J Mater Sci: Mater Med (2014) 25:2669–2676 DOI 10.1007/s10856-014-5281-6
Multilayered implantation using acellular dermal matrix into nude mice Dong Won Lee • Myung Chul Lee Hyun Roh • Won Jai Lee
•
Received: 26 December 2013 / Accepted: 16 July 2014 / Published online: 24 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Soft tissue augmentation using acellular dermal matrix has gained popularity to overcome the shortcomings of autogenous and alloplastic materials. Sometimes it needs multilayered stacking to obtain enough volume. In this study, we investigated the efficacy of multilayered implantation using acellular dermal matrix (MatriDermÒ) for soft tissue augmentation. MatriDerm was implanted subdermally on each side of the dorsum of nude mice (n = 20), stacked two layers thick in the control group and three layers thick in the experimental group. Alterations of thickness, degree of angiogenesis, and collagen and elastin fiber syntheses were observed over 40 days. Three-layered implantation with MatriDerm maintained its volume similarly as in two-layered implantation, although the thickness decreased after 30 days in both groups. At the early stage of implantation, angiogenesis and collagen and elastin fiber syntheses occurred fluently on the central portion, which is the farthest away from the surface in contact with the host tissue. Collagen and elastin fibers became more concentrated over time, and the original structure of MatriDerm could not be maintained due to being replaced with newly formed collagen and elastin fibers 40 days after implantation. Multilayered implantation with MatriDerm is considered appropriate for tissue ingrowth and can be used as a substitute for soft tissue augmentation.
D. W. Lee M. C. Lee H. Roh W. J. Lee (&) Department of Plastic and Reconstructive Surgery, Institute for Human Tissue Restoration, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea e-mail: [email protected]
1 Introduction Soft tissue augmentation is not to be overlooked in the field of plastic reconstructive surgery. Materials available for the soft tissue augmentation of contour defects continue to be developed. The ideal material should match the surrounding tissue in terms of texture, pliability, and color; neither transmit nor cause any disease in the recipient; and persist and ultimately be integrated into the host tissue [1]. Soft tissue augmentation using autogenous tissues such as fat, fascia, and dermis is preferable, since they do not trigger immunological reactions or cause inflammation [2]. However such tissues are often flawed due to donor site morbidity and the limited amount of extractable tissue. Alloplastic materials such as silicone are available but are associated with risks of infection, mobility, and extrusion [3]. On the other hand, soft tissue augmentation using allogenic or xenogenic materials stands out such that it helps to overcome the shortcomings of autogenous and alloplastic materials. Many different forms of allogenic or xenogenic materials have been introduced, and acellular dermal matrix has gained popularity because it minimizes rejection compared to other forms [4, 5]. The dermal matrix has another advantage in that integration with surrounding tissues is possible. A number of experiments using dermal matrix showed that active host cell proliferation and ingrowth of collagen fibers and vessels were observed during wound healing [1, 6–9]. Implanted dermal matrix is rapidly incorporated and maintains gross volume over a period of months [1]. Numerous dermal matrix products are available for clinical use. One of these is MatridermÒ (Dr. Suwelack Skin & Health care AG, Billerbeck, Germany), which is a highly porous collagen-elastin matrix consisting of a native bovine type I collagen fiber template coated with an
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a-elastin hydrolysate derived from bovine ligamentum nuchae [5]. The structurally intact native collagen serves as an essential component of new extracellular matrix for the migration of cells and vascularization [10]. As the healing process continues, the fibroblasts produce their own collagen matrix while Matriderm is degraded. Thick, acellular dermal matrix has been clinically used for breast reconstruction and soft tissue augmentation [3, 11, 12]. Sherris et al. [3] reported successful results of human acellular dermal matrix for augmentation rhinoplasty form from a series of 51 patients. Garramone et al. [11] demonstrated that acellular dermal matrix graft improves the long-term maintenance of projection in reconstructed nipples. However, thick dermal matrix sometimes needs multilayered stacking to obtain enough volume. Contact with the host tissue is sufficient only when the matrix is a bilayer, but multilayered matrix lacks contact. Therefore, it is doubtful whether multilayered matrix can survive as matrix with sufficient contact. There have been few experiments about the survival of matrix when implanted in multilayered stacks. In this study, we observed alterations of thickness, angiogenesis, and collagen and elastin fiber syntheses when Matriderm was implanted as two- or three-layer stacks and investigated whether multilayered Matriderm could be used for soft tissue augmentation.
