Jacob S. Hanker

Biomaterials and biomedical devices

This review discusses the factors important in the incorporation or integration of biomaterials and devices by tissue. Methods for surface modification and surface-sensitive techniques for analysis are cited. In vitro methods to evaluate the biocompatibility or efficacy of certain biomaterials and devices are presented. Present and future directions in neural prostheses, cardiovascular materials, blood or bone substitutes, controlled drug delivery, orthopedic prostheses, dental materials, artificial organs, plasma- and cytapheresis, and dialysis are discussed.

The demonstration of cholinesterases by the formation of osmium blacks at the sites of Hatchett's brown

SummarySmall amounts of Hatchetts brown (cupric ferrocyanide, Cu2Fe(CN)6·7H2O), deposited at the sites of cholinesterase activity in tissues by the procedure of Karnovsky and Roots (1964), may be enhanced by bridging to osmium through thiocarbohydrazide (TCH). Alternatively, amplification of the deposits may be attained by utilizing the cupric ferrocyanide as a catalyst to effect the oxidative coupling of 3,3′-diaminobenzidine (DAB). The resulting intensely colored osmiophilic polymer is more visible and after osmication, is electron opaque. With these procedures, the light microscope preparations are more permanent than with the Gomori modification of the Koelle and Friedenwald procedure.One of the major advantages of this method is that thick serial plastic sections (1–2 μm) may be taken and readily studied by light microscopy until the required enzymatically stained areas appear. Thus the selection of areas for ultrathin sectioning is facilitated; enzymatically stained structures usually difficult to locate, such as intraepithelial nerve endings, are readily found.The intensification procedures permit shorter incubation times at lower temperatures. This results in diminished tendency of the original amorphous, gel-like Hatchetts brown deposits to coalesce into relatively large cubic crystals.Immediate rinsing of the tissues after the formation of Hatchetts brown with a 0.1 M, pH 7.2, tris (hydroxymethyl)aminomethane (TRIS) buffer prior to intensification with either TCH or DAB was found effective in eliminating some of the smaller background deposits after the longer incubations.Studies in developing mice with an hereditary sensory neuropathy showed severe depletion of acetylcholinesterase activity concomitant with the loss of sensory endings from the rugae of hard palate, confirming the peripheral nature of the neuropathy.High levels of acetylcholinesterase found associated with afferent trigeminal components, especially sensory endings containing clear vesicles, suggest that they may be cholinoceptive.

Wafer embedding: Specimen selection in electron microscopic cytochemistry with osmiophilic polymers

SynopsisA new wafer embedding procedure is described that permits light microscopic screening of embedded tissue prior to ultrathin sectioning. It is particularly valuable when used on specimens obtained with an automatic sectioner and treated cytochemically to obtain visible intermediate or visible and electron opaque final reaction products. Aldehyde-fixed tissues are cut into sections with an automatic sectioner, incubated cytochemically including osmication if required, then embedded in epoxy resin between fluorocarbon-coated coverglasses which are supported by a platform specially designed for this purpose. The resultant wafer, less than 0.2 mm thick, is examined by light microscopy for optimal areas of cytochemical reaction and desirable structural features. Such areas are cut out and glued to blank blocks with fast curing cyanoacrylate cement for subsequent ultrathin sectioning. The usefulness of this technique is demonstrated by the location of: (1) esterase-positive lysosomes in kidney and trigeminal ganglia; (2) palatal sensory endings stained for acetylcholinesterase; and (3) phagosomes arising from the resorption of horseradish peroxidase tracer by the cuboidal parietal epithelial cells of Bowmans capsule in the male mouse.

The demonstration of dehydrogenases and monoamine oxidase by the formation of osmium blacks at the sites of Hatchett's brown.

