Chapter 5: Summary and Future prospects
B) The ways in which lipoproteins may be modified to act as contrast agents. (Adapted with permission from Cormode et al, 2010)
3.3 Results
subsequent increment of absorbance with time over a period of 10
compared to a more saturated growth in control group of 12 well tissue culture plate (Fig 3.3 G).
Fig 3.6: Electrical property of GNP
ohmic nature of the I-V characteristic of the GNP
experimental setup in inset. (Reprinted with permission from Das et al, Data in Brief, 2015
These results indicate enhanced cellular proliferation over porous nano
with higher surface area to volume ratio than conventional tissue culture plates with similar dimensions. However, no appreciable difference was observed in rate of proliferation between GNP-
3.3.4 Morphology of nerve condui proliferation
FESEM images showed that the fabricated conduits had an internal diameter of 1.25mm and a wall thickness of 0.34mm with a lamellar architecture comprising multiple layers of nanofibers (
conduits cultured with rat Schwann cell line (SCTM41) showed adherence of cells, formation of cell clusters and growth of cells in three dimensions on both inner and outer surface of GNP-SF as well as SF conduits (
subsequent increment of absorbance with time over a period of 10
compared to a more saturated growth in control group of 12 well tissue culture plate
: Electrical property of GNP-SF nanocomposite scaffold. The figure shows the V characteristic of the GNP-SF nanocomposite scaffold with the
Reprinted with permission from Das et al, Data in Brief, 2015 These results indicate enhanced cellular proliferation over porous nano
with higher surface area to volume ratio than conventional tissue culture plates with similar dimensions. However, no appreciable difference was observed in rate of
-SF and SF scaffolds.
Morphology of nerve conduits and Schwann cell adhesion and
FESEM images showed that the fabricated conduits had an internal diameter of 1.25mm and a wall thickness of 0.34mm with a lamellar architecture comprising multiple layers of nanofibers (Fig 3.2 E). Electron microscopic observations of conduits cultured with rat Schwann cell line (SCTM41) showed adherence of cells, formation of cell clusters and growth of cells in three dimensions on both inner and
SF as well as SF conduits (Fig 3.2 F).
subsequent increment of absorbance with time over a period of 10 days as compared to a more saturated growth in control group of 12 well tissue culture plate
. The figure shows the SF nanocomposite scaffold with the Reprinted with permission from Das et al, Data in Brief, 2015) These results indicate enhanced cellular proliferation over porous nano-scaffolds with higher surface area to volume ratio than conventional tissue culture plates with similar dimensions. However, no appreciable difference was observed in rate of
ts and Schwann cell adhesion and
FESEM images showed that the fabricated conduits had an internal diameter of 1.25mm and a wall thickness of 0.34mm with a lamellar architecture comprising croscopic observations of conduits cultured with rat Schwann cell line (SCTM41) showed adherence of cells, formation of cell clusters and growth of cells in three dimensions on both inner and
3.3.5 Porosity and Swelling ratio of Nerve conduits
The porosity of SF nerve conduit was found to be 15.6% ( conduits exhibited minute porosity of 5.37% (
The SF conduits saturated at a swelling ratio of 35.7% while GNP showed a maximum swelling of only 2.1% (
3.3.6 In vivo intra dermal test
The 24 hour saline extract of the GNP
not produce any abnormal clinical symptoms, oedema or erythema
The site of administration of the extract did not exhibit any abnormality as compared to the control saline group upto 72 hours from the time of intra
3.3.7 Surgical implantation of nerve conduits
The dimensions of the conduits were found to be suitable for implantation in a rat sciatic nerve. The texture and strength of the conduits were also appropriate and could withstand the implantation and suturing process without any deformation. The MRI image of the thigh region po
conduits and no deformation or delocalisation of the conduits from their implanted position was observed.
Fig 3.7: Magnetic resonance imaging of animals.
visualized (encircled area) by MRI after a week of implantation.
from Das et al, Data in Brief, 2015
3.3.5 Porosity and Swelling ratio of Nerve conduits
The porosity of SF nerve conduit was found to be 15.6% (േ 0.3 conduits exhibited minute porosity of 5.37% (10.08).
