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6.2.1. SOFT TISSUE HISTOLOGY

In chapter IV, 10% formalin was used to fix samples for 1 day, and further dehydrated with graded ethanol washes. Samples were embedded in paraffin, sectioned to 5 µm and mounted on glass slides. To proceed to distinct stainings, sections were deparaffinized with Clearite and rehydrated with a graded series of ethanol washes.

Safranin O and Alcian Blue staining were performed to detect articular cartilage extracellular matrix components, such as mucopolysaccharides and glycosaminoglycans. Briefly, safranin O staining was performed with Weigert’s iron hematoxylin, 10 min followed by running in tap water for 10 min. Slides were then immersed in 0.02% fast green solution, 5 min and rinsed 10- 15 sec in 1% acetic acid. Finally, samples were stained with 0.1% safranin O solution for 5 min.

Regarding alcian blue stain, slides were immersed 30 min in 0.01 g/mL alcian blue solution, and washed in running tap water for 2 min. A counterstain with nuclear fast red (0.5%) was performed for 5 min. Upon staining, sections were washed in running tap water, dehydrated with a graded series of ethanol, cleared with Clearite and mounted with resinous mounting media (Entellan Merck & Co., Inc. USA).

In chapter V, samples were fixed in 4% formaldyhyde solution for 1 day. Bone scaffolds were decalcified with immunocal solution (Decal Chemical, Tallman, NY) for 1 day, and further dehydrated with graded ethanol washes, concurrently with remaining silk constructs. Samples were embedded in paraffin, sectioned to 5-µm slices and mounted on glass slides. For staining, sections were deparaffinized with CitriSolv and rehydrated with a graded series of ethanol washes. Samples were stained using standard hematoxylin and eosin.

6.2.2. HARD TISSUE HISTOLOGY

Hard tissue histology was performed in samples harvested from subcutaneous implantation in NOD SCID mice, described in chapter VIII. In order to assess new bone formation, contructs were not decalcified and further processed according to hard tissue histology methods: constructs were fixed in 10% formalin for 1 day and dehydrated with sequential washes in 70 % ethanol (2 days), 100 % ethanol (2 days with twice daily solution changes), and toluene (2 days with once daily solution change). Constructs were then washed in activated methyl methacrylate (MMA) with daily changes of MMA solution for four days at 4 °C, and then placed at 32 °C until the MMA cured. Plastic-embedded sections were sectioned to 8 µm on a Leica hard tissue microtome. Staining for the new osteoid formation was done using the traditional Goldner’s Masson trichrome stain (table I and II):

Table I – Protocol for Goldner’s Masson trichrome stain

Staining step Time (minutes)

Weigert’s Hematoxylin 30

Rinse tap water 2

Ponceau working solution 5

Acetic acid 0.2% 3

Phosphotungstic-Phosphomolybdic acid 10

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Table II – Composition of solutions used in Goldner’s Masson trichrome stain

Weigert’s Hematoxylin Ferric chloride 29% aqueous solution

Solution A Ferric chloride, anhydrous 1.5 g

Hematoxylin 1 g Distilled water 99 mL

95% Ethyl alcool 100 mL Hydrochloric acid (concentrated) 1 mL

Solution B (Shelf life is 8 months.)

Ferric chloride 29% aq. solution 4 mL Masson Fuchsin Ponceau Orange G Stock

Distilled water 95 mL Ponceau 2R 2 g

Hydrochloric acid (concentrated) 1 mL Acid Fuchsin 1 g

Working solution: Orange G 2 g

Mix equal amount of A and B. Use immediately. 0.2 % Acetic acid 300 mL

(Shelf life of A and B is 8 months.) _{Working solution:}

0.2% Acetic acid Stock solution of F-P-O 10 mL

Glacial acetic acid 2 mL 0.2 % Acetic acid 90 mL

Distilled water 1 L (Shelf life of F-P-O stock is 9-12 months.)