SummaryMethods are described for the direct cytochemical demonstration by light or electron microscopy of several mitochondrial dehydrogenase enzymes and monoamine oxidase using ferricyanide as an artificial electron acceptor. Hatchetts brown, cupric ferrocyanide, is the primary reaction product produced at the sites of mitochondrial oxidative enzymes in briefly fixed tissues by the histochemical incubation. After post-fixation, the cupric ferrocyanide is utilized as a catalyst to effect the oxidative polymerization of 3,3′-diaminobenzidine (DAB), resulting in an amplification of the original deposits. Upon osmication an osmium black end-product, which is ideal for light or electron microscopy, is produced at the tissue sites of the flavoprotein enzyme.Dimethylsulfoxide (DMSO) in concentrations of approximately 14%, was found to mediate electron transfer from cysteine as well as from many reduced carriers to artificial acceptors. In these reactions DMSO, because of its basic oxygen, is an excellent acceptor for hydrogen bonding and can substitute for water in solvation phenomena. Because of its low dielectric constant, it acts to facilitate electron transfer.With these new procedures for monoamine oxidase (MAO), lactic dehydrogenase (LDH), NADH dehydrogenase, and NADPH dehydrogenase, a heterogeneity of mitochondria with respect to oxidative enzymes was noted. In addition, differential sensitivities of the enzymes to fixatives was noted.There appeared to be an inverse relationship of the localization of LDH and MAO in certain areas of gray and white matter of medulla oblongata, cerebellum and sensory ganglia. This could be due to the heterogeneity of mitochondrial populations noted with respect to these enzymes.The strong LDH activity exclusively in mitochondria in different types of fixed cells and its absence from unfixed tissues confirms a mitochondrial localization for the enzyme. These findings suggest that more caution is needed in the interpretation of results from cellfractionation cytochemical and biochemical studies; moreover, results with non-disruptive direct visual histochemical and cytochemical methods may be valid, even if they do not conform to results from use of the former indirect methods.

Biochemical and histochemical evidence for the existence of a tuberoinfundibular cholinergic pathway in the rat

Although considerable biochemical, physiological and pharmacological data indicate that cholinergic pathways exist in the hypothalamus 9,1°,13, the exact location of these circuits has yet to be determined. Choline acetyltransferase (ChAc; EC 2.3.1.6), the enzyme responsible for the synthesis of acetylcholine (ACh), is believed to be present exclusively in cholinergic neurons 10. In fact, levels of this enzyme correlate well with the amount of ACh in different brain regions 1. The enzyme is present in several diencephalic nuclei and its highest activity in the hypothalamus is in the median eminence (ME) 1. It seems reasonable to assume that the ChAc in the ME is present in nerve terminals, since this region is devoid of neuronal perikarya 8 and also because ChAc has not yet been identified in glial cells la. Previous studies have demonstrated that after Halasz deafferentation (hypothalamic islands composed primarily of the isolated arcuate nucleus and the ME), ChAc activity in the ME is not significantly reduced 19. These data suggested that an intrinsic cholinergic ventrobasal pathway exists in the rat hypothalamus. We have utilized chemical lesioning and microdissection techniques coupled with microchemical and histochemical methods to demonstrate the existence of a tuberoinfundibular ACh pathway. The administration of high doses of monosodium L-glutamate (MSG) to neonatal rodents or primates results in a central nervous system lesion, largely restricted to the arcuate nucleus of the hypothalamus16,17. Since this lesion destroys 80-90 ° o of arcuate perikarya 17, but apparently spares axons in passage, examination of the arcuate nucleus and ME of MSG-treated rats provides a convenient means to examine the arcuate contribution to cholinergic terminals in the ME. Timed-pregnant Sprague-Dawley rats (Holtzman) were housed in rooms with controlled lighting (14 h light; 10 h dark) and fed laboratory chow and water ad libitum. Their offspring were injected intraperitoneally with MSG (4 mg/g BW) on alternate days during the first ten days of life as previously described 16. Control animals received

New observations on middle term hydroxyapatite-coated titanium alloy hip prostheses

HA-coated hip prostheses were retrieved from elderly patients after death. Histological analysis, scanning electron microscopy and microanalysis by energy-dispersive X-ray spectrometry were performed on the same sections. These revealed good osseointegration of the implant material and evolution of bone and material.

The light microscopic demonstration of hydroperoxidase-positive Phi bodies and rods in leukocytes in acute myeloid leukemia.

