CELL - BASED NANOCOMPOSITES AND BIOMOLECULES FOR BONE TISSUE ENGINEERING - Advanced Biomaterials: Fundamentals, Processing, and Applications - page 562

recombinant proteins via the transfection, that is, transfer of DNA to the cell
nucleus, applicable in both an in vitro or in vivo settings. For instance, the deliv-
ery of a gene encoding for an osteoinductive factor to target the cell-of-interest
can galvanize the cells to produce the necessary factors at the location of bone
pathology. The in vivo approach of gene therapy is to inject the genetically-mod-
ifi ed vector or plasmid into the patient, although there might be interferences
such as not being able to transfect the desired cell type or recombination with a
wild-type vector. Alternatively, the patient's cells can be harvested and expanded
in vitro prior to transfection before implantation. Nevertheless, this process is
more cumbersome and time-consuming.
Bone marrow stromal cells that were isolated from rats were transfected
with an adenovirus encoding cDNA for rhBMP-2 and seeded on PLGA/HA
microspheres. In co-culture experiments (primary human MSCs and transfected
cells), BMP-2 produced by the transfected cells promoted osteogenic differentia-
tion and mineralization, with a signifi cant increase in proliferation, ALP and
calcium deposits than those MSCs which were co-cultured with non-transfected
cells 130 .
In another case, retrovirus encoding the CDNA for BMP-2 was used to trans-
fect a murine stromal cell line and the transfected cells were cultured on the
PLGA/HA material for a week. Results showed that the transfected cells attached
onto the material and proliferation was seen. BMP-2 was continually expressed
by the transfected cells. The material specimens were then implanted into the
quadriceps muscle porch of severe combined immunedefi cient (SCID) mice,
with three sample groups namely PLGA/HA with 3

g of rhBMP - 2 (positive
control), PLGA/HA with BMP-2 producing W-20 cells, and PLGA/HA with non-
transfected W-20 cells. No bone formation was observed in the PLGA/HA with
non-transfected W-20 cells, whereas bone formation occurred in the PLGA/HA
with the BMP-2 producing cells and PLGA/HA with rhBMP-2 groups, indicative
of the potential application of retroviral gene transfer in concert with an appro-
priate material for bone regeneration 131 .
Non-collagenous molecules in bone such as dentin matrix protein 1 (DMP1)
has been reported to be involved in apatite nucleation 132,133 . Using genetically-
engineered silk DMPI, combined with SBF treatment, calcium-defi cient carbon-
ated HA was formed. It was found that the carboxyl terminal domain of DMP1
induced HA nucleation. HA was absent in samples with no carboxyl terminal
domains of the DMP1. Such studies showed that other macromolecules besides
collagen were involved in HA nucleation and deposition 132 .
Recombinant collagen technology has also been investigated extensively.
It has been shown that recombinant human-like collagen synthesized from
Escherichia coli ( E.coli ) underwent mineralization when the collagen was
subjected to a series of chemical treatments involving CaCl 2 and NaH 2 PO 4
solutions. Nano-HA crystals were deposited on the recombinant collagen fi brils,
forming mineralized collagen fi bers and the crystallographic c - axis alignment
of the nano-HA crystals was parallel to the longitudinal axis of the collagen
fi brils 134 .

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