(Shelf life is indefinite.) Light green

Phosphotungstic-Phosphomolybdic acid Light green stain 0.1 g

Phosphotungstic acid 5 g 0.2 % Acetic acid 100 mL

Phosphomolybdic acid 5 g (Shelf life is 6 months.)

Distilled water 100 mL

(Shelf life is 1-2 months.)

6.2.3. IMMUNOHISTOCHEMISTRY

Immunohistochemistry (IHC) is a method for demonstrating the presence and location of proteins in tissue sections, enabling the observation of processes in the context of intact tissue. IHC staining performed with antibodies that recognize the target protein. Antibodies are highly specific, binding only to the protein of interest in the tissue section. The engineered tissue developed in the context of this thesis, was evaluated by detecting the antibody-antigen interaction using a chromogenic detection, where an enzyme conjugated to the antibody cleaves a substrate, producing a colored precipitate at the location of the protein.

In chapters V, VI and VII, silk fibroin scaffolds were to engineer bone tissue, while gellan gum hydrogels were used to engineer cartilage tissue in chapter IV. In these chapters, samples were fixed in 10% formalin for 1 day, embedded in paraffin, sectioned to 5 µm and mounted on glass slides. In chapter V and VIII, where decellularized bone scaffolds were also used to engineer bone tissue. In order to perform soft tissue histology, samples were decalcified for 2 days with Immunocal solution (Decal Chemical, Tallman, NY, USA) before embedding in

paraffin. When starting IHC procedure, sections were deparaffinized with CitriSolv and rehydrated in a graded series of ethanol washes. Antigen retrieval was performed by heat- induced method using a microwave and Citrate Buffer (0.1M Citric acid and 0.1M Sodium citrate), 20 min. Once horseradish peroxidase enzyme conjugate is used for protein detection, suppression of any endogenous peroxidase activity was performed by incubating sections 30 min in H2O2 0.3% RT with agitation, to decrease background staining (false positives).

After washing with PBS+0.025% Triton-X, normal horse serum - NHS (20 min, RT) was applied to reduce non-specific binding of primary antibody. Excess serum was blot, before incubation with diluted primary antibody (table III) overnight at 4ºC (in moist chamber). Negative controls were performed by omitting the primary antibody incubation step.

Sections were further washed with PBS+0.025% Triton-X before incubation with biotin-labeled secondary antibody, 30 min, RT, after which, VECTASTAIN ABC reagent was applied. This is an avidin-biotinylated enzyme complex (ABC) that will bind to the biotinylated secondary antibody (Vectastain Universal Elite ABC Kit, PK-6200 Vector Laboratories).

Finally, the tissue antigen is localized by incubation (5-10 min) with peroxidase substrate solution (3, 3'- diaminobenzidine), yielding a brown stain (DAB substrate kit SK-4100 Vector Laboratories). Counterstaining of cell nuclei was further performed with haematoxylin (2 min). Sections were lastly dehydrated, cleared in CitriSolv and mounted in organic mounting media. Table III – Primary anti-bodies used for immunohistochemistry in chapters IV - VIII

Target protein Primary Antibody Dilution Company / Ref Chapter

Collagen II (Col II) Rabbit anti-human

polyclonal antibody 1:50 Abcam / ab34712 IV

Collagen I (Col I) Rabbit anti-human

polyclonal antibody 1:200 Abcam / ab292 IV

CD31 Rabbit anti-human

monoclonal antibody

1:250 Millipore / 04-1074 VII, VIII

von Willebrand Factor (vWF) Rabbit anti-human polyclonal antibody

1:200 Sigma-Aldrich / F3520 VII, VIII

Collagen I (Col I) Mouse anti-human

monoclonal antibody 1:500 Abcam / ab6308 V, VIII

Osteopontin (OPN) Rabbit anti-human

polyclonal antibody 1:750 Chemicon / ab1870 V - VII

Bone Sialoprotein (BSP) Rabbit anti-human

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