An effective reactivation of alkaline phosphatase in hard tissues completely decalcified for light and electron microscopy

Yoshiki, Shusaku; Umeda, Tetsuya; Kurahashi, Y.

doi:10.1007/bf00279812

Cited by 62 publications

(17 citation statements)

References 11 publications

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“…The Ln2-LG3-P2-DN3 sequence within the LG3 domain was located in the connecting loop of the K and L strands of the human laminin α2 chain [17]. This is partially consistent with a previous report [31] showing that alignment of LG sequences was localized to the GD-6 peptide (KQNCLSSRASFRGCVRNLRLSR, residues 3011-3032) from the C-terminal LG domain of the murine laminin α1 chain to a region between the K-L and L-M loops of the mouse laminin α1 chain, which we found to be in the connecting loop between the K and L strands of the human laminin α2 chain. However, Ln2-LG3-P2-DN3 itself, a sequence of only nine amino acids, was interpreted to have no specific conformational structure from the computational structure prediction algorithm used in this study.…”

Section: Discussionsupporting

confidence: 92%

“…New bone or osteoid is known to stain red using this method [30]. Next, ALP histochemistry was performed as described previously [31]. Briefly, the sections were stained in nitroblue tetrazolium chloride/5bromo-4-choloro-3-indolyl phosphate toluidine salt solution at 37 ºC for 24 h. The slides were counterstained with methyl green for 1 min and rinsed and examined at 200 magnification using the light microscope.…”

Section: Histological Assessmentmentioning

confidence: 99%

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Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering

et al. 2015

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Finding bioactive short peptides derived from proteins is a critical step to the advancement of tissue engineering and regenerative medicine, because the former maintains the functions of the latter without immunogenicity in biological systems. Here, we discovered a bioactive core nonapeptide sequence, PPFEGCIWN (residues 2678-2686; Ln2-LG3-P2-DN3), from the human laminin α2 chain, and investigated the role of this peptide in binding to transmembrane proteins to promote intracellular events leading to cell functions. This minimum bioactive sequence had neither secondary nor tertiary structures in a computational structure prediction. Nonetheless, Ln2-LG3-P2-DN3 bound to various cell types as actively as laminin in cell adhesion assays. The in vivo healing tests using rats revealed that Ln2-LG3-P2-DN3 promoted bone formation without any recognizable antigenic activity. Ln2-LG3-P2-DN3-treated titanium (Ti) discs and Ti implant surfaces caused the enhancement of bone cell functions in vitro and induced faster osseointegration in vivo, respectively. These findings established a minimum bioactive sequence within human laminin, and its potential application value for regenerative medicine, especially for bone tissue engineering.

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Section: Discussionsupporting

confidence: 92%

Section: Histological Assessmentmentioning

confidence: 99%

Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering

et al. 2015

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“…From the bone samples of day 15, a segmental unit of callus (2 × 2 mm callus area) was harvested and fixed at +4 °C in formal calcium for 24 h, and then kept in Holt's solution for additional 24 h. From the tissue samples, 12 µm thick frozen sections were taken using the cryostat (Slee Corporation, London/ UK). Enzyme histochemical reaction was ascertained on the sections by a modified simultaneous azo-coupling method (Yoshiki et al 1972). The sections were counterstained with methyl green nuclear stain and mounted with Kaiser's glycerol jelly.…”

Section: Histologymentioning

confidence: 99%

“…The remaining tissue samples of day 15 and the samples of days 21 and 30 were fixed in the periodate-lysineparaformaldehyde (PLP) fixative (McLean and Nakane 1974), decalcified in an EDTA-G solution (M o r i et al 1988) and embedded in paraffin for histochemical demonstration of ALP (Yoshiki et al 1972), and trichrome stain (Bancroft 1992). All specimens were examined under the Nikon Eclipse E-400 light microscope (Nikon Corporation, Japan).…”

Section: Histologymentioning

confidence: 99%

Effects of zinc and aprotinin on the healing of ulnar diaphyseal fractures in rabbits

2018

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The effects of zinc and aprotinin on fracture healing in experimentally induced fractures were investigated by means of histometric analyses and alkaline phosphatase histochemistry. Healthy 54 adult New Zealand White female rabbits were separated into three groups as control, zinc, and aprotinin treatment. The control animals did not receive any medicament; zinc sulphate was given orally to the rabbits in the Zn group for 15 days. Aprotinin was postoperatively infiltrated into the fracture area at the 3rd and 24th h following operation. Immobilization of fracture ends of all groups was similar throughout the experiment. The zinc administered group displayed the highest alkaline phosphatase positive cell level through the experiment. By day 30 after the operation, fibrocartilage and osseous tissues reached the highest levels in the zinc treated group. Based on the observation of augmented osseous tissue formation and increased alkaline phosphatase positive osteoblastic cell activity in the callus, it was conluded that Zn sulphate is a potent stimulator of bone formation by increasing mineralization in the fractured bone segments.

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“…New bone or osteoid is known to be stained red using this method [26]. Next, ALP histochemistry was performed as previously described [27]. Briefly, the sections were stained in nitroblue tetrazolium choloride/5-bromo-4-choloro-3-indolyl phosphate toluidine (NBT/BCIP) salt solution at 37 o C for 24 h. The slides were counterstained with methyl green for min and rinsed and examined at 200 magnification using the light microscope.…”

Section: Histological Assessmentmentioning

confidence: 99%

The effect of the DLTIDDSYWYRI motif of the human laminin α2 chain on implant osseointegration

Kang¹,

Kim²,

Min³

et al. 2013

Biomaterials

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Considerable effort has been directed towards replacing lost teeth using tissue-engineering methods such as titanium implants. A number of studies have tried to modify bioinert titanium surfaces by coating them with functionally bioactive molecules for faster and stronger osseointegration than pure titanium surfaces. Recently, peptides have been recognized as valuable scientific tools in the field of tissue-engineering. The DLTIDDSYWYRI motif of the human laminin-2 α2 chain has been previously reported to promote the attachment of various cell types; however, the in vivo effects of the DLTIDDSYWYRI motif on new bone formation have not yet been studied. To examine whether a laminin-2-derived peptide can promote osseointegration by accelerating new bone formation in vivo, we applied titanium implants coated with the DLTIDDSYWYRI motif in a rabbit tibia model. The application of the DLTIDDSYWYRI motif-treated implant to tibia wounds enhanced collagen deposition and alkaline phosphatase expression. It significantly promoted implant osseointegration compared with treatment with scrambled peptide-treated implants by increasing the bone-to-implant contact ratio and bone area. These findings support the hypothesis that the DLTIDDSYWYRI motif acts as an effective osseointegration accelerator by enhancing new bone formation.

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An effective reactivation of alkaline phosphatase in hard tissues completely decalcified for light and electron microscopy

Cited by 62 publications

References 11 publications

Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering

Identification of a bioactive core sequence from human laminin and its applicability to tissue engineering

Effects of zinc and aprotinin on the healing of ulnar diaphyseal fractures in rabbits

The effect of the DLTIDDSYWYRI motif of the human laminin α2 chain on implant osseointegration

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