Figure B1. Circular dichroism spectroscopy to confirm secondary structure of the peptide
and to monitor assembly. Top: Dissolved KLD in 10% sucrose to 0.35 mg/mL. Added 2 or 5 µL of 0.2 N NaOH to 200 µL KLD solution to get pH 7.5 and 9, respectively. As pH increases, the peptide self-assembles. Bottom: 25 µL of PBS was added to 275 µL of 0.35% RAD.
In situ peptide assembly
Made 4 defects, front two were subchondral (5 mm diam), back two were 5 mm diam x 2 mm deep cartilage-only defects with vertical edges.
Figure B2. In situ peptide assembly and cell viability. Used 2 peptide concentrations for
subchondral defects, front-most one was 6.3 mg/mL, back one was 3.5 mg/mL. At 5 min, starting to gel but still liquid. At 8 min, the 6.3 mg/mL gel was assembled. At 12 min, the 3.5 mg/mL gel was assembled. (Used 2 µL of Coomassie blue to make the peptide purple in order to see it in the defect.) These gels stayed in the defect even after tilting the joint to fill the other defects. PBS did not wash the gels out. Used 2 peptide concentrations for non-chondral defects, front one was 6.3 mg/mL but we added 100 µL more sucrose to it since starting to clump in falcon tube, ended up looking like original 6.3 mg/mL but actually probably more like 4 mg/mL. Back defect was 3.5 mg/mL. At 8 min, both defects had assembled nicely and did not get disrupted by stream of PBS or by articulation of joint. Chondrocytes in KLD survived this procedure (Lower right).
Notes:
Blood from subchondral defect did not affect self-assembly process but these were not actively bleeding defects, so this may not reflect the surgical situation.
Joint Articulation/KLD retention study
Figure B3. In situ filling and joint articulation using 4 mg/mL KLD with Trypan blue.
Made 15 mm square defect, 1.5 mm deep on trochlear groove of bovine. Filled to edge of defect. Dripped in PBS to initiate self-assembly. Lubricated joint surface (not defect area) with PBS. Articulated joint 3 times and KLD remained in defect. Additional 3
articulations had no effect on KLD. Upper left, before fill; Upper right: fill; Lower left: after articulations.
Figure B4. In situ filling and joint articulation using 3.2 mg/mL KLD with Trypan blue.
Did same as above and peptide stayed in defect, but didn’t seem as sticky as 4 mg/mL. Left: fill; Right: after articulation.
Other conditions of Rabbit Study
Table B1. Treatment groups not included in Chapter 3.
Condition n KLD BMSCs TGF-β1 bIGF/dex HA
4 (left) + + 1.4 ng + -
4 (right) 7 + + 0.7 ng + -
5 (left) - + - - +
5 (right) 6 - - - - +
Figure B6. Gross and histologic effects comparing defects treated with
KLD+BMSCs+1.4 ng TGF-β1 in group 3 to defects treated with the same combination in group 4. Defects in group 3 had contralateral empty defects while defects in group 4 had contralateral defects treated with KLD+BMSCs+0.7 ng TGF-β1.
Figure B7. Immunohistochemistry scores (0-4). A) Aggrecan. B) Collagen II. C)
Results: Radiographic analyses pre- and post-treatment did not reveal any differences
among groups 1-3, but comparing treated defects in group 3 to the 100 ng/mL TGF-β1 defects in group 4 showed more radiographic pathology for the group 4 knees (p<0.057) demonstrating higher amounts of lysis, bony proliferation, osteophyte formation and patellar luxation in that group.
Upon necropsy, joints in groups 1-3 and 5 appeared normal, while three rabbits that received treatments in both knees (group 4) had mild to severe inflammation. Group 4 also demonstrated bony proliferation along the trochlear ridges and patellar luxation. Those receiving an intra-articular injection of BMSCs in one knee (group 5) also showed mild osteophyte formation, while rabbits with both knees treated (group 4) had mild to moderate osteophyte formation. Comparing group 3 and group 4 defects treated with the same amount of TGF-β1 (100 ng/mL), group 4 revealed significantly worse repair based on total grade (the overall quality of the repair tissue taking into account all observed factors) (p<0.019), color (p<0.019), and synovial membrane (p<0.0496), while incision appearance, inflammation and swelling, and articular surface integrity approached significance (p<0.055).
Within group 4, comparing the defect with 50 ng/mL TGF-β1 to the contralateral defect receiving 100 ng/mL TGF-β1, there were no significant differences for any of the scores. Defects receiving an intra-articular injection of BMSCs (group 5) appeared more yellow in color than those receiving HA alone (p<0.028).
Similar to gross evaluation, there were no significant differences within group 4. Knees with 100 ng/mL TGF-β1 in group 4 had worse repair tissue than treated defects in group 3 with more hypocellularity (p<0.055) and less repair tissue thickness (p<0.064). There were no significant differences within group 5, although the nature of predominant tissue and reconstitution of subchondral bone scores trended towards a worse repair for the BMSC-treated defects (p<0.07).
Comparing group 3 treated defects to group 4 defects with 100 ng/mL TGF-β1, collagen II scoring was not significant, but trended (p<0.1) towards higher scores for group 3. Within groups 4 and 5 there were no significant differences.
Discussion: In addition, treating two knees in one rabbit with BMSCs and different doses
of TGF-β1 (group 4) resulted in an increased inflammatory response and bony reaction, as seen by the comparison between this group and the treated defects in group 3 in which only one knee received TGF-β1. This finding suggests possible systemic effects of treatment and that negative effects were caused by increasing the total body dose of TGF- β1 (2.25 ng total for rabbits receiving two treatments and 1.5 ng for those with one treatment). Finally, treating defects with an intra-articular injection of BMSCs in HA (group 5) offered no advantage over injection of HA alone.