The novel use of waste animal bone from New Zealand agricultural sources as a feedstock for forming plasma sprayed hydroxyapatite coatings on biomedical implant materials.

This study presents the feasibility of using animal bone-derived hydroxyapatite (HAP) as feedstock powders for plasma spraying. Bovine, cervine and ovine bone from abattoirs was boiled in a pressure cooker to remove blood, fat and adhering meat tissue. The bone was then placed in a muffler furnace, pyrolyzed at approximately 1000 degrees C to remove collagen and resid-ual organics, cooled and subsequently ground to a powder then digested in nitric acid. Sodium hydroxide was added to the digest to reprecipitate the HAP. Ageing of the precipitate followed by filtration, extensive washing and drying produced the white powder used as the feedstock. X-ray diffractometry (XRD) and Fourier transform infrared spectroscopy (FTIR) confirmed the powder to be poorly crystalline HAP with low-level carbonate. Out of several batches of the sieved powders, one batch was plasma sprayed to produce adherent HAP coatings; therefore, demonstrating that animal bone-derived HAP powders can be seri-ously considered as a feedstock powder, subject to the powder being processed for the correct rheological characteristics for easy flowing within the plasma spray flow lines. The phase composition of the successful plasma sprayed HAP coatings on both stainless steel and titanium were found by XRD to be mainly HAP with minor contributions from á -tricalcium phosphate, tetra-calcium phosphate and CaO; therefore, demonstrating that feedstock decomposition on its passage through the plasma spray torch was insignificant under the conditions employed. Scanning electron microscopy (SEM) micrographs of the coatings indicated that their morphology featured the classical heterogeneous and splat-like appearance expected of plasma sprayed coatings. Young's modulus and Vicker's microhardness tests conducted on the coatings revealed values in the range, respectively, 22-87 GPa and 166-287 (HV200 ) indicating high strength plasma spray HAP coatings had been produced from the feedstock powder.

Wound healing: captopril, an angiogenesis inhibitor, and Staphylococcus aureus peptidoglycan.

BACKGROUND: Captopril, an angiotensin-converting enzyme inhibitor, used for treating hypertension and heart failure, inhibits angiogenesis in the corneas of rats in response to basic fibroblast growth factor, slows the growth of experimental tumors in rats, and leads to the regression of Kaposi's sarcoma. Because angiogenesis is key to wound healing, we hypothesized that captopril would impair wound healing. We hypothesized also that because local application at operation of Staphylococcus aureus peptidoglycan (SaPG) increases angiogenesis and accelerates wound healing in rats, SaPG would prevent or ameliorate the postulated captopril-impaired wound healing. MATERIALS AND METHODS: In each experiment, rats were divided randomly into two groups: one drinking tap water, and the other, tap water containing 0.5 mg captopril/ml. All ate chow and drank ad libitum, pre-operatively (4-12 days) and postoperatively (7 days). In experiments 1 and 2, bilateral paravertebral 5.5-cm skin incisions were made aseptically (intraperitoneal sodium pentobarbital), and closed with interrupted No. 35 stainless-steel sutures. On one side, the wound was immediately inoculated with 157 microliter pyrogen-free isotonic saline and on the other side the wound was inoculated with 157 microliter saline containing 4.7 mg SaPG (860 microgram SaPG/cm incision). In the third experiment, polyvinyl alcohol (PVA) sponges (16-17 mg dry wt each) containing either 50 microliter saline or 0.5 mg SaPG in 50 microliter saline were implanted subcutaneously, two on each side, via 1-cm incisions closed with a single suture. In the fourth experiment, 5.5-cm bilateral skin incisions and subcutaneous implantation of PVA sponges were done as described but all sites were instilled with saline only. All rats were euthanized (CO(2) asphyxia) 7 days postoperatively. RESULTS: Wound breaking strength (WBS) of the saline-treated incisions was significantly higher (P < 0.001) in captopril-treated rats than in controls (172 +/- 13 g vs 105 +/- 6 g) in experiment 1 and higher, but not significantly in captopril-treated rats in experiment 2 (153 +/- 8 g vs 114 +/- 6 g) (PNS). SaPG inoculation of the incisions increased WBS significantly in both control and captopril-treated rats: 187 +/- 11 g vs 105 +/- 6 g (P < 0.001) and 283 +/- 16 g vs 172 +/- 13 g (P < 0.001), respectively, in experiment 1, and 217 +/- 13 g vs 114 +/- 6 g (P < 0.0001) (controls) and 266 +/- 17 g vs 153 +/- 8 g (captopril-treated rats) (P < 0.0001) in experiment 2. In experiment 3, subcutaneous PVA saline-inoculated sponge reparative tissue hydroxyproline (OHP) content was similar in control and captopril-treated rats, and SaPG inoculation increased reparative tissue OHP significantly in both groups: 2458 +/- 218 microgram/100 mg dry sponge vs 3869 +/- 230 microgram/100 mg (P < 0.001) (controls) and 2489 +/- 166 microgram/100 mg vs 4176 +/- 418 microgram/100 mg (P < 0.001) (captopril-treated rats). Histologically, angiogenesis and reparative tissue collagen were similar in control and captopril-treated rats, in both saline-inoculated and SaPG-inoculated sponges. In experiment 4 (all incisions and subcutaneous PVA sponges were saline-inoculated), there was no significant difference in WBS between control and captopril-treated rats (107 +/- 6 g vs 96 +/- 5 g, NS). PVA sponge reparative tissue OHP was significantly higher in captopril-treated rats: 3698 +/- 170 microgram/100 mg dry sponge vs 2534 +/- 100 microgram/100 mg (P < 0.0001). CONCLUSION: Unexpectedly, in four experiments, captopril did not inhibit WBS or PVA sponge reparative tissue angiogenesis or collagen accumulation; in fact, WBS was increased significantly in one of three experiments, and PVA sponge reparative tissue OHP was increased significantly in one of two experiments. Also, captopril did not interfere with the wound healing-accelerating effect of SaPG.

