SF-RGD scaffolds1. Cocoons from B. mori were boiled for 1 h in an aqueous solution of 0.02

M Na2CO3 and rinsed with water to extract sericins.2. Purified silk was solubilized in 9 M LiBr solution and dialyzed (Pierce,

MWCO 3500 g/mol) first against water for 1 day and then against 0.1 M 2-(N-morpholino)ethanesulfonic acid buffer (MES), 0.5 M NaCl, pH 6, for 1 day.

- For conjugation with RGD sequences ### SF solution was coupled with GRGDS peptide(Arg-Gly-Asp-Ser).(Through specific peptide motifs such as RGD (ARG-GLY-ASP), extracellular

matrix (ECM) components interact with integrin and can modify the behavior of cells. We analyzed the effect of an RGD-containing peptide, RGDS, on the regulation of TGF-β1 secretion by cultured human mesangial cells (HMC) and the intracellular mechanisms implied in signal transduction.)

• The carboxyl groups on SF were first activated by reaction with EDC/NHS for 15 min at room temperature.

• To quench excessive EDC, 70 ll/ml ß-mercaptoethanol was added.

• The solution was then incubated with 0.5 g/l peptide for 2 h at room temperature.

• The reaction was stopped with 10 mM hydroxylamine. Purified SF-RGD was dialyzed against 0.1 M MES, pH 4.5–5, for 1 day, lyophilized and redissolved in hexafluoro-2-propanol (HFIP) to obtain a 17% (w/v) solution.

• Granular NaCl crystals were used as porogen.

• Sieved fractions in the range of small (106–212 lm), medium (212–300 lm), or large (300–425 lm) diameters were weighed into a Teflon container, and SF-RGD/HFIP solution was added at a ratio of 20:1 (NaCl/SF-RGD).

• HFIP was allowed to evaporate for 2 days, and NaCl/SF-RGD blocks were immersed in 90% (v/v) methanol for 30 min to induce a conformational transition to ß-sheet .

• Blocks were removed and dried, and NaCl was extracted by incubation in water for 2 days, resulting in scaffolds with >90% porosity.

• Disk shaped scaffolds (8 mm diameter, 2 mm thick) were prepared using a dermal punch (Miltey, Lake Success, NY) and steam autoclaved at 121 ?C for 15 min.

The blenled scaffold+Aloe vera

Concept Smart scaffold modelConjugateSilk Boil

Figure 1. (a) Silk processing and (b) silk scaffold fabrication flowchart.Biomacromolecules, Vol. 5, No. 3, 2004

Porous 3-D Scaffolds from Regenerated Silk Fibroin

Aloe barbadensis var miller

Extraction of the aloe vera

Take some aloe Vera leaves

Cut the thorns out from both sides

Cut all of the aloe Vera and small pieces

Extraction of the Aloe vera

Put these pieces in the grinder

The result will be a very sticky liquidThen you will take a container to store this in and put a sifter on top

Immunofluorescence for α-SMA

(a–b) Immunofluorescence for α-SMA cultured for 3 days on the tissue culture plate and the aloe on plate

α-SMA on plate the aloe on plate

Pure silk scaffold Scaffold with free Aloe in medium

the blended silk fibroin/ chitosanthe blended scaffold crosslink with Aloe

SEM micrographs of prepared scaffolds after freeze-drying with methanol treatment.

(A) Pure Silk fribroin, (B) Blenled Silk and Chitosan(C) Blenled scaffold with RGD

MTT result after osteoblasts cultured in scaffolds for 0, 7, 14, 21, and 28 days. SFSF/CSF/G. *Significant differences are from SF scaffold at p < 0.05.

Photomicrographs of osteoblasts growth on the surface and inner area of the scaffolds. The osteoblasts were

stained with hematoxylin and eosin

scale bar = 50 μm.

SEM micrograph of osteoblast growth on the surface

area of the scaffolds. (A) SF, (B) SF/C and (C) SF/G..

Magnification x200

SEM micrograph of chondrocytes growth in the inner area of the scaffolds. (A) (B) (C) Magnification x500.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3680310/

introduction• We demonstrated that gels containing the integrin ligand arginine-glycine-aspartic acid (RGD)

supported mesenechymal stem cell (MSC) incorporation. High cell viability and proliferation of the encapsulated cells showed biocompatibility of the material.

http://www.chem.ucla.edu/dept/Faculty/maynard/Highlights.htm

METHOD• RGD peptides were conjugated onto chitosan molecules via a

carbodiimide reaction. Briefly, 16 mg RGD-peptide (0.017 mmol), 3.6 mg N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride ′(0.017 mmol) and 1.9 mg N-hydroxy-2,5-pyrrolidinedione (0.017 mmol) were dissolved in 60 μL N, N-dimethylformamide and then mixed with 3 mL of chitosan solution (2%, w/v in 1% acetic acid). After 24-h incubation at room temperature, the mixture was dialyzed against deionized water. After freeze-drying, the peptide-grafted chitosan (chitosan-g-RGD) was stored at −20 °C until use. The graft ratio of RGD to chitosan was estimated as 2.75 mol% with respect to the total moles of the amino groups of chitosan molecules.

• Fabrication of UV-crosslinked chitosan scaffolds with conjugation of RGD peptides for bone tissue engineering,Wei-Bor Tsai

The SEM images of the chitosan-based scaffolds. Scale bar = 100 μm.

Scale bar = 100 μm.

Chitosan conjugate RGD

CULTURE

Mechanical property

Fig. 2 Shows the effect of aloe vera gel extract on the proliferation of bone marrow mesenchymal cells (BMSC) and MC3T3-E1 osteoblast cell line via the MTT assay. Cells were treated with the aloe vera gel extract at concentrations 1, 5,10, 20 and 50 µg/ml for 24 hours. Data shown in the form of mean±S.D. (* demonstrates the significance from the control group at p > .05, n=12)

2548;28:127-36 วง ทั�นต จุ�ฬา. 2548