STARCH/CELLULOSE ACETATE AND ITS ADEQUABILITY TO BE USED AS SCAFFOLDS FOR BONE
TISSUE ENGINEERING
AJ Salgado+, * , DW Hutmacher§, JE Davies¶, RL Reis+, *
3B’s Research Group – Biomaterials, Biodegradables and BiomimeticS, University of Minho, Campus de Gualltar, 4710-057 Braga, Portugal;
*
Department of Polymer Engineering, University of Minho, Campus de Azurém, 4800-058 Guimarães, Portugal;
§
Division of Bioengineering, Faculty of Engineering and Department of Orthopaedics, Faculty of Medicine, National University of Singapore, E3-05-15-10-10
Kent Ridge Crescent, 119260, Singapore, Singapore;
¶
Institute of Biomaterials and Biomedical Engineering, Rosebrugh Building, 4 Taddle Creek Road, Toronto, Ontario, M5S 3G9, Canada
email : [email protected]
elaborated on the scaffolds structure. In the in vivo assays, and after 1
Introduction:
Reconstruction of extensive orthopaedic bone defects is still a problem
week it was possible to observe that bone formation was occurring
affecting millions of people worldwide, which contemporary medicine
around, and within, the scaffold after 1 week. The bone/scaffold
has yet to solve. In this context tissue engineering has been emerging as
interface was occupied by a form of “connective tissue” that, in a first
a valid approach to circumvent some of the limitations of existing
analysis, could be considered as fibrous tissue. However, closer
therapies. One of the most important elements in tissue engineering is
observation revealed that the thin strands of extracellular matrix within
the scaffold, a 3D porous structure that acts as a temporary matrix for
this highly cellular tissue could not only be traced back to the matrix of
cell and tissue ingrowth, until the tissue is completely regenerated, and
the surrounding bone tissue, but also stained positive for osteocalcin.
the tissue function restored.
Thus, the initial “connective tissue” surrounding the scaffold materials
The design and fabrication of scaffolds from a material point of view
could be a very early form of bone formation. A minimal initial
can be grouped into: (I). biodegradable and bioresorbable polymers
multinucleated giant cell (MGC) response was also observed. TRAP
which have been used for clinically established products, such as
staining revealed that a small percentage of the MGC were putative
collagen, fibrin glue, PEO, polyglycolide (PGA), polylactides (PLLA,
osteoclasts. No other cellular evidence of an inflammatory response was
PDLA) and polycaprolactone (PCL); (II). recently regulatory approved
observed at the three time points employed. After 3 weeks no non-bony
polymers,
such
as
polyorthoester
(POE),
polyanhydrides,
connective tissue was found and the MGC response was almost nonpolyhydroxyalkanoate (PHA), hyaluronic acid derivatives, chitosan and
existent. The degree of bone ingrowth and bone contact was higher by
(III). as an alternatives to those the synthesis and development of
this time point. After 6 weeks there were no MGCs on the surface of
polymeric biomaterials of synthetic and natural origin. Within these,
SCA scaffolds. The degree of bone contact in both the marrow cavity
starch based biomaterials were originally proposed for several
and cortical bone compartments was significantly higher when
biomedical applications, including tissue engineering scaffolding [1,2].
compared with the other time points, and bone was in contact with the
The latter have been shown to exhibit adequate mechanical and
majority of the scaffold surface (Fig.1 is a representative example of the
degradation properties for bone tissue engineering [1,2].
results obtained for SCA). This was confirmed by SEM. In those areas
The objectives of the present study were to evaluate the cytotoxicity of
were bone was not contacting the SCA surface, marrow was seen in
the leachables released by the scaffolds, to determine osteoblasts
direct contact with the material, which indicated a highly biocompatible
adhesion and proliferation patterns when seeded on the referred
response.
scaffolds, and finally to evaluate the in vivo response to starch/cellulose
acetate (SCA) scaffolds.
+
Materials and Methods:
Scaffolds used in the present work were composed of a blend (50/50
wt%) of corn starch with cellulose acetate (SCA). Scaffolds were
processed by a previously described methodology based on extrusion
with blowing agents [2]. Cytotoxicity tests were carried out according to
ISO/EN 10993 part 5 guidelines. MEM extraction and MTS tests were
carried out, both with a 24h extraction period. Extracts from latex rubber
and culture medium with no extraction material were used respectively
as positive and negative controls. For the direct contact studies human
calvarial osteoblasts were used. Cells were seeded with a density of
3x105 cells/scaffold and allowed to grow for 3 weeks. Cellular viability
and proliferation were assessed weekly by MTS test and FDA/PI
staining. The adhesion of cells to the materials was analysed by scanning
electron microscopy (SEM) and by fluorescence microscopy through
phalloidin staining. Protein expression (osteonectin and osteocalcin)
was analysed by western blot and ELISA. For the in vivo assays the rat
femoral defect model was used. Bone defects were made in the distal
femurs of Wistar rats using a low speed dental drill (2.3 mm in
diameter). The animals were sacrificed after 1, 3 and 6 weeks and the
femurs were removed, processed and sectioned. Sections were then
stained with Masson’s trichrome stain. Bone regeneration, in growth and
bone contact with the scaffold were further studied by x-ray,
immunohistochemistry and scanning electron microscopy (SEM).
Results and Discussion:
Results showed that the materials did not cause any morphological
changes or induce any deleterious alteration to their metabolic and thus
were considered as non-cytotoxic. Furthermore the MTS test, SEM and
fluorescence microscopy (FDA/PI and phalloidin) staining showed that
cells were able to attach and proliferate within the porous structure.
Protein expression studies also showed that cells had high ALP activity
and bone extracellular matrix proteins (osteocalcin, osteonectin) were
being expressed, meaning that bone ECM was being
Figure 1- Photomicrograph of SCA scaffold after 6 weeks of
implantation: a) 4x (original magnification).
Conlusions:
SCA scaffolds, contrary to other biodegradable scaffolds, revealed noncytotoxic behaviour and allowed osteoblasts to adhere and proliferate
within the scaffold structure. Furthermore when implanted in vivo they
revealed biocompatible behaviour, allowing bone deposition both on the
surface and in the inner areas of the scaffold. Overall it can be said that
starch based scaffolds may provide an alternative to current biomaterials
used for bone tissue engineering applications.
References:
1- M.E. Gomes et al. (2001), Biomaterials 22, 883-889.; 2- M.E. Gomes
et al. (2002), Materials Science and Engineering C, 20, 19-26.
Acknowledgements:
Portuguese Foundation for Science and Technology through funds from
POCTI and/or FEDER programs (PhD scholarship to A.J. Salgado
(SFRH/BD/3139/2000)., and an Ontario Research and Development
Challenge Fund (ORDCF) grant to JED for financial support for the
work described.
51st Annual Meeting of the Orthopaedic Research Society
Poster No: 1747