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Tissue preparation for assays Total protein
Tissue preparation for hydroxyproline estimation The total protein content of the wound tissue
The granulation tissue specimens were dried at 60°C for homogenate was determined according to the method
[19]
24 h and then were weighed and kept in glass‑stoppered of Lowry et al. Absorbance was measured at 540 nm
test tubes. 6N HCl was added to each tube for a total and expressed in mg/g of tissue. Standards were treated
of 40 mg of dried granulation tissue per ml of acid. similarly using BSA at concentrations of 0, 20, 40, 60, 80,
The tubes were kept in boiling water bath for a total and 100 µg/mL in 0.1 M phosphate buffer at pH 7.4.
of 24 h (12 h and each for 2 days) for hydrolysis. The Matrix metalloproteinase‑2 and ‑9
hydrolysate was then cooled, and the excess acid was A total of 100 mg of tissue was homogenized in 1 mL
neutralized by 10N sodium hydroxide (NaOH) using of ice‑cold lysis buffer. Subsequently, homogenates were
phenolphthalein/methyl red as an indicator. The volume centrifuged at 3,000 g for 5 min at 4°C, and supernatants
of neutral hydrolysate was diluted with distilled water to were stored at ‑80°C until use. MMP‑2 and MMP‑9 were
a concentration of 20 mg/mL of dried granulation tissue measured using prefabricated ELISA kits, according to
in the final hydrolysate. The hydrolysate was used to manufacturer protocol (R and D Systems, USCN Life Science
estimate the level of hydroxyproline. [18] Inc., USA). Plates were read at 450 nm and 540 nm, and

Tissue preparation for estimation of antioxidants and concentrations were calculated using a 4‑point standard
curve and expressed as ng/mg of protein.
[20]
oxidative biomarkers
The wet weight of the granulation tissue samples was Nitric oxide estimation
noted and homogenized by a Rotex homogenizer in The level of NO was estimated as NO , an NO metabolite
−
2
ice‑cold 0.2 M phosphate buffer (pH 7.4). Homogenates in tissue samples. NO is a highly reactive free radical gas
were centrifuged at 15,000 rpm for 30 min in a which is a ready oxidizer and remains stored in tissues as
−
cooling centrifuge, and the supernatant was then used nitrates (NO ) or NO . Thus, NO concentration can be
−
2
3
−
−
to determine total protein, GSH, and an oxidative estimated by measuring concentrations of NO and NO
3
2
biomarker (MDA). in combination. The simplest technique is to monitor
the reduction of NO to NO by nitrate reductase or
−
−
For the MMP‑2 assay, the granulation tissue was rinsed a metallic catalyst, followed by the colorimetric Griess
2
3
in ice‑old phosphate buffer saline (PBS) (0.1 mol/L, reaction to measure NO levels (nitrite levels).
−
pH 7.0‑7.2) to remove all traces of excess blood and 2
−
weighed prior to homogenization. The tissues were Sample NO levels were estimated by a colorimetric assay
2
minced into small pieces and homogenized on ice in which is based on the Griess reaction. Using a two‑step
−
5 mL of PBS. The resulting suspension was sonicated diazotization reaction, acidified NO produces a nitrosating
2
with an ultrasonic cell disrupter or subjected to two agent which reacts with sulfa nitric acid to produce the
freeze‑thaw cycles to further break the cell membranes. diazonium ion. This ion is then coupled to N‑(1‑naphthyl)
The homogenates were then centrifuged for 5 min at ethylenediamine dihydrochloride to form a red azo derivative
50 g, removing the supernatant and aliquot store at which is measured at 550 nm using a spectrophotometer.
−
no more than ‑80°C. The concentrations of NO are calculated from a standard
2
curve established with serial dilutions of sodium NO .
− [21]
For the NO assay, the tissue was homogenized in an 2
isotonic solution of PBS containing 10 mM of NEM Glutathione estimation
and 2.5 mmol ethylenediaminetetraacetic acid (EDTA). The reaction mixture was prepared by adding 80 µL of
The addition of NEM/EDTA blocks the SH‑groups tissue supernatant to 0.9 mL of 0.2 M sodium phosphate
while inhibiting the transition of metal‑catalyzed buffer of pH 7.00 and 20 µL of 10 mM 5.5’‑dithiobis
trans‑nitrosation reactions, preventing artificial nitrosation (2‑nitrobenzoic acid) (DTNB) solution. The yellow‑colored
and thiolate‑ and ascorbate‑mediated degradation of substance formed by the reaction of GSH and DTNB
endogenous Nitrosothiols and nitrite (NO ). The protein was measured at 412 nm. The results were expressed as
−
2
concentration was determined according to Lowry et al. GSH µmol/mg protein. [22]
[19]
using purified BSA as the standard. Malondialdehyde
Estimation of biochemical parameters The MDA levels of wound tissue homogenate were
[23]
Hydroxyproline measured according to the method of Ohkawa et al. To
The dried tissue was added to 10 mL of 6N HCl 0.1 mL of the test sample, 1 mL of 10% TCA and 1 mL of
and stored at 110°C for 24 h. The neutralized acid 0.67% TBA were added and then heated in a boiling water
hydrolysate of the dry tissue was used to determine bath at 100°C for 30 min. The mixture was cooled under
tap water and centrifuged at 12,000 rpm for 10 min. Clear
the level of hydroxyproline. The reaction mixture supernatant was determined by the absorbance at 535 nm
contains 0.05 M copper sulfate, 2.5N NaOH, 6% H O , and expressed as nmol/mg protein.
2
2
3N sulfuric acid, and 5% p‑dimethylaminobenzaldehyde
using L‑hydroxyproline as the standard. Absorbance was Statistical analysis
measured at 540 nm and expressed as µg/mg of dry Statistical analysis for biochemical parameters was
tissue weight. [18] performed by Student’s t‑test, and data were expressed as
268 Plast Aesthet Res || Vol 2 || Issue 5 || Sep 15, 2015