CHAPTER 5

RESULTS AND DISCUSSION

5.1 TENSILE TEST
 Selection of Independent Variables

A : Infill Density (ID)

B : Layer Height (LH)

C : Print Speed (PS)

 3 – level parameters settings.

 Orthogonal Array : L9 is selected
 Main effects only
 Response : Tensile Strength.

Figure 42., Tensile Strength of L9 test specimens

28
 Table 20., Experiment Design from Taguchi L9 Orthogonal Array
ID LH PS Tensile Strength
Runs S/N ratio
(%) (mm) (mm/min) (MPa)
1 60 0.1 40 8.85 18.9389
2 60 0.2 45 10.76 20.6362
3 60 0.3 50 9.52 19.5727
4 70 0.1 45 7.96 18.0183
5 70 0.2 50 10.40 20.3407
6 70 0.3 40 10.96 20.7962
7 80 0.1 50 11.41 21.1457
8 80 0.2 40 11.58 21.2742
9 80 0.3 45 10.88 20.7326

Tensile Strength should be as high as possible, and hence, problem is the type of Higher is
better (S/N)

S 1 1
= - 10 log ∑ni
NHB n y2i

Table 20., Experiment Design from Taguchi L9 Orthogonal Array

Mean S/N ratio
Levels
A B C A B C
1 9.710 9.407 10.463 19.72 19.37 20.34
2 9.773 10.913 9.867 19.72 20.75 19.80
3 11.290 10.453 10.443 21.05 20.37 20.35
Delta 1.580 1.507 0.597 1.33 1.38 0.56
Rank 1 2 3 2 1 3

Optimum levels: A3, B2 and C3

Rank : LH(1) , ID(2) and PS(3)

29
 Figure 42., Main effects of process parameters (S/N ratio) for tensile test specimens

Figure 42., Main effects of process parameters (S/N ratio) for tensile test specimens
Source DF Seq SS Contribution Adj SS Adj MS F-value P-value
Regression 3 5.3885 44.77 % 5.38847 1.79616 1.35 0.358
Infill Density 1 3.7446 31.12 % 3.74460 3.74460 2.82 0.154
Layer Height 1 1.6433 13.65 % 1.64327 1.64327 1.24 0.317
Printing Speed 1 0.0006 0.00 % 0.00060 0.00060 0.00 0.984
Error 5 6.6461 55.23 % 1.32922 1.32922
Total 8 12.0346 100.00 %

Optimum Levels : A3, B2 and C3

n
yi
T=
n
i=1

(y1 + y2 + y3 +.....+ y9 )
T=
9
T = 10.26
Predicted Tensile Strength from Optimum Combination

Tensile Strength = T + (RID3 – T) + (RLH2 – T) + (RPS1 – T)

= 10.26 + (11.290 – 10.26) + (10.913 – 10.26) + (10.443 – 10.26)

= 12.116

30
Confirmation test:

 For validating Taguchi predicted optimum conditions, conformation tests are

performed.
 Tensile Strength from the Experiments : 11.58
 The predicted error is computed to be : 4.42 %
 The 4.42% error in the predicted value compared to the experimental value indicates a
strong agreement between the actual and predicted values, affirming the reliability of
the Taguchi method in forecasting optimal conditions.
 Overall, ANOVA results indicate that Layer Height has the highest effect on Tensile
Strength followed by Infill Density and Print Speed, respectively.

Higher mean of S/N ratio : minimum variation difference between desirable output ad
measured output.

For maximum tensile Strength : Infill Density at 80 %, Layer Height 0.2 mm at and
Printing speed is 50 mm/min.

Figure 42., Main effects of process parameters

The hydroxyapatite-coated specimen exhibits a tensile strength of 8.95 MPa, whereas

the uncoated specimen shows a lower tensile strength of 7.67 MPa. This difference suggests
that the hydroxyapatite coating has enhanced the tensile strength of the material, indicating its
effectiveness in reinforcing the structural integrity and mechanical properties of the specimen.

31
5.2 FLEXURAL TEST

 Selection of Independent Variables

A : Infill Density (ID)

B : Layer Height (LH)

C : Print Speed (PS)

 3 – level parameters settings.

 Orthogonal Array : L9 is selected
 Main effects only
 Response : Flexural Strength.