Electrostatic channeling in the bifunctional enzyme dihydrofolate reductase-thymidylate synthase.

The bifunctional enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) carries out two distinct reactions, with the dihydrofolate produced by the TS-catalyzed reaction acting as the substrate for the DHFR-catalyzed reaction. Brownian dynamics simulation techniques were used to investigate the possible role of electrostatics in determining efficient channeling of the substrate, by explicitly simulating substrate diffusion between the two active sites. With a substrate charge of -2, almost all (> 95%) substrate molecules leaving the TS active site reached the DHFR active site at zero ionic strength. Under the same conditions, but in the absence of electrostatic effects, successful channeling was reduced to only around 6%: electrostatic effects therefore appear essential to explain the efficient channeling observed experimentally. The importance of substrate charge, the relative contributions of specific basic residues in the protein, the role played by the second monomer of the dimer in channeling and the effects of changing ionic strength were all investigated. Simulations performed for substrate transfer in the opposite direction suggest that channeling in DHFR-TS is not strongly directional and that the role of electrostatics is perhaps more one of restricting diffusion of the substrate than one of actively guiding it from the TS to the DHFR active site. The results demonstrate that electrostatic channeling can be a highly efficient means of transferring charged substrates between active sites in solvent-exposed environments.

The effect of the platelet derived wound healing formula and the nerve growth factor on the experimentally injured spinal cord.

The main purpose of this study is to investigate the effect of platelet derived wound healing formula (PDWHF) and nerve growth factor (NGF) in the treatment of experimental spinal cord injury. PDWHF is a conglomerate of growth factors which include platelet derived growth factor (PDGF), platelet derived angiogenesis factor (PDAF), transforming growth factor-beta (TGF beta) and platelet factor IV (PF4). Complete spinal cord transection was performed at T12 in rats and the treatment of the spinal cord injury was achieved by filling the dead space with type 1 collagen gel impregnated with PDWHF, or with 2.5S-NGF. Controls were treated with only type 1 collagen gel. Animals were sacrificed at 1, 2 or 3 months. Histopathologically, tissue autolysis and cavity formation by phagocytosis expanded 1-3 mm into the cord stumps and the volume of cavitation was less in the two treated groups. In the NGF group, a greater number of surviving nerve cells were observed in this region. Most of the control animals formed only thin, short axonal bundles, however, increased axonal regrowth was noted in animals treated with trophic factors, especially in the NGF group. The NGF group formed thick axonal bundles and abundant neuroma. Increased angiogenesis was observed in the collagen gel matrix and the injured spinal cord parenchyma, in the PDWHF group. Recent studies have shown that mammalian adult CNS possesses the ability for structural and/or functional plasticity following injury under appropriate circumstances. In this in vivo study, exogenous NGF appeared to induce axomal outgrowth and nerve cell survival. PDWHF produced notable angiogenesis which seemed to improve the extracellular microenvironment. This may be important for the delivery of exogenous trophic factors, nutrients and for the changes of extracellular matrices to support nerve cells and axons.

