Experiment (1)

(a) (b)

(c) (d)

(e) (f)

Figure 3.16 The progress of BMSC after adding osteogenic differentiated media. (a), (b) and (c) Images for not- ready cells for Day 2 Well1, Day 5 Well1 and Day 7 Well1 respectively. (d), (e) and (f) Images for ready cells for

Day 10 Well1, Day 13 Well1 and Day 15 Well1 respectively.

Results: It started to form differentiated stem cells as in Figure 3.16(d), where all the cells are connected all together through the whole well to give an indication that stem cells are formed and are ready for implantation from Day 10.

3.6.2 Experiment (2)

(a) (b)

(c) (d)

(e) (f)

Figure 3.17 (a),(b),(c) Images for not-ready cells of Day2 Well2, Day5 Well2, and Day7 Well2 respectively. (d),(e) and (f) Images for ready cells for Day 11 Well2, Day 13 Well2 and Day 15 Well2 respectively.

Results: It started to form differentiated stem cells as in Figure 3.17(d), where all the cells are connected all together through the whole well to give an indication that stem cells are formed and are ready for implantation from Day 11.

3.6.3 Experiment (3)

(a) (b)

(c) (d)

Figure 3.18 (a) and (b) Images for not-ready cells for Day 5 Well3 and Day 7 Well3 respectively. (c) and (d) Images for ready cells for Day 10 Well3 and Day 13 Well3 respectively.

Results: The differentiated stem cells forms a colony from Day 10 to give an indication of ready osteogenic differentiated stem cells to be implanted as shown in Figure 3.18(c) and Figure 3.18(d).

3.6.4 Experiment (4)

(a) (b)

(c) (d)

(e)

Figure 3.19 (a), (b),(c) and (d) Images for not-ready cells for Day 2 Well4, Day 5 Well4, Day 7 Well4 and Day 9 Well4 respectively. (e) Image for ready cells Day 13 Well4.

Results: The differentiated stem cells forms a colony at Day 13 to give an indication of ready osteogenic differentiated stem cells to be implanted as shown in Figure 3.19(e).

3.6.5 Experiment (5)

(a) (b)

(c) (d)

Figure 3.20 (a) and (b) Images for not-ready cells for Day 4 Well5 and Day 6 Well5 respectively. (c) and (d) Images for ready cells for Day 10 Well5 and Day 13 Well5 respectively.

Results: It started to form differentiated stem cells from Day 10 as in Figure 3.20(c). All cells are connected together to indicate that they are ready for implanting.

3.6.6 Experiment (6)

(a) (b) (c)

(d) (e) (f)

(g)

Figure 3.21 (a),(b) Images of not-ready cells for Day 5 Well6 and Day 7 Well6 respectively. (c), (d), (e), (f) Images for ready cells for Day 9 Well6, Day 10 Well6, Day 13 Well6, Day 15 Well6 and Day 17 respectively.

Results: The differentiated stem cells forms a colony from Day 9 to give an indication of ready osteogenic differentiated stem cells to be implanted as shown in Figure 3.21(c).

3.6.7 Experiment (7)

(a) (b)

(c) (d)

Figure 3.22 (a), (b) Images for not-ready cells for Day 2 Well7 and Day 6 Well7 respectively. (c) and (d) Images for ready cells for Day 10 Well7 and Day 12 Well7 respectively.

Results: The differentiated stem cells forms a colony from Day 10 to give an indication of ready osteogenic differentiated stem cells to be implanted as shown in Figure 3.23.

The question that arises is when these cells will be ready for implantation. These are ready when they form colonies as in Figure 3.18(c),Figure 3.19(e), Figure 3.21(c) and Figure 3.22(c) or when all cells are connected together to be all spread in the well as in Figure 3.16(d), Figure 3.17(d),and Figure 3.20(c). If these osteogenic differentiated stem cells were left for few more days, they could be affected by bacteria. If this happened, everything should be discarded, get rid of the wells, flask and also the cell culture should be sterilized to start all over again.

Seven experiments (seven wells) were done at the Tissue Engineering Center to give a total of 36 images, Table 3.1 shows each experiment with it’s dates that the images were taken at. “” stands for not-ready cells and “√” stands for ready cells.

Table 3.1 Clinician’s classification of the stem-cell images. Number of Experiments Number of Days Day 2 Day 4 Day 5 Day 6 Day 7 Day 9 Day 10 Day 11 Day 12 Day 13 Day 15 Day 17 Experiment 1    √ √ √ Experiment 2    √ √ √ Experiment 3   √ √ Experiment 4     √ Experiment 5   √ √ Experiment 6   √ √ √ √ √ Experiment 7   √ √

It can be seen that in most of the experiments the stem-cell started to be ready after Day 7. Experiments 1, 3, 5, and 7 they were considered ready on Day 10, Experiment 6 on Day 9, and Experiment 2 on Day 11. The clinicians were undecided about the image of Day 9 in Experiment 4, so decided to classify it as not-ready for another day.

It was observed for each of the experiments that images became classified as ready for implantation after approximately 10days, and then remained ready in the ready state for the duration of the experiment.

As stated earlier, the total number of images from the seven experiments that were done in the Tissue Engineering Center were 36 images of dimension 400×400. The osteogenic differentiated stem cells images are predominantly textural, and do not rely on definite shape to be detected, which lead to the idea of dividing each image into four equal parts as shown in Figure 3.24 and Figure 3.25 on the next page. This will increase the number of images in the dataset base to 144 images.

(a) (b)

Figure 3.24 An image for not-ready osteogenic differentiated stem cells Day 4 well 5. (a) The original image with a dimension of 400×400. (b) The four equal parts after dividing the original image each part of

dimension 200×200.

(a) (b)

Figure 3.25 An image for ready osteogenic differentiated stem cells Day 13 Well 5. (a) The original image with a dimension of 400×400. (b) The four equal parts after dividing the original image each part of

3.7 Conclusion

This Chapter described the Methodology that has been used in this study, and then concentrated on the preparation of the cell cultures and the collection of the Image Dataset. The seven experiments that were done gave a total of 36 images where 18 images were for the ready osteogenic differentiated stem cells while the other 18 were not-ready osteogenic differentiated stem cells. As the images were predominantly textural, the size of the dataset was increased by a factor of 4 by splitting each image into four sub-images of 200×200 pixels. The next Chapter will deal with the image pre-processing and feature extraction techniques that were applied to the images of differentiated stem cells.