Gerald J. Pinero

Citric and lactic acids as root canal irrigants in vitro

The efficacy of solutions of lactic acid, three concentrations of citric acid, sodium hypochlorite, and physiologic saline used as root canal irrigants was evaluated. With use of extracted, single-rooted human teeth, the amounts of hydroxyproline and calcium removed by these irrigants were measured. The treated teeth were then examined with a scanning electron microscope. It was concluded that a 10% solution of citric acid as a lubricant, followed by a 2.5% solution of sodium hypochlorite as an irrigant, and then again use of the citric acid solution, will produce clean canal walls with patent dentinal tubules.

Bone matrix proteins in osteogenesis and remodelling in the neonatal rat mandible as studied by immunolocalization of osteopontin, bone sialoprotein, α2HS-glycoprotein and alkaline phosphatase

The neonatal rat mandible was used as a model to study bone formation, mineralization, quiescence, and resorption, using immunolocalization and a variety of tissue-processing techniques. Monospecific antibodies for osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (AP) and alpha 2HS-glycoprotein (alpha 2HS-GP) were used on fixed paraffin-embedded tissue, fixed frozen tissue and unfixed frozen tissue. Immunostaining was correlated with mineral content by two procedures, the von Kossa and the morin techniques. Morin fluorescence was used with secondary immunostaining to provide a way of closely correlating bone matrix proteins and matrix mineralization. Co-immunolocalization procedures were used to compare the sites of bone proteins in the matrix. AP was found earliest during osteogenic cell differentiation, appearing in the preosteoblasts, followed by OPN and BSP, which first appeared in osteoblasts. alpha 2HS-GP expression was not observed in cells. The results provide clear evidence for the presence of OPN in osteoid, while BSP and alpha 2HS-GP were confined to the mineralized matrix. Immunostaining of bone proteins is highly technique-dependent: immunolocalization investigations required several methods of approach to ensure adequate demonstration of these proteins in cells and matrix. The results support the contention that osteopontin is multifunctional in bone metabolism, and that alpha 2HS-GP, though produced in the liver, is abundant in bone matrix and may also have a function in bone metabolism.

Molecular Analysis of Rat Dentin Sialoprotein

Dentin sialoprotein (DSP) is a noncollagenous protein originally isolated from rat dentin. Because it is made by odontoblasts that are actively synthesizing dentin. DSP may play an important role in dentinogenesis. We have isolated a full length DSP cDNA from a rat odontoblast/dental pulp cDNA library (Ritchie et al. [1994] J. Biol. Chem. 269:3698-3702) which codes for a 17 residue signal peptide and a 366 residue, 53 kDa mature protein. In situ hybridization revealed DSP mRNA expression by odontoblasts, but no other cells, in jaws from newborn rat. Northern analysis of various rat tissues demonstrated the presence of DSP transcripts in newborn tooth germs and 21 day old rat incisors. Moreover, multiple transcripts of 4.6 kb and 1.5 kb were found in these two tissues. To better understand the origin of these DSP mRNA multiple transcripts, we have isolated two rat genomic clones. Digestion of each clone with EcoRI followed by Southern analysis revealed that DSP cDNA hybridized to a 4 kb fragment in a lambda dash clone and to a 6 kb fragment in a cosmid clone. Since DSP cDNA hybridized to a 6 kb EcoRI fragment and a 4 kb EcoRI fragment obtained from a rat liver genomic cDNA digested with EcoRI, the multiple DSP mRNA transcripts are most likely derived from two related DSP genes which coexist in the rat genome.

An ultrastructural study of human epithelial rests of malassez maintained in a differentiated state in vitro

Healthy human periodontal ligaments (PDL), obtained from the extracted teeth (premolars and third molars), were cultivated for 1-35 days, using a multi-purposes culture chamber (MPCC) equipped with various transparent membranes. The resting state of the epithelial rests of Malassez (ERM), similar to their in vivo counterparts, appeared as small islands or strands with scant cytoplasm containing poorly developed organelles. This state was most effectively maintained in MPCC with a cellophane sheet. MPCC with a Sartorius membrane filter permitted proliferation and emigration of ERM. Proliferating ERM were characterized by more profiles of rough endoplasmic reticulum and free ribosomes, new formation of actin-containing microfilaments, less prominent tonofilaments and desmosomes and loss of gap junctions. Most of these ultrastructural changes are manifested in epithelial cells during wound healing. The emigrating ERM from PDL explants, as well as occasional proliferating ERM within explants, consisted of two cell types--outer basal-like cells, as described above, and inner tonofilament-rich prickle-like cells, suggesting a propensity for differentiation of ERM. The results show the possibility of controlling the growth and differentiation of ERM through the MPCC culture environment.

Expression and localization of PG-Lb/epiphycan during mouse development.

We have examined the expression pattern of the PG‐Lb/epiphycan gene that encodes a small leucine‐rich repeat proteoglycan during mouse embryonic development. PG‐Lb/epiphycan mRNA transcripts were first detected at E12.5 days postcoitus (dpc) at high levels in structures that were developing cartilage elements. The gene is expressed in a very specific temporal and spatial fashion in cartilaginous structures. To examine PG‐Lb/epiphycan gene expression during cartilage development in more detail, we performed in situ hybridization on hindlimb sections at specific stages of mouse embryonic development. The expression of PG‐Lb/epiphycan was compared to that of collagen type II and collagen type X, which are early and late markers for cartilage development, respectively. The expression of PG‐Lb/epiphycan occurs later than collagen type II in cartilage development, but its expression appears in the growth plate before and is excluded from the zone of hypertrophic chondrocytic cells expressing collagen type X. An antibody against PG‐Lb/epiphycan localized the protein within the entire growth plate of the E17.5 dpc embryonic hindlimb cartilage including the hypertrophic zone where PG‐Lb/epiphycan gene expression is turned off. Our results show that PG‐Lb/epiphycan gene expression is an intermediate marker for chondrogenesis, and that the protein can be localized to the extracellular matrix surrounding resting, proliferating, and hypertrophic chondrocytes by immunofluorescence histochemistry. Dev Dyn 1999;216:499–510. ©1999 Wiley‐Liss, Inc.

