Barbieri Thesis - BioMedical Materials program (BMM)

Chapter 5 – Alkali surface treatment effects
temperature in dark for 20 minutes. A standard curve of known concentration of DNA
was generated concurrently and used to determine sample concentrations. To
evaluate the DNA content, the samples were excited with 480 nm wavelength and
fluorescence emission was read at 520 nm with a spectrophotometer (AnthosLabtec
Instruments GmbH). To evaluate cell differentiation, alkaline phosphatase (ALP)
activity was measured at the time points 1, 4, 7 and 14 using the same lysate than for
DNA assay. A 50 L cell lysate was mixed with 50 L p–nitrophenylphosphate (p–
NPP) (Sigma–Aldrich) in 1M diethanolamine buffer containing 1 mM MgCl2 (pH 9.8)
and incubated at 37°C for 15 minutes. The reaction was stopped by the addition of 50
L of 0.1N NaOH. A standard curve of known concentration of p–nitrophenol (Sigma)
was generated concurrently and used to determine sample concentrations. Enzyme
activity was quantified by absorbance measurements at 405 nm (AnthosLabtec
Instruments GmbH). All experiments were conducted in quintuplicate.
Table 1. Composition of the two culture mediums used. All the components were purchased from
Invitrogen.
Component Expansion Osteogenic
Minimum essential medium (–MEM)
[%vv.] 90 90
with ribonucleosides but without ascorbic acid
Foetal bovine serum (FBS) [%vv.] 10 10
L–glutamine [mM] 2 2
L–ascorbic acid 2–phosphate (ASAP) [mM] 0.2 0.2
Penicillin [IU mL –1 ] 100 100
Streptomycin g mL –1 ] 100 100
Basic fibroblast growth factor (bFGF) [ng mL –1 ] 1 0
Dexamethason (DEX) [nM] 0 10
5.2.9. In vivo tissue response
With the permission of the local animal care committee (Animal Center, Sichuan
University, Chengdu, China; protocol #P11029), granules of the three composites (0.5
g) were implanted in the paraspinal muscles of six skeletally mature mongrel dogs
(male, 1–4 years old, weight 10–15 kg) for 12 weeks to evaluate the tissue reaction
and the osteoinductive property of the composites. The surgical procedure was
performed under general anaesthesia (pentobarbital sodium, Organon, the
Netherlands; 30 mg kg –1 body weight) and sterile conditions. The back of the dogs
was shaved and cleaned with iodine. A longitudinal incision was made and the
paraspinal muscle was exposed by blunt separation. Longitudinal muscle incisions
were subsequently made with a scalpel and separate muscle pouches were created
by blunt separation. The granules were then placed in the pouches and the wound
was closed in layers using silk sutures. After surgery, the animals received
intramuscular injections of penicillin for three consecutive days to prevent infection.
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