Bilayered Scaffolds (Loofah/PLLA/cellulose/chitin) for Osteochondral Defect Repair: Synthesis and In-vitro
Characterization
Berivan CECENa,
Leyla Didem KOZACIb, Mithat YUKSELc, Aylin KARAa, Nevin ERSOYd, Alper BAGRIYANIKd, Hasan
HAVITCIOGLUa,d
a*Department
of Biomechanics/Dokuz Eylül University, Izmir, Turkey, bDepartment of Medical Biochemistry/Yildirim Beyazit University,
Ankara, Turkey, cDepartment of Chemistry Engineering/Ege University, Izmir, Turkey, dDepartment of Histology&Embryology/Dokuz
Eylül University, Izmir, Turkey, eDepartment of Orthopedics and Traumatology/Dokuz Eylül University, Izmir, Turkey
Statement of Purpose: The objectives of the present study
were; (a) to investigate characteristics of natural and novel
loofah based bilayer scaffolds (b) to determine their effects
on the morphology, adherence and proliferation capacities
of chondrocytes and osteoblasts in vitro. In this study, we
produced a bilayer scaffold and investigated the ability of
co-cultures of chondrocytes and osteoblasts to repair
articular cartilage in osteochondral defects. For this
purpose, fibrin glued loofah+PLLA+cellulose scaffold
with MG-63 cells and loofah+PLLA+chitin scaffold with
SW-1353 cells were used to promote bone and cartilage
regeneration, respectively.
Methods: Loofah was soaked to swell and washed with
water, then dried with NaOH (2 M). It was first coated with
PLLA and dried. PLLA coated loofah was then either
soaked in 4% cellulose (in 4% PLLA mixture) and dried at
500C (Loofah+PLLA+cellulose) or
4%
Chitin
(Loofah+PLLA+chitin). SW-1353 and MG-63 cells were
seeded on previously glued loofah+PLLA+chitin and
loofah+PLLA+cellulose scaffolds, respectively to form
double layers. Scanning Electron Microscopy (SEM),
Energy Dispersive X-ray Spectrometry (EDS), XTT, LDH
and ALP Activity Assays and Histologic staining
(hematoxylin and eosin, masson trichrom, alizarin red,
alcian blue and toluidine blue) and immunohistochemistry
were performed to demonstrate cellularity and extracellular
matrix (ECM) deposition. The data of XTT, ALP and LDH
assays were analyzed by using parametric repeated
measures ANOVA and non-parametric Kruskal-Wallis
test. p<0.05 was considered statistically significant.
Results: The morphologies of loofah scaffolds, were
characterized by SEM analysis. The cells adhered on
bilayer scaffolds maintaining their spherical and spindle
forms. Furthermore, cells produced ECM on the scaffolds.
According to the EDS analysis, the C and O elements were
observed among the main elements of PLLA and cellulose.
The N element (main element of the chitin) has also been
detected (Figure 1).
Figure 1
Cell numbers were significantly higher on the 8th day
compared to the 3rd and 5th days by XTT assay (p<0.05).
LDH activity slightly increased at 5th day which might be
the result of release of PLLA polymer which may be toxic
during its degradation. During the 8-day assay period, ALP
activity significantly increased (p<0.05), Figure 2. H&Estaining revealed heterogeneous cell morphologies with
ECM formation inside the bilayer scaffolds. Masson's
trichrome staining confirmed the existence of the cells in
scaffold structures.
Figure 2
Histological analyses using TB confirmed well-formed
chondrocytes 8 days after seeding. (Figure 3). Similar to
the results of MT staining, immunohistochemical analyses
revealed the presence of both collagen types I and type II
expression in cultures. TEM showed osteoblast and
chondrocyte interactions. Ultrastructural morphology
showed that cells developed bud-like extensions and were
in contact with each other.
Figure 3
Conclusions: Tissue engineered osteochondral grafts must
be designed to repair and integrate both the injured
cartilage and the subchondral bone (1). Adhesion of cells
to the surface of a biomaterial is one of the major factors
mediating its biocompatibility. Experimental methods to
determine cell adhesion rely on morphological and
biomechanical approaches. While porosity of scaffold
mimics the microstructure of cancellous bone or cartilage
the surface properties of biomaterials play a major role in
cellular interactions, such as cell adhesion, infiltration and
proliferation (2,3). This study shows that cultivation of
chondrocytes and osteoblast on loofah based scaffolds may
have potential for applications in the field of cartilage
tissue engineering to repair osteochondral defects. Further
studies are required to use these constructs to diminish
complications
caused
by
currently
available
osteochondral-materials.
References
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Staudenmaier, R. Biomaterials 2006;27:3955. 2. Marquesa, A.P.,
Reisa, R.L., Hunt, J.A. Biomaterials 2002;23:1471. 3. LópezRuiz, E., Jiménez, G., García, M.Á., Antich, C., Boulaiz, H.,
Marchal, J.A., Perán, M. Expert Opin Ther Pat 2016;8:877.