SummaryUnique fusiform or spindle-shaped particles (Phi bodies) and rods with hydroperoxidase (catalase and/or peroxidase) activity are present in human granulocyte precursors only in acute myelogenous leukemia (AML). These newly recognized particles are much more numerous and prominent than Auer rods. They may be rapidly and readily identified using the microscope in marrow or peripheral blood films when the procedures recommended in this paper for fixation, incubation for hydroperoxidase demonstration in 3,3′-diaminobenzidine (DAB)/H2O2 medium, copper salt treatment and counterstaining (optional) with the Papanicolaou method are employed. Films prepared in the same manner but treated with benzidine/H2O2 medium for myeloperoxidase did not reveal these particles.We believe that Phi bodies are pathognomonic of AML since they are almost invariably present in AML patients with active disease. Their presence serves to distinguish AML from acute lymphocytic leukemia and from chronic granulocytic leukemia in blast crisis. Since the particles disappear in disease remission and reappear upon relapse, the recommended procedure is not only useful in diagnosis but in guiding therapy.When a very rapid diagnosis is needed, it is not necessary to counterstain the preparations, but the nuclei, cytoplasm and plasmalemma can readily be observed in the granulocyte precursors when they are counterstained by the Papanicolaou method. This treatment does not diminish the clarity of the Phi bodies and rods which stain by virtue of their peroxidatic activity.This cytochemical diagnostic procedure should be considered for adoption by hematology laboratories.

New methods for the demonstration of lysosomal hydrolases by the formation of osmium blacks

SummaryMethods are described for the direct cytochemical demonstration of the enzymes nonspecific esterase and acid phosphatase based on synthetic substrates which initially deposit Hatchetts brown (cupric ferrocyanide, Cu2Fe(CN)6·7 H2O) at their subcellular sites. The small amounts of Hatchetts brown deposited as a result of the enzymes activity may be intensified by bridging to osmium through thiocarbohydrazide. Alternatively, even greater amplification of the sites of activity may be attained by utilizing the Hatchetts brown as a catalyst to effect the oxidative coupling of 3,3′-diaminobenzidine resulting in the formation of an osmiophilic indamine-type polymer.One of the major advantages of this new approach is that it permits the study of acid hydrolase localization without lead in the incubation medium. Studies were performed with these methods having identical incubation media except for synthetic substrate in many different cell types and tissues. They verify a frequent nonlysosomal localization for acid phosphatase and the heterogeneity of lysosomes and lysosomal populations with respect to hydrolase content.These methods give information obtained by direct cytochemical observation an advantage not previously held, in comparison with information from cell-fractionation cytochemical or biochemical studies. Initial studies with these methods on many tissues reinforce previous suggestions of the involvement of acid hydrolases in extralysosomal sites in subcellulur anabolic processes.

PERIPHERAL NEUROPATHY IN MOUSE HEREDITARY DIABETES MELLITUS. II. ULTRASTRUCTURAL CORRELATES OF DEGENERATIVE AND REGENERATIVE CHANGES

Carson K.A., Bossen E.H. & Hanker J.S. (1980) Neuropathology and Applied Neurobiology 6,361–374

Peripheral neuropathy in mouse hereditary diabetes mellitus. I. Comparison of neurologic, histologic, and morphometric parameters with dystonic mice.

SummaryC57BL/KsJdb/db inbred mice have an hereditary autosomal recessive disease resembling in some respects maturity onset human diabetes mellitus. At 8–11 months of age, they displayed intermittent symptoms suggestive of a mild sensory neuropathy. These symptoms consisted of adduction of their hind limbs and flexing hind paws when raised by the tail, and inability to maintain their position on the roto wheel. Peripheral nerves and sensory ganglia of the diabetic mice were compared with those of the unafflicted littermates and studied with respect to Schwann cell counts and myclinated nerve fiber diameter measurements. In addition, teased fibers of peripheral nerves were compared for obvious changes in internodal distance and demyelination. Chromatolytic neurons were more abundant in lumbosacral spinal ganglia of diabetic mice than in corresponding ganglia of controls or in more anterior spinal ganglia and trigeminal ganglia of diabetics. Histologic studies showed an increase in Schwann cell counts in longitudinal sections of peripheral nerves. A similar but larger increase was observed in peripheral nerves of mice affected with an hereditary sensory neuropathy, dystonia musculorum. A small but general decrease in myelinated fiber diameter was observed in sensory and motor nerves.