bone cubes in water, (2) pressure cooking at 15 psi in water, (3) soaking in 0.1 mol l(-1) NaOH followed by a thorough rinse under running water, (4) microwave heating of the bone cubes in water, (5) refluxing in methyl acetate and finally (6) removal of internal liquid from the cubes by shaking and then air drying. Subsequent deproteination of the defatted bone cubes was optimally achieved by (1) soaking in 5% sodium hypochlorite solution at ambient temperature using ultrasonication, (2) thorough rinsing of the cubes in water followed by drying. The final product is a defatted/deproteinated, bleached material that can be molded into various shapes for implant use in the body. The bone specimens were characterized by a suite of analytical techniques (i.e. infrared, 31P and 13C solid magic-angle spinning (MAS) nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopies, atomic absorption (AA) spectrometry, inductively coupled plasma (ICP) spectrometry, differential scanning calorimetry (DSC), and scanning electron microscopy (SEM)) in order to follow compositional changes during the various stages of processing. In general, bovine condyles proved to be the best source of xenograft materials with condyles from other animal species (i.e. deer, sheep and ostrich) being too small to constitute a utilizable source of cancellous bone. This study shows how value can be added to a hitherto underutilized abattoir by-product by using simple processing techniques.

The processing and characterization of animal-derived bone to yield materials with biomedical applications. Part II: milled bone powders, reprecipitated hydroxyapatite and the potential uses of these materials.

Further studies on the processing and use of animal-bone-derived calcium phosphate materials in biomedical applications are presented. Bone powders sourced either from the direct crushing and milling of bovine, ovine and cervine bone or after being subjected to defatting and acid digestion/NaOH reprecipitation and sodium hypochlorite hydrogen peroxide treatment of animal bones were characterized using Fourier transform infra-red (FTIR) spectroscopy, 13C solid state magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, atomic absorption (AA) and inductively coupled plasma (ICP) spectrometric techniques. Bone powders were trialled for their potential use as a substrate for phosphine coupling and enzyme immobilization as well as a feedstock powder for plasma spraying on titanium metal substrates. Results indicated that enzyme immobilization by phosphine coupling could be successfully achieved on milled cervine bone with the immobilized enzyme retaining some activity. It was found that the presence of impurities normally carried down with the processing of the bone materials (viz., fat and collagen) played an important role in influencing the adsorbency and reactivity of the powders. Plasma spraying studies using reprecipitated bovine-derived powders produced highly adherent coatings on titanium metal, the composition of which was mostly hydroxyapatite (Ca10(PO4)6(OH)2) with low levels of alpha-tricalcium phosphate (alpha-Ca3(PO4)2) and tetracalcium phosphate (Ca4P2O9) also detected. In general, animal derived calcium phosphate materials constitute a potentially cheaper source of calcium phosphate materials for biomedical applications and make use of a largely under-utilized resource from abattoir wastes.

The processing and characterization of animal-derived bone to yield materials with biomedical applications. Part III: material and mechanical properties of fresh and processed bovine cancellous bone.

Conversion of bovine cancellous bone to a useful biomedical xenograft material involves several processing steps which include boiling, defatting and deproteination (i.e. bleaching). This study has shown how these processes can influence cancellous bone modulus and strength. It was found that prolonged boiling in water for six hours followed by NaOCl bleaching had a deleterious effect on the overall strength of the bovine bone. In contrast, bone samples subjected to only moderate boiling (1.5 hours) exhibited a 22% stiffness increase due mainly to the effects of drying. The same stiffened samples, when subjected to the bleaching procedure, retained some strength with only a small reduction in moduli values. It can be concluded that careful control of defatting and bleaching procedures on bovine bone is able to give a strong, albeit, brittle material with preservation of the original bone architecture. The bone xenograft materials are worthy of further investigation in in vivo clinical trials to assess their performance in contact with biological fluids.

A simple silver-staining technique for detecting Bence Jones proteins in unconcentrated urine.