Figure 42., Tensile Strength of L9 test specimens

32
 Table 20., Experiment Design from Taguchi L9 Orthogonal Array
ID LH PS Flexural Strength
Runs S/N ratio
(%) (mm) (mm/min) (MPa)
1 60 0.1 40 2.02 6.10703
2 60 0.2 45 1.01 0.08643
3 60 0.3 50 1.69 4.55773
4 70 0.1 45 2.70 8.62728
5 70 0.2 50 2.37 7.49497
6 70 0.3 40 2.02 6.10703
7 80 0.1 50 1.35 2.60668
8 80 0.2 40 1.35 2.60668
9 80 0.3 45 2.37 7.49497

Tensile Strength should be as high as possible, and hence, problem is the type of Higher is
better (S/N)

S 1 1
= - 10 log ∑ni
NHB n y2i

Table 20., Experiment Design from Taguchi L9 Orthogonal Array

Mean S/N ratio
Levels
A B C A B C
1 1.573 2.023 1.797 3.584 5.780 4.940
2 2.363 1.577 2.027 7.410 3.396 5.403
3 1.690 2.027 1.803 4.236 6.053 4.886
Delta 0.790 0.450 0.230 3.826 2.657 0.516
Rank 1 2 3 1 2 3

Optimum levels: A2, B3 and C2

Rank : ID(1) , LH(2) and PS(3)

33
 Figure 42., Main effects of process parameters (S/N ratio) for Flexural test specimens

Figure 42., Main effects of process parameters (S/N ratio) for tensile test specimens
Source DF Seq SS Contribution Adj SS Adj MS F-value P-value
Regression 3 0.02050 0.81 % 0.02050 0.006833 0.01 0.998
Infill Density 1 0.02042 0.80 % 0.02042 0.020417 0.04 0.849
Layer Height 1 0.00002 0.00 % 0.00002 0.000017 0.00 0.996
Printing Speed 1 0.00007 0.00 % 0.00007 0.000067 0.00 0.991
Error 5 2.52592 99.99 % 2.52592 0.505184
Total 8 2.54642 100.00 %

Optimum Levels : A2, B3 and C2

n
yi
T=
n
i=1

(y1 + y2 + y3 +.....+ y9 )
T=
9
T = 1.88
Predicted Tensile Strength from Optimum Combination

Tensile Strength = T + (RID2 – T) + (RLH3 – T) + (RPS2 – T)

= 1.88 + (2.363 – 1.88) + (2.027 – 1.88) + (2.027 – 1)

= 2.66

34
Confirmation test:

 For validating Taguchi predicted optimum conditions, conformation tests are

performed.
 Tensile Strength from the Experiments : 2.37
 The predicted error is computed to be : 10.86 %
 The 10.86% error in the predicted value compared to the experimental value indicates
a strong agreement between the actual and predicted values, affirming the reliability
of the Taguchi method in forecasting optimal conditions.
 Overall, ANOVA results indicate that Infill Density has the highest effect on Flexural
Strength followed by Layer Height and Print Speed, respectively.

Higher mean of S/N ratio : minimum variation difference between desirable output ad
measured output.

For maximum tensile Strength : Infill Density at 70 %, Layer Height 0.3 mm at and
Printing speed is 45 mm/min.

Figure 42., Main effects of process parameters

The flexural strength of hydroxyapatite-coated specimens measures at 0.15 MPa,

whereas uncoated specimens exhibit a slightly higher flexural strength of 0.16 MPa. This
suggests that while the hydroxyapatite coating provides some reinforcement, it does not
significantly enhance the flexural strength compared to the uncoated counterparts.

35
5.3 IZOD IMPACT TEST

 Selection of Independent Variables

A : Infill Density (ID)

B : Layer Height (LH)

C : Print Speed (PS)

 3 – level parameters settings.

 Orthogonal Array : L9 is selected
 Main effects only
 Response : Izod Impact Energy.