The SF conduits saturated at a swelling ratio of 35.7% while GNP showed a maximum swelling of only 2.1% (Fig 3.4).
intra dermal test
The 24 hour saline extract of the GNP-SF (Fig 3.5A) and SF conduits ( not produce any abnormal clinical symptoms, oedema or erythema
The site of administration of the extract did not exhibit any abnormality as compared to the control saline group upto 72 hours from the time of intra-dermal injection.
Surgical implantation of nerve conduits
nduits were found to be suitable for implantation in a rat sciatic nerve. The texture and strength of the conduits were also appropriate and could withstand the implantation and suturing process without any deformation. The MRI image of the thigh region post implantation reveals accurate implantation of the conduits and no deformation or delocalisation of the conduits from their implanted
: Magnetic resonance imaging of animals. The implanted conduit could be (encircled area) by MRI after a week of implantation. (Reprinted with permission from Das et al, Data in Brief, 2015)
3) while GNP-SF
The SF conduits saturated at a swelling ratio of 35.7% while GNP-SF conduits
) and SF conduits (Fig 3.5B) did not produce any abnormal clinical symptoms, oedema or erythema in the animals.
The site of administration of the extract did not exhibit any abnormality as compared dermal injection.
nduits were found to be suitable for implantation in a rat sciatic nerve. The texture and strength of the conduits were also appropriate and could withstand the implantation and suturing process without any deformation. The st implantation reveals accurate implantation of the conduits and no deformation or delocalisation of the conduits from their implanted
The implanted conduit could be Reprinted with permission
3.3.8 Functional analysis of regenerated sciatic nerve
A normal nerve was found to exhibit NCV of 59m/sec, CMAP of 17.5mV, MUP of 152μV and an ideal SFI of 0. The results of electrophysiological studies and walking track analysis are presented in
of the conduits a minute MUP of 32
after implantation the animals with nanocomposite conduits without Schwann cells (GNP-SF) exhibited NCV of 54m/sec, CMAP of 3.4mV, MUP of 105
55. Animals having pre-seeded nanocomposite conduits with Schwann cells (GNP SF cell) after 9 months showed NCV of 58m/sec, CMAP of 7.8mV, MUP of 112 and SFI of -47. The silk fibroin conduits without Schwann cells (SF) exhibited NCV of 22m/sec, CMAP of 4.3mV, MUP of 85
silk fibroin conduits with Schwann cells (SF cell) after 9 months showed NCV of 29m/sec, CMAP of 4.4mV, MUP of 105
Fig 3.8: Surgical implantation and having a 10mm gap was created.
gap and sutured to the proximal and distal ends of the nerve stump.
for conducting NCV and CMAP studies with the nerve stimulator (white) and recording electrode (black). The nerve stimulator is placed once at the distal end (
proximal end (ii) of the implanted conduit (apparent position shown in red) to record NCV through the conduit. The recording electrode is kept fixed near the ankle of the animal.
(Reprinted with permission from Das et al, Data in Brief, 2015
After 18 months, the animals implanted with GNP 42m/sec, CMAP of 16.5mV, MUP of 11
cell conduits showed NCV of 50m/sec, CMAP of 29.7mV, MUP of 133
-38.4 after 18 months. Implantation of SF conduits led to a NCV of 50m/sec, CMAP of 6mV, MUP of 87μV and SFI of
cell nerve conduits exhibited NCV of 50m/sec, CMAP of 10mV, MUP of 123 Functional analysis of regenerated sciatic nerve
A normal nerve was found to exhibit NCV of 59m/sec, CMAP of 17.5mV, MUP of nd an ideal SFI of 0. The results of electrophysiological studies and walking track analysis are presented in Fig 3.9 and Fig 3.10. Immediately post implantation of the conduits a minute MUP of 32μV was observed in all the groups. At 9 months after implantation the animals with nanocomposite conduits without Schwann cells
SF) exhibited NCV of 54m/sec, CMAP of 3.4mV, MUP of 105
seeded nanocomposite conduits with Schwann cells (GNP SF cell) after 9 months showed NCV of 58m/sec, CMAP of 7.8mV, MUP of 112
47. The silk fibroin conduits without Schwann cells (SF) exhibited NCV of ec, CMAP of 4.3mV, MUP of 85μV and SFI of -75 after 9 months. Pre
silk fibroin conduits with Schwann cells (SF cell) after 9 months showed NCV of 29m/sec, CMAP of 4.4mV, MUP of 105μV and SFI of -73.
