Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies.

Anionic sites in articular cartilage revealed by polyethyleneimine staining.

Articular cartilage is a unique tissue that contains neither blood vessels nor nerves, and that performs mechanical loading during joint movement. These properties are endowed by abundant glycosaminoglycans (GAGs), which are capable of retaining water-soluble substances. The GAGs attach to core proteins and form proteoglycans. Although many studies have focused on proteoglycans and collagen fibrils in cartilage, little is known about the nature of the negative charge of GAGs. Recently, we investigated this subject using a cationic dye, polyethyleneimine (PEI), with several different techniques such as pre-embedding, post-embedding, and quick-freezing and deep-etching methods. In addition, we investigated whether the anionic charge is altered at low pH, using PEI and cationic colloidal gold (CCG) labeling. The shapes of PEI-positive structures revealed by the pre-embedding method varied at different pHs. Three-dimensional analysis using the quick-freezing and deep-etching method demonstrated that meshwork structures composed of fine filaments were decorated with tiny PEI granules. Additionally, the meshwork structure was broken down after chondroitinase ABC digestion. These data indicate that the large PEI deposits observed in pre-embedding preparations are, at least in part, artificial images, and that the meshwork structure consists of chondroitin sulfate-retaining anionic sites. Low pH conditions changed PEI or CCG labeling patterns, showing that negative charges of GAGs in articular cartilage are altered under environmental pH conditions. These findings demonstrate that binding capacities of anionic sites to water-soluble or ionic substances are greatly affected by pH alterations without actually decreasing the number of anionic sites. Therefore, to understand cartilage dynamics and the pathogenesis of joint diseases in greater detail, alterations of anionic charge during mechanical loading or under pathological conditions should be examined in future studies.

A histochemical study of anionic sites in the intermediate layer of rat femoral cartilage using polyethyleneimine at different pH levels.

Anionic sites in the intermediate layer of young rat hyaline cartilages were examined using a cationic dye, polyethyleneimine (PEI), at different pH levels. Femoral heads were resected and fixed in 2.5% glutaraldehyde and treated with 0.5% PEI at pH 7.4, pH 2.5 or pH 1.0. Some cartilage samples were first digested with chondroitinase ABC or hyaluronidase. The PEI deposits at pH 7.4 appeared to be irregular shapes. Their sizes seemed to be larger than those at pH 2.5 or pH 1.0. The PEI deposits were also found on the surface of collagen fibrils at both pH 7.4 and pH 2.5 even after the chondroitinase ABC digestion, but were not found at pH 1.0. Moreover, they disappeared after hyaluronidase digestion. Accordingly, it is suggested that PEI-positive structures varied depending on pH levels. In addition, hyaluronan may be localized near collagen fibrils, but most sulphated proteoglycans may not.

Home lead-work as a potential source of lead exposure for children.

Health examinations for lead poisoning were made on 62 family members from 15 families of homes carrying on lead work, such as quench-hardening in a molten lead bath and type-printing, as work at home. The most interesting findings concern the occurrence of cases with an unduly high lead absorption among children, but not among adult family members other than home lead-workers. The home environments of the children with an unduly high lead absorption represented contamination with housedust high in lead contents. The ingestion of the contaminated housedust by hand-to-mouth is probably responsible for the excessive lead exposure of the affected children. The results of the present study suggest that contamination of housedust with lead due to home lead-work constitutes a possible hazardous source of lead exposure for children.

Proteoglycans in articular cartilage revealed with a quick freezing and deep etching method.

OBJECTIVES: To clarify the three dimensional ultrastructure of proteoglycans, and their relationship with other matrix components in articular cartilage. METHODS: Specimens from rat femoral heads were examined using three techniques: (1) Histochemical staining with cationic polyethyleneimine (PEI), using a pre-embedding or a postembedding method. Some tissues were pretreated with chondroitinase ABC or hyaluronidase. (2) Quick freezing and deep etching (QF-DE). Some specimens were fixed with paraformaldehyde and washed in buffer solution before quick freezing; others were frozen directly. (3) Ultrathin sections were studied after conventional preparation. RESULTS: Proteoglycans were observed as aggregated clumps with PEI staining by the pre-embedding method, but as fine filaments by the postembedding method. They were lost with enzyme digestion; this was also demonstrated by the QF-DE method. The ultrastructure was well preserved by the QF-DE method when fixation and washing procedures were included, but not without these procedures. A fine mesh-like structure was connected to the cell membrane in the pericellular matrix. Filamentous structures suggestive of aggrecans were observed among collagen fibrils. They had side chains, approximately 50 nm in length, which branched from the central filaments at intervals of 10-20 nm, and were occasionally linked to other structures. Many thin filaments were also attached to the collagen fibrils. CONCLUSIONS: The QF-DE method incorporating paraformaldehyde fixation and buffer washing procedures revealed three dimensional, extended structures suggestive of proteoglycans.