Improved Immunohistochemical Staining of Osteopontin (OPN) in Paraffin-Embedded Archival Bone Specimens Following Antigen Retrieval: Anti-Human OPN Antibody Recognizes Multiple Molecular Forms

Abstract. Studies to assess osteopontin (OPN) localization in adult human bone using immunochemical techniques produce conflicting results due to variations in tissue processing or antibody immunoreactivity. The present study was designed to resolve these discrepancies using well-characterized antibodies and improved antigen detection. An anti-osteopontin (α-OPN) antiserum was developed that recognizes various soluble molecular weight forms of human OPN, including monomeric, cleaved, and dimerized products. An affinity column of full length recombinant human OPN (rOPN) coupled to support was used to purify α-OPN antibodies. Western analysis showed that the affinity-purified antibodies recognized numerous molecular weight forms of OPN. These antibodies were used to study the distribution of OPN in adult human bone using immunohistochemical techniques combined with an antigen retrieval protocol utilizing a newly developed antigen retrieval solution, Retriev-All™ (Bronco Technologies Inc, Pasadena, TX). Immunolocalization of OPN in archival bone specimens prior to antigen retrieval produced no demonstrable immunostaining even at high concentrations of α-OPN. Use of the antigen retrieval protocol restored OPN immunoreactivity, with strong staining apparent in cement lines, osteoblasts, osteocytes, canaliculi, osteoid, and bone matrix. We conclude that antigen retrieval restores immunochemical recognition of OPN in archival specimens containing bone without increasing nonspecific binding.

The effect of endotoxin on the synthesis of connective tissue matrix components by pulp fibroblasts in vitro

Human and bovine pulp fibroblasts were treated with low levels of endotoxin and assayed for the utilization of various isotopes to measure synthesis of DNA, collagen, and sulfated and nonsulfated glycosaminoglycans. Endotoxin at 5 to 125 μg/ml stimulated the uptake of 3 H-thymidine by both cell lines. Utilization of the other isotopes also increased but varied with the cell lines and endotoxin concentrations. The results suggest that pulp fibroblasts are able to respond to low levels of endotoxin by increased cell division and synthesis of connective tissue matrix.

Continuously infused calcium hydroxide: Its influence on hard tissue repair*

To study the ability of calcium hydroxide to promote hard tissue repair, Alza Alzet Osmotic Pumps, implanted in Sprague-Dawley rats, were used to deliver either calcium hydroxide and glycerol, barium hydroxide and glycerol, tetracycline and glycerol, or glycerol only to a standardized round bur defect in a rat femur. The pumps infused one of the reagents into the defects continuously over a 4-wk experimental period. The effects of each reagent on the healing of the bony defects were compared by histological evaluation. The Alza Osmotic Pump proved to be an effective method to deliver an agent to an experimental site. Our preliminary findings from a limited sample size indicated that calcium hydroxide contributed to a more complete osseous repair than either barium hydroxide or tetracycline. Barium hydroxide with a sustained pH equivalent to calcium hydroxide showed no greater healing than the controls. Tetracycline results were also similar to controls.

Clinical Science Scanning Electron Microscopic and Radiographic Correlation of Articular Surface and Supporting Bone of the Mandibular Condyle

Specimens of mandibular condyle from human cadavers were employed for scanning electron microscopic and radiographic observations of the articulating surface and underlying bone. Smooth articular surface supported by smooth bone was most frequently observed. Craters and depressions in the articular surface were associated with resorption of underlying bone. Lateral radiographs proved to be of limited value in detecting defects of this type.Specimens of mandibular condyle from human cadavers were employed for scanning electron microscopic and radiographic observations of the articulating surface and underlying bone. Smooth articular surface supported by smooth bone was most frequently observed. Craters and depressions in the articular surface were associated with resorption of underlying bone. Lateral radiographs proved to be of limited value in detecting defects of this type.

Human Gingival Fibroblast Production of Interferon

Three human gingival fibroblast cell lines were used to determine whether they could be induced by a synthetic RNA and super-induced by metabolic inhibitors to produce interferon (IFN-β). When established procedures were followed for human fetal or newborn skin fibroblast cell lines, the adult gingival fibroblasts produced comparable amounts of IFN-β. It was shown that the superinducers alone would not cause an IFN-β production response, and that the absence of serum in the production medium also inhibited the production of IFN-β. The effect of IFN-β on cell growth was carried out in T-flasks seeded with 105 HEp-2 cells. After one and two wk, the cells of triplicate control flasks and triplicate flasks containing various dilutions of the production media were enumerated to determine a cell multiplication inhibition (CMI) value. A correlation between the IFN-β content and the CMI effect, however, was not obtained, and it was concluded that other CMI agents, possibly more potent than the IFN-β, were being produced by the stimulated human gingival fibroblasts. Cell protein assays which gave a high ng/ cell protein content correlated with TEM micrographs which showed clusters of complex lysosomes, primarily in cells cultured in the IFN-β-containing nutrient. However, since commercial IFN-β initiated no such lysosomal response, it was further concluded that the complex lysosomes were due to CMI agent(s) other than IFN-β.