Tumor necrosis factor alpha (rhTNF) fails to stimulate angiogenesis in the rabbit cornea.

BACKGROUND: The objective of this study was to thoroughly examine the in vivo angiogenesis activity of human recombinant tumor necrosis factor alpha (rhTNF). METHODS: rhTNF (0.5 ng to 1.0 microgram) was incorporated into the slow release polymers Hydron or HYPAN and implanted into the rabbit cornea. Release of biologically active rhTNF from the polymers was determined with the L929 cytotoxicity assay. RESULTS: All concentrations tested failed to elicit capillary formation beyond that observed for controls. Less than 2% of the rhTNF was released from the Hydron over 7 days. HYPAN released five times the amount of rhTNF in vitro, but even at doses of 500 ng (104.3 ng suggested release) no angiogenesis was stimulated. CONCLUSIONS: Under the circumstances tested, rhTNF is not angiogenic in vivo.

Structure-based design of novel, urea-containing FKBP12 inhibitors.

The structure-based design and subsequent chemical synthesis of novel, urea-containing FKBP12 inhibitors are described. These compounds are shown to disrupt the cis-trans peptidylprolyl isomerase activity of FKBP12 with inhibition constants (Ki,app) approaching 0.10 microM. Analyses of several X-ray crystal structures of FKBP12-urea complexes demonstrate that the urea-containing inhibitors associate with FKBP12 in a manner that is similar to, but significantly different from, that observed for the natural product FK506.

Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex.

Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.

Rabbit cornea assay of angiogenesis: methodology and evaluation.

The rabbit cornea angiogenesis assay has been used extensively to determine the capacity of cells, tissues, growth factors, and a variety of other compounds to stimulate the formation of capillaries. Improper use of the assay coupled with lack of thorough evaluation has generated disparate, perplexing, and even erroneous results. The objective of this article is to review the corneal angiogenesis assay, paying close attention to specific techniques, modes of evaluation, and considerations that investigators need to make before drawing any conclusions.

Age-related alterations in the morphology of femoral artery vasa vasorum in the rat.

The objective of this study was to explore any age-related morphological changes in the vasa vasorum of the rat femoral artery. Vascular corrosion casts were prepared from 2, 12 and 24-month-old rats. Examination of the casts with the scanning electron microscope revealed dramatic differences in the appearance of the vessels of young and aged rats. The vasa vasorum of 2-month-old rats consisted of a dense network of capillaries. These vessels were dramatically reduced in number by 12 months, and even fewer capillaries were present at 24 months. This reduction in capillary density is consistent with the observed age-related decreases in oxygen tension and may explain why the aged are more prone to atherosclerosis.

Proliferation of wound derived capillary endothelial cells: young versus aged.

The objective of this study was to compare the proliferative potential of wound derived capillary endothelial cells (WCEC) from aged and young rats. Endothelial cells were isolated from subcutaneously implanted sponges in 2- and 24-month-old rats. The identity of the cells as endothelial was confirmed by staining for Ac-LDL uptake. Aged and young WCEC (20,000/well) were stimulated with increasing concentrations of fetal calf serum (0, 2.5, 5, 10 and 15%). The increase in cell number was determined with a Coulter counter. At all serum concentrations, the proliferative capacity of WCEC from aged rats was significantly higher than that of WCEC from young rats.

Vascular endothelial growth factor (rhVEGF165) stimulates direct angiogenesis in the rabbit cornea.

The object of this study was to test vascular endothelial growth factor (VEGF) for angiogenic activity in the rabbit corneal assay. VEGF doses ranging from 20 ng to 1000 ng were incorporated into a slow release polymer and implanted into the avascular rabbit cornea. Capillary formation in the cornea was visually analyzed on a daily basis and examined with histology, transmission electron microscopy and scanning electron microscopy of vascular corrosion casts on days 2 and 7 post-implantation. VEGF implants (200ng to 1000ng) consistently stimulated angiogenesis. This neovascularization occurred in the absence of inflammation. We conclude that VEGF acts directly on endothelial cells, initiating and mediating the formation of capillaries.

