Shinichiro Oida

Response of the Mouse Femoral Muscle to an Implant of a Composite of Bone Morphogenetic Protein and Plaster of Paris

A composite consisting of 1 mg of bone morphogenetic protein (BMP) and 5 mg of plaster of Paris (PLP) was implanted into the mouse femoral muscle. Control PLP without BMP was implanted into the contralateral muscle. The BMP/PLP composite induced cartilage formation within two weeks, trabecular bone within three weeks, and lamellar bone including bone marrow within six weeks. PLP did not disturb BMP-induced bone formation. In addition, the BMP/PLP composite was resorbed early after implantation. These results suggest that PLP composite system is one of the most suitable BMP delivery systems currently available.

Expression of prostaglandin E receptor subtypes in bone : expression of EP2 in bone development

Prostaglandin (PG) E2 displays physiological and pharmacological action in various tissues including bone. It increases intracellular Ca, and stimulates or inhibits cAMP production through the PGE receptor subtypes EP1, EP2, and EP3, respectively. These receptor subtypes have been recently cloned. In the present study, we investigate the expression of these receptor subtypes in bone tissue. RT-PCR revealed that EP1, EP2, and EP3 were expressed in rat calvariae and that osteoblastic cells (MC3T3-E1) expressed EP1 and EP2. In situ hybridization analysis using cryosection of neonatal calvariae revealed that EP2 was expressed by osteoblasts and cells not in contact with bone, probably including preosteoblasts. EP2 expression was observed at an early stage in calvarial development, at 14 days prenatal. EP2 expression was also observed at day 3 in rat bone marrow cell culture in which bone-like mineralized nodules are formed at day 8. It has been established that PGE2 response accompanying cAMP production is one of the characteristics of osteoblasts. The present results indicate that this phenotype appears at an early stage of osteoblastic differentiation and bone development.

Expression of bone morphogenetic protein genes in the human dental pulp cells

Dental pulp has a potential to induce ectopic bone formation, but little is known about its mechanism. We thought that bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-beta) superfamily, are involved in the osteoinductive activity of dental pulp. In order to prove this assumption, we constructed a cDNA library from primary culture cells of human dental pulp (HDP cells), and screened the library with previously cloned cDNAs for mouse BMP-2 and -6 as probes. Three distinct cDNA clones encoding human BMP-2, -4 and -6 were isolated. By Northern blot analysis, specific transcripts of the genes of those BMPs were detected in the HDP cells. It was concluded that the BMPs were expressed in a certain population of dental pulp cells and might play some roles in ectopic bone formation by dental pulp.

Properties of Alkaline Phosphatase of the Human Dental Pulp

Enzymatic and immunological properties of alkaline phosphatase [ALP; orthophosphoric monoester phosphohydrolase, alkaline optimum, EC 3.1.3.1.] in the human dental pulp were investigated. In inhibition and thermal inactivation studies, dental pulp ALP showed properties of universal-type ALP (kidney/bone/liver type). Dental pulp ALP cross-reacted with polyclonal and monoclonal antibodies against purified swine-kidney ALP, and with monoclonal antibody against ALP of human osteoblast-like cells in the same manner as ALPs of human bone and kidney. The sodium dodecyl sulfate-gel electrophoretic pattern showed a 140, 000-Mr native protein band. These data suggest that dental pulp ALP can be classified as a universal-type ALP having antigenic determinants common to ALP of the kidney and bone.

Ectopic induction of cartilage and bone by bovine bone morphogenetic protein using a biodegradable polymeric reservoir

Two kinds of bone morphogenetic protein (BMP) containing polymeric reservoirs made from poly(lactide-co-glycolide) (PLG) and a mixture of PLG/poly(ethylene glycol) (PEG) (PLG/PEG) were implanted into mice femoral muscles. In the PLG/PEG reservoir with BMP, bone formation was induced within 3 weeks. On the other hand, in the PLG reservoir with BMP scarcely any bone formation could be observed. The results suggest that the carrier system is most important for the development of BMP-induced bone formation and the PLG/PEG reservoir system is available for BMP delivery.

