Pengaruh Dimensi Benda Uji Terhadap Kuat Tekan Beton

Kuat tekan adalah karakteristik mekanik utama dari beton yang dapat diketahui melalui penelitian uji tekan di laboratorium terhadap benda uji. Baik dalam bentuk kubus ataupun silinder dengan ukuran standar: 10cm x 10cm x 10cm dan 15cm x 15cm untuk kubus dan 10cm x 20cm dan 15cm x 30cm untuk silinder. Untuk mendapatkan informasi mengenai kecendrungan harga kuat tekan beton dengan variasi dimensi benda uji, telah dilakukan penelitian-penelitian di laboratoriun untuk mendapatkan komposisi campuran tertentu pada umur beton 28 hari, variasi ukuran benda uji dibuat: 10cm x 10cm x 10cm, 12,5cm x 12,5cm x 12,5cm dan 15cm x 15cm x 15cm untuk kubus dan 10cm x 20cm, 12,5cm x 25cm dan 15cm x 30cm untuk silinder. Dengan jumlah benda uji masing-masing 20 buah untuk setiap ukuran benda uji. Melalui prosedur standar pengujian kuat tekan dan menggunakan formula-formula baku perhitungan tekan rata-rata diperoleh informasi bahwa peningkatan ukuran dimensi benda uji menghasilkan penurunan kuat tekan rata-rata, untuk benda uji kubus dengan ukuran masing-masing: 10cm x 10cm x 10cm, 12,5cm x 12,5cm x 12,5cm dan 15cm x 15cm x 15cm diperoleh kuat tekan rata-rata masing-masing: 32,86MPa, 31,26MPa dan 31,036MPa. Sedangkan untuk silinder dengan kururan 10cm x 20cm, 12,5cm x 25cm dan 15cm x 30cm diperoleh kuat tekan rata-rata masing-masing: 31,47MPa, 30,85MPa dan 30,44MPa

The PP-delivered peptide list.

<p>Closed RGD, closed 7-RGD-7, closed 5-RGD-5, closed 3-RGD-3, and 1-RGD-1 contained 9, 7, 5, 3, and 1 juxta-amino acids at the amino- and carboxyl-terminal sides of RGD, respectively. Closed 2-RGD-2 had 2 amino acids at both sides of RGD. Closed 2-RGD-3 had two and three, and closed 3-RGD-2 had three and two amino acids at the amino- and carboxyl-terminal sides of RGD. Close RGE was the RGD-inactivated mutant of closed RGD. C-opened RGD included the RGD domain with an open carboxyl-terminal side, and C-opened RGE was the RGD-inactivated mutant of C-opened RGD. N-opened RGD included the RGD domain with an open amino-terminal side and N-opened RGE was the RGD-inactivated mutant of N-opened RGD. SSD repeats had representative SSD repeats. DSP was the well-conserved glycosaminoglycan attachment site in the dentin sialoprotein (DSP), which was the amino-terminal cleaved product of DSPP.</p><p>The PP-delivered peptide list.</p

Replacement of the 1^st and/or 2^nd carboxyl-terminal amino acids of the PP-RGD domain altered its ability.

<p>Ninety-six-well plates were precoated with 250 nM or 1 µM of normal rPP-ΔSSD and various mutated rPP-ΔSSD proteins, seeded with MG63 cells in serum-free medium, and incubated for 1 hr. The number of attached cells was evaluated as described above. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. *p<0.05, **p<0.01, and ***p<0.001 indicate significantly higher than rPP-ΔSSD-coated wells at the same concentration.</p

Six other mesenchymal cell lines were unable to attach to rPP.

<p>Three hDPC (human dental pulp cells) cells from different donors, Saos2 (human osteosarcoma cell line), parental MC3T3-E1 (heterogeneous cell population), and C2C12 (mouse myoblast cell line) were seeded onto rPP, rC-DMP-1, and vitronectin (100 nM) with MnCl₂ (1 mM). The number of attached cells was evaluated as described before. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 indicates significantly lower and ⁺⁺p<0.01 and ⁺⁺⁺p<0.001 indicate significantly higher than rC-DMP-1-coated wells.</p

Nominal effects of rPP on MG63 and MC3T3-E1 cell adhesion.

