Hilda K. Lo

Cryopreservation of articular cartilage. Ultrastructural observations and long-term results of experimental distal femoral transplantation.

Ultrastructural changes associated with the freeze-preservation of human articular cartilage have been investigated and related to changes in transplanted distal femoral allografts in nonhuman primates. Human osteoarticular specimens were frozen at 2 degrees/minute in the presence of 15% glycerol and kept in liquid nitrogen freezers (vapor phase) from one day to two years. Ultrastructural changes were confined primarily to chondrocytes and were related to the freezing phenomenon, not to the time of storage. The cartilage matrix was affected little, explaining why articular cartilage initially survives clinical transplantation, but later undergoes degenerative changes. Osteoarticular allografts of baboons were frozen in an identical fashion to the human articular cartilage and transplanted into adult baboons. Long-term observations (five years) on these animals showed healing and replacement of the osseous portion of cryopreserved allografts. Fractures that appeared to coincide with maximum revascularization of the graft were the principal complication. Articular surfaces of the cryopreserved allografts underwent degenerative changes over five years. These degenerative changes were also manifested radiologically and appeared similar to those observed in humans. By contrast, fresh osteoarticular allografts healed poorly through fibrous union. However, in one of two fresh allografts, the articular cartilage remained intact five years after transplantation.

Hypothermic storage and cryopreservation of cartilage. An experimental study.

Osteochondral autografts of femoral condyles in dogs were frozen at different cooling velocities after exposure to either glycerol or dimethyl sulfoxide to determine the freezing regimen best suited for preservation of intact cartilage. Autografts were also subjected to hypothermic storage in tissue culture media for ascertainment of how long they can be stored under these conditions. Autografts maintained in vitro under various conditions of storage were examined after transplantation. Autografts were chosen for this portion of the investigation in order to study the effects of storage uncomplicated by immunologic interactions. No differences were found between the cryoprotective actions of glycerol and dimethyl sulfoxide. The freezing rate that produced the least damage in the cartilage was 2 degrees/min. Cartilage survived ten days of hypothermic storage in tissue culture medium. Allografts frozen by the standard method of glycerolization and cooling at 1 degree/min were also studied. These showed eventual loss of chondrocytes and conversion of hyaline cartilage to fibrocartilage.

A modified periodic acid-thiocarbohydrazide-silver proteinate staining sequence for enhanced contrast and resolution of glycogen depositions by transmission electron microscopy.

Oxidation of araldite-embedded liver sections by 1% w/v aqueous H5IO6 for 15 min and a 5-min reaction of carbonyls with 1% w/v thiocarbohydrazide in 10% v/v acetic acid was employed for subsequent staining of glycogen with silver-proteinate (S-P). The network of branching intracellular glycogen aggregates was revealed by 15-min staining with S-P, whereas 24 hr incubation in S-P was necessary to enhance the contrast of glycogen inclusions. We conclude that the proposed modification of glycogen staining readily affords the means for its localization at a desired level of contrast and resolution.

Turnover of phospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to long-wave UV light, psoralen, and UV light and psoralen

The turnover of 32P-labeled phospholipids in HUT 102 lymphoblasts was determined after a 2 h interaction of lymphoblasts with 8-methoxypsoralen (8-MOP) (15 micrograms ml-1), longwave UV light (UVA) irradiation and PUVA (8-MOP and UVA). In parallel experiments, micellar suspensions of lyso-phosphatidylcholine (PtdC), dipalmitoyl-PtdC and dilinoleoyl-PtdC, treated in a similar manner, served for the correlative assessments of cellular lipid changes. The dark reaction, UVA irradiation and PUVA all depressed total phospholipid levels in HUT 102 cells, although only PUVA induced a statistically significant decline. Thin layer chromatography (TLC) analysis revealed that neither UVA nor 8-MOP alone triggered any significant changes in the cellular content of phosphatidylinositol (PtdI), phosphatidylinositol 4-monophosphate (PtdIP) and phosphatidylinositol 4,5-bisphosphate (PtdIP2), whereas the lyso-PtdC and PtdI content of lymphoblasts showed a two-fold increase after PUVA. The TLC analysis of lyso-PtdC and micelles of dipalmitoyl-PtdC did not reveal any detectable changes after the dark reaction with 8-MOP, UVA irradiation and PUVA. In contrast, the derivatives of dark and UVA mediated reactions of 8-MOP with dilinoleoyl-PtdC were detected by TLC. These results suggest that the formation of 8-MOP derivatives of cellular phospholipids effected by PUVA, modulates the turnover of phosphoinositides and the rate of cellular proliferation.