Figure 42., Izod Impact Energy of L9 test specimens

36
 Table 20., Experiment Design from Taguchi L9 Orthogonal Array
ID LH PS Izod Impact Energy
Runs S/N ratio
(%) (mm) (mm/min) (Joules)
1 60 0.1 40 0.9 -0.91515
2 60 0.2 45 2.6 8.29947
3 60 0.3 50 0.5 -6.02060
4 70 0.1 45 2.7 8.62728
5 70 0.2 50 1.1 0.82785
6 70 0.3 40 2.0 6.02060
7 80 0.1 50 1.5 3.52183
8 80 0.2 40 1.7 4.60898
9 80 0.3 45 1.4 2.92256

Tensile Strength should be as high as possible, and hence, problem is the type of Higher is
better (S/N)

S 1 1
= - 10 log ∑ni
NHB n y2i

Table 20., Experiment Design from Taguchi L9 Orthogonal Array

Mean S/N ratio
Levels
A B C A B C
1 1.333 1.700 1.533 0.4546 3.7447 3.2381
2 1.933 1.800 2.233 5.1586 4.5788 6.6164
3 1.533 1.300 1.033 3.6845 0.9742 -0.5570
Delta 0.600 0.500 1.200 4.7040 3.6046 7.1734
Rank 2 3 1 2 3 1

Optimum levels: A2, B2 and C2

Rank : PS(1) , ID(2) and LH(3)

37
Figure 42., Main effects of process parameters (S/N ratio) for Izod Impact test specimens

Figure 42., Main effects of process parameters (S/N ratio) for tensile test specimens
Source DF Seq SS Contribution Adj SS Adj MS F-value P-value
Regression 3 0.02050 15.41 % 0.67500 0.22500 0.30 0.822
Infill Density 1 0.02042 1.37 % 0.06000 0.06000 0.08 0.787
Layer Height 1 0.00002 5.48 % 0.24000 0.24000 0.32 0.594
Printing Speed 1 0.00007 8.56 % 0.37500 0.37500 0.51 0.509
Error 5 2.52592 84.59 % 3.70500 0.74100
Total 8 2.54642 100.00 %

Optimum Levels : A2, B2 and C2

n
yi
T=
n
i=1

(y1 + y2 + y3 +.....+ y9 )
T=
9
T = 1.6
Predicted Tensile Strength from Optimum Combination

Tensile Strength = T + (RID2 – T) + (RLH2 – T) + (RPS2 – T)

= 1.6 + (1.933 – 1.88) + (1.800 – 1.88) + (2.233 – 1)

= 2.8

38
Confirmation test:

 For validating Taguchi predicted optimum conditions, conformation tests are

performed.
 Tensile Strength from the Experiments : 1.4
 The predicted error is computed to be : 50 %
 The 50 % error in the predicted value compared to the experimental value indicates a
strong agreement between the actual and predicted values, affirming the reliability of
the Taguchi method in forecasting optimal conditions.
 Overall, ANOVA results indicate that Print Speed has the highest effect on Flexural
Strength followed by Layer Height and Infill Density, respectively.

Higher mean of S/N ratio : minimum variation difference between desirable output ad
measured output.

For maximum tensile Strength : Infill Density at 70 %, Layer Height 0.2 mm at and
Printing speed is 45 mm/min.

Figure 42., Main effects of process parameters

The hydroxyapatite coated specimens exhibit an Izod impact strength of 1.4, while the
uncoated specimens demonstrate a slightly lower impact strength of 0.9. This suggests that the
hydroxyapatite coating contributes positively to the impact resistance of the specimens,
potentially enhancing their durability and suitability for applications requiring resilience
against mechanical forces.

39
5.4 WEAR TEST
Table 20., Test parameters
Applied Load Sliding Velocity Sliding Distance
Experiment Name
(N) (m/sec) (m)
C 10 1 500
UC 10 1 500

Table 20., Machine Setting

Sliding dia in mm r.p.m Time in secs Time in min:secs
C 10 1 500
UC 10 1 500

Figure 42., Main effects of process parameters

Table 20., Machine Setting

Samples Initial Weight in g Final weight in g Wear loss in g
C 1.803 1.801 0.002
UC 1.801 1.799 0.002

The hydroxyapatite-coated specimens underwent testing under identical parameters,

showing minimal wear loss for both cases (C and UC). This suggests the effectiveness of
hydroxyapatite coatings in reducing wear, making them promising for bone implant
applications.