<p>Ninety-six-well plates were precoated with 20 or 100 nM rPP, rC-DMP-1, and vitronectin, seeded with MG63 (A) and MC3T3-E1 (B) cells in serum-free medium, and incubated for 1 hr. MG63 (C) and MC3T3-E1 (D) cells were seeded onto 100 nM rPP, rC-DMP-1, and vitronectin with either MnCl₂, CaCl₂, or MgCl₂ (1 mM) in serum-free medium, and incubated for 1 hr. After washing non-adherent cells, the attached cells were stained with 0.2% crystal violet and dissolved in 1% SDS solution. Absorbance was measured at 570 nm. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 indicates significantly lower and ⁺p<0.05 and ⁺⁺⁺p<0.001 indicate significantly higher than rC-DMP-1-coated wells at the same concentration (A and B) or rC-DMP-1-coated wells with the same divalent cation (C and D).</p

Truncated rPP terminating with Ala⁴⁸² (PP-(Ala terminal)) induced cell adhesion and migration.

<p>(A) The fluorescence intensity (FI) of Alexa-Fluor 488 conjugated rPP-ΔSSD and rPP-(Ala terminal). One hundred microliters of serially-diluted Alexa-Fluor 488 conjugated rPP-ΔSSD (Alexa-rPP-ΔSSD) and rPP-(Ala terminal) (Alexa-rPP-(Ala terminal)) were added onto black 96-well plates and FI was measured on a fluorescence microplate reader. Their FI values were equivalent. (B) Flow cytometry analysis of Alexa-rPP-ΔSSD and Alexa-rPP-(Ala terminal) binding to MG63 cells. MG63 cells were incubated with Alexa-rPP-ΔSSD and Alexa-rPP-(Ala terminal) (250 nM). rPP-(Ala terminal) binding to MG63 cells was apparently higher than that of rPP-ΔSSD and control cells, which were incubated with medium only. (C) MG63 and MC3T3-E1 cells were seeded onto rPP, rPP-(Ala terminal), rPP-RGE-(Ala terminal), and rC-DMP-1 (100 nM) with MnCl₂ (1 mM) in serum-free medium. The number of attached cells was evaluated as described above. Each value represents the mean of triplicate determinations; bars mean ±SD. (D) Organization of actin stress fibers on rPP, rPP-(Ala terminal), rPP-RGE-(Ala terminal), and rC-DMP-1. MG63 cells were seeded onto glass plates precoated with rPP (box 1), rPP-(Ala terminal) (2), rPP-RGE-(Ala terminal) (3), rC-DMP-1 (4), or none (5) (100 nM) in serum-free medium. Actin fibers were visualized by FITC-phalloidin 1 hr after seeding. Bar  = 10 µm. (E) rPP-(Ala terminal) was able to induce haptotaxis cell migration. Haptotaxis cell migration was measured in MG63 cells toward rPP, rPP-(Ala terminal), rPP-ΔSSD, and rC-DMP-1 in serum-free medium. After the 12-hr incubation, cells were fixed and stained. Migration was quantified by counting the number of cells that migrated through filters. Each value represents the mean of 10 randomly selected fields at 200× magnification; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p<0.001 significantly lower than PP-(A terminal)-coated wells.</p

The inability of rPP, rPP-ΔSSD, and rPP-RGE to bind to integrin αvβ3 or αvβ5.

<p>(A) MG63 cells were preincubated with the neutralizing antibodies against integrin αvβ3 (LM609), αvβ5 (P1F6), and β1 (4B7) and control IgG (10 µg/ml) for 15 min, and were then seeded onto rC-DMP-1 and vitronectin (100 nM) in serum-free medium. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. **p<0.01 and ***p<0.001 indicate significantly lower than the cells preincubated with control IgG. (B) The binding of integrin αvβ3 and αvβ5 to rPP, rPP-ΔSSD, rPP-RGE, rC-DMP-1, and vitronectin was analyzed using solid phase binding assays. Various amounts (0∼400 ng) of integrin αvβ3 and αvβ5 were added to 96-well plates precoated with either 100 nM of PP, PP-ΔSSD, PP-RGE, rC-DMP-1, or vitronectin. Bound integrin αvβ3 and αvβ5 were then detected with the anti-integrin αv antibody, an appropriate HRP-conjugated secondary antibody, and TMB. Each value represents the mean of triplicate determinations; bars mean ±SD.</p

Ala⁴⁸²-Ser⁴⁸³ was the key peptide bond that allowed the PP-RGD domain to become inactive.