: Surgical implantation and electrophysiological study. A, Rat sciatic injury model having a 10mm gap was created. B, The fabricated nerve conduits were implanted within the gap and sutured to the proximal and distal ends of the nerve stump. C, Experimental setup nd CMAP studies with the nerve stimulator (white) and recording electrode (black). The nerve stimulator is placed once at the distal end (
) of the implanted conduit (apparent position shown in red) to record NCV the conduit. The recording electrode is kept fixed near the ankle of the animal.
Reprinted with permission from Das et al, Data in Brief, 2015)
After 18 months, the animals implanted with GNP-SF conduits exhibited NCV of 42m/sec, CMAP of 16.5mV, MUP of 116μV and SFI of -43.4. Animals with GNP cell conduits showed NCV of 50m/sec, CMAP of 29.7mV, MUP of 133
38.4 after 18 months. Implantation of SF conduits led to a NCV of 50m/sec, CMAP V and SFI of -70 after 18 months. Animals implanted with SF cell nerve conduits exhibited NCV of 50m/sec, CMAP of 10mV, MUP of 123
A normal nerve was found to exhibit NCV of 59m/sec, CMAP of 17.5mV, MUP of nd an ideal SFI of 0. The results of electrophysiological studies and walking . Immediately post implantation V was observed in all the groups. At 9 months after implantation the animals with nanocomposite conduits without Schwann cells SF) exhibited NCV of 54m/sec, CMAP of 3.4mV, MUP of 105μV and SFI of -
seeded nanocomposite conduits with Schwann cells (GNP- SF cell) after 9 months showed NCV of 58m/sec, CMAP of 7.8mV, MUP of 112μV
47. The silk fibroin conduits without Schwann cells (SF) exhibited NCV of 75 after 9 months. Pre-seeded silk fibroin conduits with Schwann cells (SF cell) after 9 months showed NCV of
Rat sciatic injury model The fabricated nerve conduits were implanted within the , Experimental setup nd CMAP studies with the nerve stimulator (white) and recording electrode (black). The nerve stimulator is placed once at the distal end (i) and again at the ) of the implanted conduit (apparent position shown in red) to record NCV the conduit. The recording electrode is kept fixed near the ankle of the animal.
SF conduits exhibited NCV of 43.4. Animals with GNP-SF cell conduits showed NCV of 50m/sec, CMAP of 29.7mV, MUP of 133μV and SFI of 38.4 after 18 months. Implantation of SF conduits led to a NCV of 50m/sec, CMAP als implanted with SF cell nerve conduits exhibited NCV of 50m/sec, CMAP of 10mV, MUP of 123μV and
SFI of -64 after 18 months. The control group showed a more or less constant SFI of around -100 throughout their life span.
The locomotory activities of the animals implanted with nanocomposite conduits after 10 months are presented in Fig 3.11 A-D.
3.3.9 Morphological analysis of regenerated sciatic nerve
All the conduits were found to be structurally intact up to 18 months maintaining adherence to micro-sutured proximal and distal nerve ends. In the animals implanted with GNP-SF and GNP-SF cell conduits, growth of nerve along the nerve gap was complete 18 months post implantation and the regenerated nerve appeared morphologically normal (Fig 3.11 E-F). However, even after 18 months in the animals with silk fibroin conduits (both SF and SF cell) the gap along the conduit was found to be filled with tissue which appeared to be fibrous in nature (Fig 3.11 G-H).
Histological analysis of the regenerated tissue in the nanocomposite group by H&E staining revealed large recruitment of Schwann cells inside the lumen and as well as within the interlayer gaps of the conduits (Fig 3.12 A-B). Upon closer observation Schwann cells were found to be aligned in characteristic wave like fashion in the GNP-SF cell group. Fewer Schwann cells and more inflammatory cells were found inside the silk fibroin conduits (Fig 3.12 C-D). TEM analysis of the regenerated sections after 18 months showed presence of many myelinated fibers with thick myelin deposition in the nanocomposite group. However, few myelinated axons with fragmented myelin sheath was observed within the silk fibroin conduits (Fig 3.13).