2 Materials and methods 2.1 Nude mouse model and experimental protocols This study was approved by the Yonsei University Medical Center’s institutional animal care and use committee. The laboratory animals were housed in a room with regulated airflow, where temperature, humidity, and light were controlled. Four- to five-week-old nude mice (BALB/cAnNCrjBgi-nu/nu, n = 20) were anesthetized by injecting ZolazepamÒ intraperitoneally. The left sides of their dorsal skin composed the control group, and the right sides composed the experimental group. After anesthesia was administered, 15-mm incisions were made on both sides, 5 mm parallel to the central line on the dorsal skin. Adequate space was secured for implantation by dissecting a 15 9 15-mm2 region subdermally. A 2-mm thickness of Matriderm was cut into a square, 10 mm on each side. Two layers of Matriderm were inserted on the left side (control group) and three layers were implanted on the right (experimental group). After insertion, the incised skin was repaired with nylon #5–0 (Johnson & Johnson, New Brunswick, US). After the insertion of two- or three-layered Matriderm, the dorsal skin with implanted Matriderm and the
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surrounding tissues were harvested at 3, 10, 20, 30, and 40 days (n = 4, all time periods). With the specimens acquired from each time period, paraffin slices, including the skin, dermal substitute, and subcutaneous tissue, were made. Specific staining methods were carried out to analyze each item: (1) hematoxylin and eosin (H&E) stain for alteration of thickness; (2) CD31 immunohistochemistry stain for angiogenesis; (3) Masson’s trichrome stain for collagen fiber synthesis; and (4) Verhoeff van Gieson (VVG) stain for elastin fiber synthesis. 2.2 Thickness of implanted multilayed dermal matrix The tissue samples were stained with H&E for histological examination. The thickness of the multilayered dermal matrix (Matriderm) was defined as the vertical distance from the innermost to the outermost surfaces of the Matriderm, and was measured at five points in each specimen and averaged. Thickness was presented according to the time period (3, 10, 20, 30, and 40 days after implantation) for the control and experimental groups, respectively. Furthermore, measured thickness was bisected for the control group and trisected for the experimental group to calculate average thickness of a single layer in each group, and the measurements were presented and compared to identify the rate of maintenance. 2.3 Neovascularization assessment within the dermal matrix To measure the extent of angiogenesis within the dermal matrix, CD31 immunohistochemistry staining was performed. Tissue sections were pretreated with a 3 % hydrogen peroxide solution for 10 min to block endogenous peroxidase and treated with protein block serum-free reagent (X0909; DAKO, Carpinteria, CA) for 30 min to prevent non-specific reactions. Sections were incubated at 4 °C overnight with primary antibodies [rabbit anti-vascular endothelial growth factor, RB-222-P, Laboratory Vision, Fremont, CA; anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM/CD31) polyclonal antibody, M20, Santa Cruz Biotechnology, Santa Cruz, CA, USA] and incubated at room temperature for 20 min with DAKO Envision Kit (DAKO) secondary antibodies. To calculate the amount of neovascularization, the CD31 positively stained vessels, which were covered with a single layer of endothelial cells and without a muscular layer, were counted, and a comparative analysis of the number of vessels at each high-power field (9200) was performed. For each slide, the number of blood vessels was measured on five different slides in the central area, which is the farthest away from the surface of the dermal matrix (in the three-layer group, the middle layer was included),
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Fig. 1 Thickness of implanted dermal matrix. a Comparison of microscopic views between the two- and three-layer groups (H&E stain, 940). b Averaged thickness according to the time period. The thickness of the three-layer group was maintained better than the two-layer group overall