Definitions and guidelines for assessment of wounds and evaluation of healing.

Chronic wounds represent a worldwide problem. For laboratory and clinical research to adequately address this problem, a common language needs to exist. This language should include a system of wound classification, a lexicon of wound descriptors, and a description of the processes that are likely to affect wound healing and would healing end points. The report that follows defines wound, acute wound, chronic wound, healing and forms of healing, wound assessment, wound extent, wound burden, and wound severity. The utility of these definitions is demonstrated as they relate to the healing of a skin wound, but these definitions are broadly applicable to all wounds.

Definitions and guidelines for assessment of wounds and evaluation of healing.

BACKGROUND: Chronic wounds represent a worldwide problem. For laboratory and clinical research to adequately address this problem, a common language needs to exist. OBSERVATION: This language should include a system of wound classification, a lexicon of wound descriptors, and a description of the processes that are likely to affect wound healing and wound healing end points. CONCLUSIONS: The report that follows defines wound, acute wound, chronic wound, healing and forms of healing, wound assessment, wound extent, wound burden, and wound severity. The utility of these definitions is demonstrated as they relate to the healing of a skin wound, but these definitions are broadly applicable to all wounds.

Structure of and kinetic channelling in bifunctional dihydrofolate reductase-thymidylate synthase.

The bifunctional enzyme dihydrofolate reductase-thymidylate synthase catalyses both the reductive methylation of 2'-deoxyuridylate and the subsequent reduction of dihydrofolate to yield 2'-deoxythymidylate and tetrahydrofolate at two spacially discrete sites situated on different protein domains. The X-ray structure of dihydrofolate reductase-thymidylate synthase from Leishmania major indicates that transfer of dihydrofolate between these sites does not occur by transient binding at both sites but rather by movement of dihydrofolate across the surface of the protein. The enzyme has an unusual surface charge distribution that could account for this channelling of dihydrofolate between active sites.

Platelet derived wound healing factors (PDWHF) accelerate and augment wound healing angiogenesis in the rat.

The object of this study was to determine the effect of PDWHF on wound healing angiogenesis. Sponges saturated with PDWHF were implanted in rats. Six hours to 14 days post-implantation, vascular corrosion casts were prepared and examined by SEM. Leukocyte margination and angiogenesis was accelerated by 24-48 hours. Vessel number was also greater than in the controls. Another series of PDWHF treated sponges were implanted. Fourteen days later the vasculature was perfused with Evans blue. Sponges were homogenized, and absorbance of the supernatants was determined. Absorbances of PDWHF supernatants was 2X greater than controls. PDWHF enhanced wound healing angiogenesis in the rat.

Crystal structures of the myristylated catalytic subunit of cAMP-dependent protein kinase reveal open and closed conformations.

Three crystal structures, representing two distinct conformational states, of the mammalian catalytic subunit of cAMP-dependent protein kinase were solved using molecular replacement methods starting from the refined structure of the recombinant catalytic subunit ternary complex (Zheng, J., et al., 1993a, Biochemistry 32, 2154-2161). These structures correspond to the free apoenzyme, a binary complex with an iodinated inhibitor peptide, and a ternary complex with both ATP and the unmodified inhibitor peptide. The apoenzyme and the binary complex crystallized in an open conformation, whereas the ternary complex crystallized in a closed conformation similar to the ternary complex of the recombinant enzyme. The model of the binary complex, refined at 2.9 A resolution, shows the conformational changes associated with the open conformation. These can be described by a rotation of the small lobe and a displacement of the C-terminal 30 residues. This rotation of the small lobe alters the cleft interface in the active-site region surrounding the glycine-rich loop and Thr 197, a critical phosphorylation site. In addition to the conformational changes, the myristylation site, absent in the recombinant enzyme, was clearly defined in the binary complex. The myristic acid binds in a deep hydrophobic pocket formed by four segments of the protein that are widely dispersed in the linear sequence. The N-terminal 40 residues that lie outside the conserved catalytic core are anchored by the N-terminal myristylate plus an amphipathic helix that spans both lobes and is capped by Trp 30. Both posttranslational modifications, phosphorylation and myristylation, contribute directly to the stable structure of this enzyme.

A three-dimensional model of the Cdc2 protein kinase: localization of cyclin- and Suc1-binding regions and phosphorylation sites.

