The latter were at least twice as long as wide. In order to determine the thickness of the collagen layer in the resorption lacunae, maximum resorption depths were measured before and after treatment with NaOCl and the difference between these PD0325901 nmr two depth measurements was calculated as an assessment of the thickness of the collagen

layer, as previously described [17]. Removal of organic matrix prior to seeding of OCs for resorption was performed in a similar fashion. Each bone slice was transferred into 500 μl 5–7% NaOCl and incubated at room temperature for 15 min while shaking in a thermomixer. Subsequently, the bone slices were washed individually in 50 ml sterile ddH2O while shaking for 30 min. The bone slices were stored for

up to a week in sterile ddH2O at 4 °C until use. Differentiated OCs were obtained as explained above. The cells were lyzed, RNA purified, cDNA generated and TaqMan Q-RT-PCR performed as described previously [24]. The following kits and primer/probes were used: Trizol Plus RNA Purification kit selleck chemical (RNA purification) (Invitrogen, Taastrup, Denmark), iScript kit (cDNA synthesis) (Bio-Rad, Copenhagen, Denmark), hGUS, Hs99999908_m1, hAbl, Hs00245443_m1, and hCatK Hs00166156_m1. All TaqMan primer⁄probe sets were inventoried and used according to the instructions by the supplier (Applied Biosystems, Naerum, Denmark). Each Q-RT-PCR run was normalized to the cDNA preparation of one single randomly chosen donor to enable comparisons between donors and between the different Q-RT-PCR runs. In addition to this, the expression levels of CatK were subsequently normalized to the average expression level of the house Bumetanide keeping genes hGUS and hAbl. All Q-RT-PCR reactions were run as triplicates. Excavations were generated by OCs in three different conditions: (i) the control condition; (ii) the presence of a low concentration of ethoxyzolamide to slightly decrease the demineralization rate; and (iii)

the presence of E64 to decrease the collagenolysis rate. The maximum resorption depths were measured both before and after removal of collagen left in the excavations, by using NaOCl (Figs. 2A and B). This procedure allowed us to monitor both the thickness of the layer of collagen left-over (Figs. 2C and D) and the depths to which bone was demineralized (Figs. 2A and B, hatched bars). NaOCl treatment of the excavations obtained under control conditions, made depths increasing from 5 to 9 μm (Fig. 2A), thus revealing a layer of collagen left-over of about 4 μm thickness (Fig. 2C), and a demineralization depth of 9 μm (Fig. 2A). This effect of NaOCl is in accordance with the repeated reports that demineralized collagen is left behind by the OC under control conditions, and means that the rate of collagen degradation is on average slower than the rate of demineralization in control conditions (Fig. 1, average control condition).