CDNA CLONING OF S100 CALCIUM-BINDING PROTEINS FROM BOVINE PERIODONTAL LIGAMENT AND THEIR EXPRESSION IN ORAL TISSUES

The periodontal ligament (PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. To characterize PDL cells at the molecular level, we constructed a cDNA library from bovine PDL tissue. We then focused on the isolation of S100 calcium-binding proteins (CaBPs), because they mediate Ca2+ signaling and control important cellular processes such as differentiation and metabolism. We screened the PDL cDNA library with a mouse S100A4 cDNA, and cloned the bovine cDNAs of two S100 CaBPs (S100A4 and S100A2). In Northern blotting analysis, the highest expression of S100A4 was detected in PDL from erupted teeth (PDLE). PDL from teeth under eruption (PDLU) showed a lower expression of S100A4, and its expression in gingiva was faintly detectable. S100A4 expression was also high in the pulp tissue followed by the dental papilla of the tooth germ. S100A2 expression was high in PDLE and gingiva. Interestingly, only PDLE exhibited a high expression of both S100A4 and S100A2. PDLE also expressed the highest level of β-actin, a target cytoskeletal protein for S100A4. It is conceivable that the high expression of S100A4 in PDLE is a result of the maturation of the PDL and/or a response to mechanical stress generated by mastication. Since there was a marked difference of S100A4 expression between PDL and gingiva, we propose that S100A4 could be a useful marker for distinguishing cells from these two tissues.

Purification of swine kidney alkaline phosphatase by immunoaffinity chromatography

A homogeneous alkaline phosphatase preparation was obtained from swine kidney cortex by a simple purification step of immunoaffinity chromatography. The enzyme was purified 426 times that of the initial acetone powder with a recovery of 69.6% and a specific activity of 1206 units/mg of protein. The sodium dodecyl sulfate-gel electrophoretic pattern showed a single 80,000-Mr protein band as the monomer of the purified enzyme.

Purification and subunit structure of alkaline phosphatase from bovine enamel organ.

A highly-purified alkaline-phosphatase preparation having a specific activity of 703 U/mg protein was obtained from bovine enamel organ by a series of procedures: butanol extraction, isoelectric and acetone precipitation, ion-exchange, concanavalin A affinity and gel-filtration chromatography. The purified enzyme showed the same properties as kidney-type isozyme and contained carbohydrate moieties which react with concanavalin A. Analysis by polyacrylamide gel electrophoresis showed that the purified enzyme split into half the size of the native molecule (160,000 in mol. wt) after being heated in sodium dodecyl sulphate (SDS) solution and the subunit had no catalytic activity. The results indicate that the enzyme is a dimeric glycoprotein comprised of two identical subunits, each having a molecular weight of 80,000.

EXPRESSION OF MRNA ENCODING TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE IN HUMAN DENTAL TISSUES

Abstract. Tissue-nonspecific-type alkaline phosphatase (TNSALP) is found in the bone, liver, kidney, and other tissues, and its gene consists of 12 exons with the coding sequence beginning in the second exon. Recently, a noncoding first exon was identified in the liver message (liver type) which differed from that of the previously known osteoblast-derived cDNA sequence (bone type). Although these two mRNAs produce an identical protein, they have different promoter regions. It is known that ALPs in dental pulp and periodontal ligament are classified into TNSALP by their enzymatic and immunological properties, but little is known about the expression of ALP mRNAs and the transcriptional mechanisms. In order to examine the expression of their mRNA type, specific oligonucleotide primers corresponding to two types of mRNAs of human TNSALP were designed and amplified by reverse transcription-polymerase chain reaction (RT-PCR). It was found that bone-type mRNA was expressed in the human dental tissues such as dental pulp, periodontal ligament, and dental sac, whereas liver-type mRNA was not expressed. Thus, it was concluded that the human dental tissues express the bone-type isozymes and are regulated by the same transcriptional mechanism as in the bone.

Determination of Bovine Enamel Protein by Enzyme-Linked Immuno-Adsorbent Assay

A specific enzyme-linked immuno-adsorbent assay for enamel protein of the developing tooth was devised, using antibody against antigen prepared from immature bovine enamel. Samples containing 5-500 ng of amelogenin protein in 50 microliters gave consistent results. Bovine enamelin cross-reacted with the antibody but was less reactive than amelogenin. Proteins in other tissues or fluids did not react to the antibody.