Percutaneous collagen induction–regeneration in place of cicatrisation?  M.C. Aust, K. Reimers, H.M. Kaplan, F. Stahl, C. Repenning, T. Scheper, S. Jahn, N. Schwaiger, R. Ipaktchi, J. Redeker, M.A. Altintas, P.M. Vogt  Journal of Plastic, Reconstructive & Aesthetic Surgery  Volume 64, Issue 1, Pages 97-107 (January 2011) DOI: 10.1016/j.bjps.2010.03.038 Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 1 a) Medical Roll-Cit (made by Vivida C.C. Renaissance Body Science Institute, Cape Town South Africa). (b) Schematic image of the procedure. (c) Rat with shaved back: Preoperative: untreated skin. (d) Rat with shaved back: 1h postoperative. An intradermal bleeding and bruising is visible. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 2 Microphotographs taken of skin samples stained with Haematoxylin–Eosin. The size of the scale bar is 200μm (representative example). (a) Untreated animal (control). (b) Unneedled animal with 8 weeks of skincare. (c) Needled animal without skincare. (d) Needled animal with 8 weeks of skincare. The epidermal thickening (up to 140% after 8 weeks in group (D)) coincided with increased thickening of the granular layer, an increased number of epidermal cell layers and a more compact stratum corneum. The control group (A) showed a uniform mean epidermal thickness of 13.0μm through the entire set. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 3 Masson’s Trichrome staining. The size of the scale bar is 2050μm (representative example). (a) Untreated animal (control). (b) Unneedled animal with 8 weeks of skincare. (c) Needled animal without skincare. (d) Needled animal with 8 weeks of skincare Collagen fibere bundles were increased, thickened, and more loosely woven in both the papillary and reticular dermis most prominently in the needled plus skincare group (D). Elastin fibres in the dermis highly linear and the epidermal–dermal interface showed regular dermal papillae; cellular polarity and normal epidermal differentiation appeared to be maintained; and the elastin network within the reticular dermis was regularly thickened and organized in all groups. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 4 Selected genes of group (D) against untreated control rats (group (A)). Shown are ratios of the expression values of different time points after needling against untreated rats. Ratios between 0 and 1 have been transformed to negative values for a better comparison. Standard errors are marked by error bars. A bold border shows the significant change in gene expression. Please see also significant modification (calculated by t-test) in Table 2. Collagen I levels were quantitatively increased in treated skin (group (D)) in comparison to normal untreated samples (group (A)) with very high significance (p value less than 10e-5) at all time points. In contrast to that collagen III is only significantly increased at time point 4 weeks (p-value=0.00183) and not significantly changed 2 and 8 weeks after treatment. FGF7 was found to be strongly increased in group (D) 2 weeks after the treatment and modestly increased 4 weeks and not significantly changed 8 weeks after needling, whereas EGF was not significantly increased. The results support our hypothesis that the expression levels of collagens qualitatively increase after PCI treatment. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 5 Immunofluorescence visualization of collagen I: Staining with antibodies directed against Collagen I (Alexa488) and DAPI. The size of the scale bar is 100μm (representative example). (a) Unneedled animal with 8 weeks of skincare areas of the dermis failed to react with the antibodies. (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare abundant collagen present throughout the dermis. (d) Needled animal with 8 weeks of skincare stained without primary antibody. The amount of type I collagen was qualitatively increased in treated group (Figure 5c) compared to their controls judged by the brighter fluorescence. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 6 Immunofluorescence visualization of collagen III demonstrated in the dermal zone just beneath the epidermis (Alexa488-conjugated antibody) and DAPI. The size of the scale bar is 100μm (representative example). (a) Unneedled animal with 8 weeks of skincare Collagen III is still detectable 8 weeks after treatment. (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare only small amounts of type III collagen were found 8 weeks after needling. (d) Needled animal with 8 weeks of skincare stained without primary antibody. While the amount of type III collagen was qualitatively increased particularly in the needled group 2 weeks and 4 weeks after the operation compared to their controls (data not shown), only small amounts of type III collagen were found in set 8 weeks postoperatively (Figure 6c). In the unneedled sample collagen III was still detectable 8 weeks after treatment (Figure 6a). Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 7 Microphotographs taken of skin samples stained with PAS. The size of the scale bar is 2100μm (representative example). (a) Untreated animal (control). (b) Unneedled animal with 8 weeks of skincare. (c) Needled animal without skincare. (d) Needled animal with 8 weeks of skincare. The epidermal thickening coincided with increased thickening of the granular layer, an increased number of epidermal cell layers, and a more compact stratum corneum. The staining intensity observed in samples derived from needled skin (7c and 7d) was deepened compared to the unneedled samples (7a and 7b). The staining pattern was most regular and intensified in the needling plus skincare group (7d). Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 8 Immunofluorescence visualization of GAGs (Alexa488-conjugated) and DAPI. The size of the scale bar is 100μm (representative example). (a) Unneedled animal with 8 weeks of skincare. GAGs showed dense deposits occupying much of the dermis, leaving only isolated collagen bundles visible (white arrows). (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare GAGs were observed, appearing at the dermal–epidermal junction and interspersed between the abundant collagen bundles in the papillary and reticular dermis (white arrows). (d) Needled animal with 8 weeks of skincare stained without primary antibody. As observed in the PAS staining a marked increase in the amount of GAGs was observed throughout the different needled groups in comparison to the unneedled groups. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 9 Immunofluorescence visualization of fibronectin in the epidermis and dermis (Alexa488-conjugated) and DAPI. The size of the scale bar is 100μm (representative example). (a) unneedled animal with 8 weeks of skincare. (b) unneedled animal with 8 weeks of skincare stained without primary antibody. (c) needled animal with 8 weeks of skincare increase in the amount of fibronectin in the epidermis and dermis. (d) needled animal with 8 weeks of skincare stained without primary antibody. Positive staining for fibronectin was observed in the papillary dermis of all groups, but again there is an intense staining in the needled samples and a comparably weaker signal in the unneedled ones. Together with the quantitative data this indicates that fibronectin expression was markedly increased in the dermis of all needled groups compared to the unneedled groups. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions

Figure 10 Immunofluorescence visualization of VEGF observed in the epidermis and dermis (Alexa488-conjugated) and DAPI. The size of the scale bar is 100μm (representative example). (a) Unneedled animal with 8 weeks of skincare. (b) Unneedled animal with 8 weeks of skincare stained without primary antibody. (c) Needled animal with 8 weeks of skincare increase in the amount of VEGF in the epidermis and dermis. (d) Needled animal with 8 weeks of skincare stained without primary antibody. VEGF showed a membranous staining pattern along the intercellular junctions in the basal and suprabasal layers of the epidermis. A brighter fluorescence indicates that the amount of VEGF in the dermis is augmented in the needled groups compared to the unneedled groups. Journal of Plastic, Reconstructive & Aesthetic Surgery 2011 64, 97-107DOI: (10.1016/j.bjps.2010.03.038) Copyright © 2010 British Association of Plastic, Reconstructive and Aesthetic Surgeons Terms and Conditions