Periodic acid-induced blastogenesis of lymphocytes is independent of phosphoinositide turnover.

The blastogenic transformation of lymphocytes by periodic acid was investigated to determine if blastogenesis induced by this mitogen was preceded by phosphoinositide turnover as previously shown for the lectins. Although periodate oxidation stimulated nucleic acid synthesis and interleukin-2 production, no changes in phosphoinositide turnover could be detected when compared to control lymphocyte cultures. These data indicate that increased phosphoinositide turnover is not an absolute prerequisite for lymphocyte proliferation.

TRANSMISSION LIGHT MICROSCOPY OF STRUCTURALLY COLORED SEMITHIN CARTILAGE SECTIONS

Abstract Conversion of osmified tracheal cartilage constituents into an array of laminar interference gratings has been attained by three tandem reactions. Oxidation of semithin, LR white-embedded cartilage sections by acetic anhydride in dimethyl sulfoxide is the first step in the conversion process. Subsequent addition reactions of oxidized cartilage pyranoses and furanoses with thiocarbohydrazide constitutes the second step. Reduction of silver proteinate by thiocarbohydrazones and the concomitant coating of cartilage constituents with silver gratings completes the conversion of cartilage sections into a system of layered interference filters. In transmitted light, all components of converted cartilage display vivid structural colors, which allow detailed microscopic analysis of structurally colored cellular and extracellular cartilage constituents.

Lead Tetraacetate-Thiocarbohydrazide-Silver Proteinate Method for Light Microscopy of Polysaccharides

Lead tetraacetate-thiocarbohydrazide-silver proteinate reaction sequence for light microscopy of polysaccharides was evaluated on Carnoys fixed rat liver sections. The results of this evaluation suggest that, on the light microscopic level, the lead tetraacetate-thiocarbohydrazide-silver proteinate method may serve as a practical and histochemically specific alternative to the lead tetraacetate-Schiff reaction for the localization of tissue carbohydrates.

Ultrastructural cytochemistry of hepatic lysosomes and their protein components is selectively revealed by the ninhydrin-dimethyl sulfoxide-thiocarbohydrazide-silver proteinate reaction

SummaryProteins in lysosomal membranes, lysosomes and within the transtubular network are readily accessible for electron microscopic analysis by a new three-step method. Oxidative deamination of tissue-bound amino acids by ninhydrin in a queous dimethyl sulfoxide and the concomitant formation of corresponding carbonyl groups comprise the first step. The addition reaction of thiocarbohydrazide to tissue-bound carbonyl groups comprises the second step, while the reduction of silver proteinate by tissue-bound thiocarbohydrazones is the final step of this sequential method. Glutaraldehyde-fixed and osmified ultrathin sections of rat liver embedded in LR White were oxidatively deaminated for 24 h by 1% w/v ninhydrin in aqueous 75% v/v dimethyl sulfoxide (DMSO). They were then incubated for 40 min in aqueous 1% w/v thiocarbohydrazide (TCH) and stained for 30 min at 50° C with silver proteinate (SP). The ninhydrin-dimethyl sulfoxide-thiocarbohydrazidesilver proteinate (N-DMSO-TCH-SP) reaction proved to be chemically specific and highly selective for ultrastructural resolution of the internal structure of lysosomes and their protein components. We conclude that the N-DMSO-TCH-SP reaction is the method of choice for cytochemical elucidation of the protein ultrastructure of lysosomes and their enzymatic aggregates.