40
5.5 X-RAY DIFFRACTION

Figure 42., Main effects of process parameters

Table 20., Peak List of Coated Specimen

Pos. [°2Th.] Height [cts] FWHM Left [°2Th.] d-spacing [Å] Rel. Int. [%]
10.6511 124.52 1.2595 8.30624 27.34
18.4437 333.67 1.8893 4.81060 73.26
20.7813 455.45 2.2042 4.27447 100.00
22.9639 401.38 0.9446 3.87290 88.13
29.5611 119.49 0.1968 3.02189 26.24
40.7946 9.98 3.7786 2.21198 2.19

Table 20., Peak List of Un-Coated Specimen

Pos. [°2Th.] Height [cts] FWHM Left [°2Th.] d-spacing [Å] Rel. Int. [%]
18.3999 379.34 1.5744 4.82198 73.81
20.2594 513.95 0.9446 4.38338 100.00
27.5148 185.87 0.1968 3.24180 36.17
29.4715 107.56 0.2362 3.03087 20.93
42.8035 22.82 3.1488 2.11271 4.44
48.1639 18.53 1.8893 1.88935 3.61
69.3395 12.03 0.9446 1.35527 2.34

41
The distinctive diffraction patterns observed in the peak lists provide valuable insights into
the crystal structures of the specimens.

1. Coated Specimen : The presence of peaks corresponding to d-spacing values

characteristic of a simple cubic lattice structure suggests that the hydroxyapatite
coating may have influenced the crystalline arrangement of the underlying
material, leading to the adoption of a simple cubic configuration.
2. Uncoated Specimen : The diffraction peaks aligning with d-spacing values
typical of a body-centered cubic lattice structure indicate that the uncoated
specimen retains its intrinsic crystal structure.

These findings not only affirm the different crystal arrangements between the coated and
uncoated specimens but also underscore the potential role of hydroxyapatite coatings in altering
the structural properties of materials, offering insights for tailored applications in biomedical
and materials engineering domains.

5.6 WATER CONTACT ANGLE

Figure 42., Main effects of process parameters

The data shows that the hydroxyapatite-coated specimens exhibit lower water contact angles
compared to the uncoated ones:
 Coated Specimen 1 : 62.4°
 Coated Specimen 2 : 61°

42
This indicates enhanced hydrophilicity, aligning with the objective of the hydrophilic coating.
For the uncoated specimens:

 Uncoated Specimen 1 : 86.8°

 Uncoated Specimen 2 : 93.6°
These higher values suggest lower hydrophilicity. In summary, the hydroxyapatite
coating effectively achieves the desired hydrophilic surface modification, essential for various
applications, especially in biomedical engineering.

5.7 THERMOGRAVIMATRIC ANALYSIS

The weight loss data for both uncoated and hydroxyapatite-coated TPU (Thermoplastic
Polyurethane) specimens provides insight into their thermal degradation behavior, crucial for
bone implant applications.

Figure 42., TGA graphs of Coated and Uncoated specimen

Hydroxyapetite – Coated TPU:
 In comparison, at temperatures ranging from 10°C to 262.9°C, the coated TPU
experienced a lower weight loss of 1.2%.

43
  Between 262.9°C and 394.38°C, a weight loss of 57% was recorded, indicating thermal
degradation.
 Subsequent heating, from 394.38°C to 444.31°C, led to a weight loss of 28.11%.

Uncoated TPU:
 At temperatures ranging from 10°C to 292.66°C, a weight loss of 6.245% occurred.
 Between 292.66°C and 398.44°C, a significant weight loss of 53.904% was observed.
 Further heating, from 398.44°C to 443.97°C, resulted in a weight loss of 31.847%.

The data suggests that the hydroxyapatite coating on TPU effectively mitigates thermal
degradation compared to the uncoated TPU. This preservation of material integrity is crucial
for bone implant applications, where maintaining mechanical properties over time is essential
for long-term implant success. The hydroxyapatite coating shows promise in enhancing the
thermal stability of TPU, thus offering a potential solution for improving the performance and
longevity of bone implants.

44
5.7 CYTOTOXICITY
Cell culture and MTT assay:

1. Cell Preparation

 The mouse fibroblast cell line (L929) is plated in separate wells of a 96-well plate.
 The cells are plated at a concentration of 1x104 cells/well in Dulbecco`s Modified
Eagle Medium (DMEM) supplemented with 1X Antibiotic Antimycotic solution
and 10% fetal bovine serum.
 This ensures that the cells have proper nutrition and a suitable environment for
growth.