<p>(A) MG63 cells were preincubated with closed RGD, closed 7-RGD-7, closed 5-RGD-5, closed 3-RGD-3, and 1-RGD-1 peptides (1 mM) for 10 min, and were then seeded onto vitronectin (100 nM) in serum-free medium. (B) The minimum amino acid sequences needed to sequester the ability of PP-RGD were narrowed down based on the results of (A). MG63 cells were preincubated with closed RGD, closed 3-RGD-3, 1-RGD-1, closed 2-RGD-2, closed 2-RGD-3, and closed 3-RGD-2 peptides (1 mM). (C) The various PP peptides exhibited different inhibitory activities on cell adhesion to vitronectin. MG63 cells were preincubated with the various PP peptides: closed RGD, closed RGE, C-opened RGD, C-opened RGE, N-opened RGD, N-opened RGE, SSD repeats, or DSP (1 mM) as described, and were then seeded onto vitronectin. C-opened RGD significantly inhibited the adhesion of these cells to vitronectin. The number of attached cells was evaluated as described above. Cell attachment in the presence of closed RGD was assigned a value of 100%. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Tukey's test. *p<0.05 and ***p<0.001 significantly different from the cells preincubated with any other peptide. (D) The various PP peptides showed different binding capacities to integrin αvβ3. The biotinylated GRGDS peptide (5 nM) was added to integrin αvβ3-coated wells with various PP peptides or with serially-diluted cyclic-(GRGDP). The bound biotinylated GRGDS peptide was valued using a streptavidin-HRP conjugated polymer. Dose-dependent decreases in biotinylated GRGDS binding to integrin αvβ3 were regressed to a sigmoid curve. The inhibitory values of the various PP peptides were determined by converting their absorbance to the inhibitory value of cyclic-(GRGDSP). Each value represents the mean of triplicate determinations; bars, mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. **p<0.01 and ***p<0.001 indicate significantly lower than the competitive addition of closed RGD.</p

Gene and protein structures of mouse DSPP and DMP-1.

<p>(A) SIBLING location. SIBLING proteins were located next to each other on human chromosome 4 and mouse chromosome 5. (B) The amino acids sequences of PP (the carboxyl-terminal region of DSPP) and C-DMP1 (the carboxyl-terminal region of DMP-1). The SSD repeats are colored green and the RGD domains are colored red. The amino acids are numbered from the amino-terminal of DSPP and DMP-1 after signal peptide removal. (C) The exon-intron and protein structures of mouse DSPP and DMP-1. Two glycosaminoglycans (GAG) sites have been reported in mouse DSPP (serine242 and serine254) and one GAG site in DMP1 (serine 89). SSD repeats; Serine–serine–aspartic acid repeat sequence. RGD; Arg-Gly-Asp integrin-binding site.</p

The removal of SSD repeats had no apparent effect on the adhesive ability of rPP.

<p>(A) Comparative deduced amino acid sequences of rPP and rPP-ΔSSD. The RGD domain is colored red. The amino acid sequences excluded in rPP-ΔSSD are colored green. MG63 (B) and MC3T3-E1 (C) cells were seeded onto rPP, rPP-ΔSSD, rC-DMP-1, and vitronectin (100 nM) with MnCl₂ (1 mM). The number of attached cells was evaluated as described above. Each value represents the mean of triplicate determinations; bars mean ±SD. Statistical analysis was performed by a one-way ANOVA, followed by Dunnett's test. ***p <0.001 indicates significantly lower and ⁺p<0.05 and ⁺⁺p<0.01 indicate significantly higher than rC-DMP-1-coated wells.</p