The Cdc2 protein kinase requires cyclin binding for activity and also binds to a small protein, Suc1. Charged-to-alanine scanning mutagenesis of Cdc2 was used previously to localize cyclin A- and B- and Suc1-binding sites (B. Ducommun, P. Brambilla, and G. Draetta, Mol. Cell. Biol. 11:6177-6184, 1991). Those sites were mapped by building a Cdc2 model based on the crystallographic coordinates of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) (D. R. Knighton, J. Zheng, L. F. Ten Eyck, V. A. Ashford, N.-H. Xuong, S. S. Taylor, and J. M. Sowadski, Science 253:407-414, 1991). On the basis of this model, additional mutations were made and tested for cyclin A and Suc1 binding and for kinase activity. Mutations that interfere with cyclin A binding are localized primarily on the small lobe near its interface with the cleft and include an acidic patch on the B helix and R-50 in the highly conserved PSTAIRE sequence. Two residues in the large lobe, R-151 and T-161, influence cyclin binding, and both are at the surface of the cleft near its interface with the PSTAIRE motif. Cyclin-dependent phosphorylation of T-161 in Cdc2 is essential for activation, and the model provides insights into the importance of this site. T-161 is equivalent to T-197, a stable phosphorylation site in cAPK. On the basis of the model, cyclin binding very likely alters the surface surrounding T-161 to allow for T-161 phosphorylation. The two major ligands to T-197 in cAPK are conserved as R-127 and R-151 in Cdc2. The equivalent of the third ligand, H-87, is T-47 in the PSTAIRE sequence motif. Once phosphorylated, T-161 is predicted to play a major structural role in Cdc2, comparable to that of T-197 in cAPK, by assembling the active conformation required for peptide recognition. The inhibitory phosphorylation at Y-15 also comes close to the cleft interface and on the basis of this model would disrupt the cleft interface and the adjacent peptide recognition site rather than prevent ATP binding. In contrast to cyclin A, both lobes influence Suc1 binding; however, the Suc1-binding sites are far from the active site. Several mutants map to the surface in cAPK, which is masked in part by the N-terminal 40 residues that lie outside the conserved catalytic core. The other Suc1-binding site maps to the large lobe near a 25-residue insert and includes R-215.

Alloxan diabetes alters the rabbit transarterial wall oxygen gradient.

PURPOSE: Atherosclerotic vascular occlusive disease is the most common complication of diabetes mellitus and accounts for 75% of deaths in diabetic patients. Determining the initiator and continuing stimulus for the cellular events in the formation of atherosclerotic lesions in diabetic patients could lead to the prevention of this common and deadly complication. Diabetes-induced arterial wall hypoxia is proposed as an initiator and continuing stimulus for atherosclerotic vascular occlusive disease. METHODS: Transarterial wall oxygen gradient measurements were performed on the infrarenal aorta with an oxygen microelectrode 14 to 16 weeks after the induction of alloxan diabetes in rabbits. RESULTS: Both insulin-treated and untreated alloxan diabetic rabbits revealed significantly decreased oxygen tensions throughout the arterial wall compared with control rabbits. There was no significant difference in the transarterial wall oxygen gradient between the two groups of diabetic rabbits. This effect was noted despite no difference in the partial pressure of oxygen in arterial blood or visual evidence of atherosclerotic lesion formation in the three groups. CONCLUSIONS: These findings suggest that diabetes induces arterial wall hypoxia independent of insulin therapy and before the formation of atherosclerotic lesions. Diabetes-induced arterial wall hypoxia may contribute to the formation of atherosclerotic lesions.

Structural features that specify tyrosine kinase activity deduced from homology modeling of the epidermal growth factor receptor.

To identify structural features that distinguish protein-tyrosine kinases from protein-serine kinases, a molecular model of the kinase domain of epidermal growth factor receptor was constructed by substituting its amino acid sequence for the amino acid sequence of the catalytic subunit of cAMP-dependent protein kinase in a 2.7-A refined crystallographic model. General folding was conserved as was the configuration of invariant residues at the active site. Two sequence motifs that distinguish the two families correspond to loops that converge at the active site of the enzyme. A conserved arginine in the catalytic loop is proposed to interact with the gamma phosphate of ATP. The second loop provides a binding surface that positions the tyrosine of the substrate. A positively charged surface provides additional sites for substrate recognition.

cAMP-dependent protein kinase defines a family of enzymes.

The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E. coli, the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thr197 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.