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1. Testing Accommodation
A. The physis was extracted from the calf distal ulna and sliced into .5 mm thick sections
B. One section of the physis was stained using the Live/Dead Assay (Thermo Fisher, Catalog number: L3224) and imaged immediately after extraction. Another section of the physis was stained and imaged after being cultured for 7 days (Figure 5).
C. The amount of live and dead chondrocytes were counted using the ImageJ Software (NIH, version 1.51j8) and are tabulated below (Table 3).
Figure 5: Representative Live/Dead images of physeal tissue sections. (A) A region of live and (B) dead chondrocytes in the reserve zone of a physeal tissue section that was stained and imaged immediately after extraction (0 days in culture). (C) A region of live and (D) dead chondrocytes in the reserve zone of a physeal tissue section that was stained and imaged immediately after 7 days of culture.
Table 3: Percent of Live and Dead chondrocytes in physeal tissue sections after 0 days and 7 days of culturing
2. Testing Mechanical Loading
A. The mechanical loading system was constructed using slide covers, silicone sheets, clamps, Teflon, and a spring. (Figure 6)
B. A 1mm thick distal calf ulnar physis was placed into the system and imaged before and after loading.
C. Lengths of the unloaded and loaded sides of the tissue were compared before and after loading using the ImageJ software (NIH, version 1.51j8). (Table 4)
D. Asymmetric compression of the tissue was observed and quantified. (Figure 7)
Figure 6: Conceptualization of mechanical loading mechanism within the bioreactor
Table 4: Mechanical Loading Displacement Results
Figure 7: Test of the mechanical loading system. (A) Mechanical loading system with the physis before loading (unloaded). (B) Mechanical loading system with the physis being asymmetrically loaded (unloaded).
3. Verifying Chemical Modulation
A. To mimic arterial invasion normally found on both sides of the physis, an O2 gradient had to be formed from the metaphyseal to epiphyseal ends.
B. Using Fick’s 2nd law, a series of governing equations was used to determine the O2 profile within the physis as well as the media. (Figure 8)
Governing Equations
Figure 8: Conceptualization of O2 gradient within the bioreactor
4. Verifying Visualization
A. To visualize the physis with a microscope as it sits vertically, the line of sight was redirected at a 90 degree angle. (Figure 9)
B. This was to be achieved by using a prism to reflect the line of sight from the objective lenses towards the sample sitting upright.
Figure 9: Conceptualization of integrated mechanical loading/visualization bioreactor components
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