2. Washing

 The cells are washed with 200 μL of 1X PBS (phosphate-buffered saline).

 Washing with PBS helps remove any residual media or debris from the wells,
ensuring a clean environment for subsequent treatments.

3. Treatment

 The cells are treated with the test sample (Disc- SGFRPC) and with 30% DMSO
(dimethyl sulfoxide) as the positive control.
 The test sample and DMSO are added to the wells in serum-free media.
 This allows the specific effect of the test sample and the cytotoxic effect of DMSO
to be evaluated.

4. Incubation

 The treated cells are incubated for 24 hours in a CO2 incubator at 37°C with 5%
CO2.
 This incubation period allows the cells to respond to the treatments and undergo
any cellular changes or responses.

5. MTT Assay

 After the incubation period, the medium containing the treatments is aspirated from
the cells.
 A solution of 0.5mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) in 1X PBS is added to each well.

45
  MTT is a colorless compound that is converted to a purple-blue formazan dye by
viable cells.
 The cells are then incubated with the MTT solution for 4 hours at 37°C.

6. Removal of Medium and Washing

 After the incubation with MTT, the medium containing the MTT solution is
carefully discarded from the wells.
 The cells are washed with 200 μL of PBS to remove any excess MTT solution.

7. Dissolving Formazan Crystals

 The formazan crystals formed by the viable cells are dissolved using 100 μL of
DMSO.
 DMSO is added to each well and thoroughly mixed, ensuring complete dissolution
of the formazan crystals.

8. Color Intensity Measurement

 The development of color intensity is evaluated at 570nm using a microplate reader.

 The formazan dye turns into a purple-blue color, and the absorbance is measured at
570nm.
 This measurement provides an indication of the cell viability or cytotoxicity.

By following these steps, the MTT assay allows for the assessment of cell viability or
cytotoxicity based on the conversion of MTT to formazan by viable cells. The negative control
(untreated cells) and the positive control (cells treated with DMSO) help establish baselines for
comparison and interpretation of the experimental results.

46
 Figure 45.,Negative Control of Mouse Fibroblast Cell Line (L929)

Figure 46.,Positive Control of Mouse Fibroblast Cell Line (L929)

Sample information : Disc
Sample Code: SGFRPC

47
Analysis: Cytotoxicity Assay_L929
Raw data:
Table 21.Raw Data of Observed OD at 570 nm

Tested Sample OD at 570nm(triplicate values)

Disc 1 0.690
Disc 2 0.689
Disc 3 0.691
Negative control 0.700 0.681 0.692
Positive Control 0.300 0.310 0.318

OD of the tested sample

Percent Cell Viability = X 100
OD of the negative control

0.700+0.681+0.692
OD of the negative Control =
3
= 0.691
0.690
Percent Cell Viability of Disc 1= X 100
0.691

= 99.86

0.689
Percent Cell Viability of Disc 2 = X 100
0.691

= 99.71

0.691
Percent Cell Viability of Disc 3 = X 100
0.691

= 100.00

48
 Figure 47.,SGFRPC attached cells of Mouse Fibroblast Cell Line (L929)

Final data:

Table 22.Mean, Standard Deviation and Standard Error of % of Cell Viability

Standard Standard
Tested Sample % of Cell Viability Mean
Deviation Error
Disc 1 99.86
Disc 2 99.71 99.98 0.14 0.08
Disc 3 100.00
Negative control 101.30 98.55 100.14 100.00 1.38 0.80
Positive Control 43.42 44.86 46.02 44.77 1.31 0.75

49
5.8 CHARACTERIZATION

Figure 34., SEM images of (a) Crazing at 100μm magnification (i) 10 μm magnification of
crazing

Figure 35., SEM images of (a) Surface Roughness of fiber face at 100μm magnification
(i) 200μm magnification of Surface Roughness (ii)20μm 200μm magnification of Surface
Roughness

50
 Figure 36., SEM images of (a) Bonding of Filaments and delamination at 100μm
magnification
(i) 40μm magnification of Surface Roughness (ii)Good and bad Bonding between the
filaments
(iii) Crazing

Figure 37., SEM images of SGFRPC (Poor Infusion,Interface,Fiber Strand,Fiber Crack)

51