Wake Forest Institute for Regenerative Medicine
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1 Evaluation of Algeness as a Dermal Filler Material November 2014 Final Report John D. Jackson, PhD, Associate Professor, Institute for Regenerative Medicine Institute, Wake Forest Baptist Medical Center Introduction This is a final report of the comparison between the injection of Algeness and Juvederm in a mouse model. The objective of the study was to compare the longevity/persistence and adverse effects of the dermal filler materials. Methods Female C57Bl/6 mice were used in this study. Time points included in the study were 1 week, 1 month, 3 months and 6 months post-injection. Six mice were included at each time point for a total of 24 mice. Each mouse received one injection of Juvederm Ultra Plus ( Juvederm ), one injection of Algeness, and one injection of phosphate-buffered saline (PBS) subcutaneously. The injection volume was 0.25 ml for each material. At each time point, three mice received the Juvederm injection in the left craniodorsal quadrant, the Algeness injection in the mid-laterodorsal quadrant, and PBS in the left caudodorsal quadrant. The remaining three mice received the Algeness injection in the left craniodorsal quadrant, the Juvederm injection in the mid-laterodorsal quadrant, and PBS in the left caudodorsal quadrant. Changes in the site of injections were performed to reduce any site specific effects on the injected material. At the time of retrieval, the material was measured using digital calipers to determine the volume. The volume was calculated using the formula: VV = ππ 6 LL WW HH where L is length, W is width and H is the height of the recovered material. The retrieved material was also weighed. The material pliability was scored on a sale of 1 to 4 with 1 being the softest and 4 being the hardest. The pliability of the injected material was determined by the same individual for all samples to maintain consistency during the study. After determining size, weight and pliability, the harvested material was processed for histology. Part of the material was fixed in 10% formalin and prepared for paraffin embedding. These samples were sectioned and stained with hematoxylin and eosin (H&E) to show the overall appearance of the injected material and with Masson s Trichrome to identify collagen deposition. The remaining material was processed for frozen sections that were used for the CD68 and Ly6g immunohistochemistry staining. CD68 is expressed by macrophages and Ly6g is expressed by neutrophils. 1
2 Results The injections were completed without any adverse events. There were adequate amounts of both the Juvederm and the Algeness filler material. Algeness filler had the consistency similar to petroleum jelly. This consistency did not allow the use of the supplied 27 gauge needle. A 26 gauge needle was used that allowed for better extrusion of the material. The Algeness syringes did not have calibrations and required transfer to marked syringes for appropriate measurement. There was some product loss due to this step. Additionally, this transfer and the high viscosity of the material caused some air entrapment in the materials that reduced the uniformity of the injection. The Juvederm was more smoothly and consistently injected. The calibrations on the syringe barrel were helpful in determining the volume administered. The PBS injection was rapidly absorbed and showed negligible volumetric change. Generally, Juvederm appeared to migrate to a more extent than Algeness, particularly at the later time points. Because of this migration and spreading, it became more difficult to locate the injected material at the later time points. Therefore, the measurements (volume and weight) have increased error associated with their values. Physical Measures of the Injected Material At harvest, the volume of Juvederm and Algeness decreased over time (Figure 1). Juvederm had a greater volume initially but decreased to similar values for Algeness. The weight of the harvested Juvederm material decreased over time that matched the volume measurements (Figure 2). However, the weight of the harvested Algeness material maintained a consistent level over time. The pliability of the Juvederm decreased over time; whereas the Algeness maintained a more consistent pliability score (Figure 3). 2
3 Volume of Dermal Filler Juvederm Algeness 400 Volume (mm 3 ) Figure Weeks Post-Injection Weight of Dermal Filler Juvederm Algeness Weight (g) Figure Weeks Post-Injection 3
4 Material Pliability Juvederm Algeness 2.5 Pliability Rating (1 to 4) Figure Weeks Post-Injection Overall, these results suggest that Algeness maintained a more consistent weight and pliability than Juvederm, even though the volume of both materials decreased over time. The injected Algeness material did not migrate post-injection to the extent that occurred with Juvederm. Histological analysis By week 1 post-injection, cellular infiltration is seen in both materials that includes some immune cells (macrophages) and what appear to be stromal elements (probably fibroblasts) (Figures 4 and 5). No collagen deposition was seen (Figure 4). At week 4 post-injection, a lattice work of cells can be seen in the injected material from both Algeness and Juvederm (Figure 6). These cells appear to be fibroblasts since collagen deposition is seen (Figure 6). Immune cells are present in both injected materials (Figure 7). At week 12, Algeness has maintained its lattice of stromal cells; however, Juvederm has become more disorganized (Figure 8), potentially due to the increased migration of Juvederm. Collagen is also present in both materials (Figure 8). Algeness does not appear to have an immune infiltration by week 12 (Figure 9). A few CD68 positives cells can be seen in the Juvederm sample (Figure 9). At week 24 post-injection, Algeness has maintained the same morphological appearance that was seen at the earlier time points (Figure 10). Juvederm appears to have significant collagen deposition (Figure 10). No immune cells can be seen in either of the materials at this time point (Figure 11). 4
5 Figure 4. H&E and Masson s Trichrome staining of paraffin embedded sections of Algeness and Juvederm at 1 week post-injection (200x magnification). Figure 5. CD68 staining of frozen sections at 1 week post-injection. (200x magnification) 5
6 Figure 6. H&E and Masson s Trichrome staining of paraffin embedded sections of Algeness and Juvederm at 4 weeks post-injection (200x magnification). 6
7 Figure 7. CD68 and Ly6g staining of frozen sections at 4 weeks post-injection. (200x magnification) 7
8 Figure 8. H&E and Masson s Trichrome staining of paraffin embedded sections of Algeness and Juvederm at 12 weeks post-injection (200x magnification). 8
9 Figure 9. CD68 and Ly6g staining of frozen sections at 12 weeks post-injection. (200x magnification) 9
10 Figure 10. H&E and Masson s Trichrome staining of paraffin embedded sections of Algeness and Juvederm at 24 weeks post-injection (200x magnification). 10
11 Figure 11. Ly6g staining of frozen sections at 24 weeks post-injection. (200x magnification) Conclusion Algeness maintained a more consistent presence at the injection site. Although there was some migration of the Algeness ; significantly more migration of Juvederm was seen at the later time points. Juvederm had a more disorganized appearance at 12 and 24 weeks post-injection. Both materials had collagen deposition by week 4 post-injection. Immune cells infiltrated both materials during the early time points, but no overt immune response was seen at the later time points in either injected materials. No immune cells were detected by 24 weeks post-injection in either material. Algeness is a product of Advanced Aesthetic Technologies, Inc., Suite 427, One Brookline Place, Brookline, MA, USA Juvederm is a product of Allergan Inc, Irvine, CA USA 11
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