69-72 - Polskie Stowarzyszenie Biomateriałów

The aim of this study was to develop scaffolds for cartilage
regeneration to be used in reconstructive and plastic
surgery. The porous scaffolds were produced from biocompatible
and resorbable polymer – poly(L-lactide-co-glycolide)
(PLG) acting as a supportive matrix. A thin layer of sodium
hyaluronate (Hyal) was also deposited on the surface as
well in the pore walls of PLG scaffolds in order to provide
biologically active molecules promoting differentiation and
regeneration of the tissue. Physical and chemical properties
of the scaffolds were evaluated and the scaffolds were
implanted in the rabbit auricular cartilage.
Materials and methods
Materials
A copolymer of L-lactide and glycolide (PLG), with the
molar ratio of L-lactide to glycolide 85:15 (molecular masses:
M n =80kDa, M w =152kDa), was synthesised in the Centre
of Polymer and Carbon Materials (Polish Academy of Sciences,
Zabrze, Poland) according to the method described
previously [5].
The PLG scaffolds were produced by solvent casting/
particulate leaching technique. Sieved sodium chloride
particles (POCh, Gliwice, Poland) of defined grain fraction:
250µm-320µm, were mixed with 10% (w/v) copolymer solution
in methylene chloride (POCh, Gliwice, Poland) in such
proportions that a salt volume fraction of 85% was obtained.
The mixture was transferred into polypropylene vials
(diameter 12mm, 5ml volume) and dried overnight in the air,
followed by vacuum treatment at a reduced pressure for 48h.
Next, the vials with the rigid salt/polymer mixture were cut
into slices of the thickness of 2mm and placed into 100ml
of ultra high purity water (UHQ-water, produced by Purelab
UHQ-PS apparatus, Elga, UK). The water was exchanged
several times until the conductivity of the water after washing
was about 2µS/cm, which took 2-3 days. The samples were
then dried in a vacuum oven at 35 o C for 72h.
The PLG-Hyal scaffolds were produced according to
the following procedure. UHQ-water was used to dissolve
hyaluronic acid (Na salt powder, CPN Spol. s r.o., Czech
Republic) to the concentration of 80µg/ml. Subsequently,
each scaffold was put in 5ml of the solution in the glass vial
and placed in a vacuum oven to apply a vacuum of 0.08MPa
for 10min, in order to impregnate the whole scaffold with
sodium hyaluronian solution. The scaffolds were then dried
in air for 24h followed by drying under vacuum for 72h, and
stored in a desiccator prior to use.
Materials characterization
The microstructure of the scaffolds was studied with the
use of a scanning electron microscope (Nova NanoSEM
200, FEI, U.S.A.); accelerating voltage 5kV, magnification:
600x, vacuum 60 Pa, without any coating with a conductive
layer.
Fourier transform infrared spectroscopy analysis (FTIR)
was performed in the attenuated total reflection mode (ATR)
with the use of Digilab FTS60 (BioRad).
Implantation
The experiment was performed according to the EU ISO
10993-6 guidelines and the study protocol was approved by
the local bioethics committee in Katowice (No 17/2007, 21
Feb 2007). Before implantation the samples were sterilized
with hydrogen peroxide plasma method (Sterrad 120, ASP,
Johnson & Johnson). Four adult white New Zealand rabbits
were used in the experiment. The animals were operated in
general anaesthesia. Superichondrically 6 mm x 4 mm fragment
of the auricular cartilage was removed and replaced
with the scaffold (6x4x1.5 mm). Each animal received two
implants: PLG or PLG-Hyal scaffolds were implanted into
the right and left ears. The tissues were sutured with polyglycolide
3-0 Safil. After 1 and 4 weeks from the surgery
the animals were painlessly euthanized by an overdose of
Morbital (200 mg/kg body weight).
Histological and histochemical evaluation
Implants and surrounding tissues were excised, frozen
in carbon dioxide ice and subsequently sectioned with
a cryostat microtom into slices 10 µm thick. Obtained slices
were stained by the May-Grünwald Giemsa (MGG) method
to estimate the tissue morphology, number of inflammatory
cells, and degradation process of the scaffolds. The relative
number of inflammatory cells and the activity of hydrolytic
enzyme acid phosphatase (AcP) revealed by Goldberg and
Barka method [6] were used to assess the extent of inflammation
in tissues around the implants.
Observations were made using an optical microscope
(Olympus BH2, objectives 4x and 10x), and pictures were
taken with a digital camera.
Results and discussion
The microstructure of the scaffolds observed under
scanning electron microscope is presented in FIG.1 A, B.
The pore walls of the PLG-Hyal scaffolds (FIG.1B) are much
rough and textured than those of PLG (FIG.1A) because of
the presence of hyaluronate layer on their surface.
Presence of hyaluronate was also proved by infrared spectroscopy
(FTIR-ATR) (FIG. 2 A, B). It can be seen that all peaks
characteristic of poly(L-lactide-co-glycolide) at 1730 cm -1
assigned to C=O and peaks in the range of 1000-1400 cm -1
attributed to C-O and C-O-C stretching vibrations [7] appeared
in both PLG and PLG-Hyal materials. However extra peaks:
at 1530 cm -1 , at 2800 cm -1 and at 3600 cm -1 were observed in
the spectra of PLG-Hyal scaffolds. These peaks attributed
to amide II, C-H, and O-H stretching vibrations, respectively,
are characteristic of hyaluronic acid and hyaluronates [8].
A
B
FIG.1. SEM micrographs of PLG (A) and PLG-Hyal
scaffolds (B)
FIG.2. FTIR-ATR spectra of PLG (a) and PLG-Hyal
scaffolds (b).