Detection of Bence Jones proteins in urine usually involves a concentration step, followed by electrophoresis and, if necessary, immunofixation. The time-consuming and expensive concentration step can be eliminated by use of the silver-stain technique described here. This procedure, routinely used for staining unconcentrated urine, is inexpensive, sensitive, and easily performed in a clinical laboratory. Bence Jones proteins can be detected in concentrations as low as 5 mg/L.

A case of partial deficiency of alpha 1-antichymotrypsin.

A 20-year-old woman, admitted to a neurological ward with a diagnosis of benign intracranial hypertension, was found on specific protein electroimmunoassay to have a consistently decreased concentration of alpha 1-antichymotrypsin in her plasma. Serum from her father showed the same result. Further investigation of her family demonstrated that this partial deficiency was transmitted in an autosomal dominant fashion and was not associated with any obvious specific clinical abnormalities.

An evaluation of cerebrospinal fluid oligoclonal banding confirmed by immunofixation on agarose gel.

The cerebrospinal fluid (CSF) from 115 consecutive patients undergoing diagnostic lumbar puncture or myelography was examined to determine the usefulness of immunofixation, following agarose gel electrophoresis, in the detection of oligoclonal IgG. All electrophoretic patterns were evaluated with and without immunofixation, and the interpretation of 9% of specimens was altered by immunofixation. The demonstration of oligoclonal IgG was shown to be more reliable in the diagnosis of multiple sclerosis than other indices of intrathecal synthesis of IgG. It is concluded that immunofixation should be used routinely when examining CSF for oligoclonal banding.

alpha 1-Antitrypsin microheterogeneity. Isolation and physiological significance of isoforms.

alpha 1-Antitrypsin has a microheterogeneity evident on isoelectric focusing as three major and several minor bands. We have identified the carbohydrate structures of the major bands; band 6 (isoform I) has three bi-antennary sidechains, band 4 (isoform II) has two bi- and one tri-antennary and band 2 (isoform III) has one bi- and two tri-antennary sidechains. The identity of the isoforms with the bands permitted their measurement in plasma by photometric scanning of the electrofocused gels. In healthy controls the levels of isoforms I, II and III were relatively constant and in the proportions of 5, 4 and 1, respectively. A marked change occurred during inflammation and oestrogen stress with isoforms II and III accounting for most of the increase in alpha 1-antitrypsin. One possible consequence of the changed proportions was shown to be the increased catabolism of the partially desialylated tri-antennary isoforms compared to that of the predominant bi-antennary form of the healthy individual.

Immunocytoma, cancer and other associations of monoclonal gammopathy: a review of 224 cases.

The clinical, haematological and biochemical correlations of 224 consecutive unselected examples of monoclonal gammopathy have been studied. The paraprotein frequency detedted was IgG 62 percent, IgA 15.2 percent, IgM 10.3 percent, Bence Jones protein 8.9 percent and in 3.6 percent the paraproteins were not identified. In half the monoclonal gammopathy was associated with an immunocytoma (myeloma in 82 and lymphoma in 30). In three cases the associated clinical disease was amyloidosis. In 36 cases (16.1 percent) the associated clincial disease was a nonlymphoproliferative malignant tumour. Monoclonal gammopathy may be a significant marker of malignancy in such cases. In 73 cases (32.6 percent) the associated clinical conditions were unrelated to the gammopathy although only 55 of these cases were sufficiently investigated to warrant classification as examples of benign monoclonal gammopathy. There was a strong correlation between Bence Jones proteinuria and malignancy. Sixty-five patients demonstrated Bence Jones proteinuria and in 59 of these a malignancy was detected. The association was strong between hypercalcaemia and malignancy as this was present in all 27 of the cases who had hypercalcaemia. The relationship between Bence Jones proteinuria and hypercalcaemia was also strong and Bence Jones proteinuria was detected in 73 percent of the hypercalcaemic patients as opposed to 36.7 percent in the whole series. Hypercalcaemia and Bence Jones proteinuria, when found in a patient with monoclonal gammopathy have a grave clinical connotation.