and the average vascular density (the number of vessels/ high-power field) was calculated. The vascular density was presented according to the time period in each group. 2.4 Collagen and elastin fiber syntheses within the dermal matrix To analyze the elastin and collagen fibers, Masson’s trichrome and VVG staining were performed. The staining solution for Masson’s trichrome stain was prepared with Bouin’s solution (picric acid solution, 75 ml; 37 % formalin, 25 ml; glacial acetic acid, 5 ml), Weigert’s iron hematoxylin solution (hematoxylin, 1 g; 95 % ethanol, 100 ml; ferric chloride, 2 g; concentrated HCl, 1 ml; distilled water, 95 ml), Biebrich scarlet-acid fuchsin solution (1 % Biebrich scarlet, 90 ml; 1 % acid fuchsin, 10 ml; Glacial acetic acid, 1 ml), phosphomolybdic–phosphotungstic acid (phosphomolybdic acid, 2.5 g; phosphotungstic acid, 2.5 g) and an aniline blue
solution (aniline blue, 2.5 g; distilled water, 100 ml; glacial acetic acid, 2 ml); the staining solution for VVG stain was prepared with Weigert’s iron hematoxylin solution and van Gieson solution (picric acid solution, 100 ml; 1 % acid fuchsin, 10 ml). Both the arrangement and presence of collagen and elastin fibers were analyzed in the central area (in the threelayer group, the middle layer was included). A semi-quantitative analysis of the density of the collagen and elastin fibers was executed using MetaMorphÒ image analysis software (Universal Image Corporation, Buckinghamshire, UK). Results are expressed as the average optical density (OD) for five different digital images. OD quantifies the opacity of objects when exposed to transmitted light, and it can be thought of as analogous to the inverse of the grayscale values, which pertain to the amount of spectral, or reflected, light. Matriderm sheets that were not implanted were also stained to observe the arrangement and density of collagen and elastin fibers within the matrix alone.
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Fig. 2 Capillary density of implanted dermal matrix. a Comparison of microscopic views in the central area implanted dermal matrix between the two- and three-layer groups (CD31 immunohistochemistry stain, 9200). b Averaged vascular density according to the time
period. There was no significant difference between the two- and three-layer groups (P [ 0.05), meaning adequate angiogenesis of the all layers in both groups
2.5 Statistical analysis
three layers of Matriderm were implanted under the assumption that the middle layer would survive with vascular and connective tissue ingrowth throughout the upper and lower layers, which have homogeneous characteristics. The original structure of Matriderm was stable 10 days after implantation, but could not be maintained for 40 days (Fig. 1a). The average thickness of the control (two-layer group) and experimental (three-layer group) groups were 2.55 ± 0.12 and 3.86 ± 0.11 mm, respectively, after 3 days. After 40 days, the average thickness of the control and experimental groups were 1.76 ± 0.14 and 2.45 ± 0.14 mm, respectively. While three-layer grafts were thicker than two-layer grafts significantly overall (P \ 0.05), thickness decreased with time in both groups, and the difference between days 20 and 30 was statistically significant (P \ 0.05). However, there were no significant differences between thicknesses on days 30 and 40 (Fig. 1b). To compare the average thickness of a single layer in each group, measured thickness was bisected in the control group and trisected in the experimental group. As a result, the average thickness of a single layer in the control and
Paired t test was used to compare the thickness, number of new blood vessels, and quantity of collagen and elastin fibers between the two- and three-layer groups. One-way analysis of variance was also used to test whether there was any difference between time periods on those variables in each group. Significant analysis of variance results were followed by the post hoc test for pairwise comparisons, adjusted by Bonferroni correction. The statistical significance was evaluated with a 95 % confidence interval.
3 Results 3.1 Thickness of implanted dermal matrix decreases with time, but was maintained for 30 days We assumed that two layers of Matriderm could be maintained, because each layer was supplemented from the adjacent tissues above and below the implanted matrix. However,
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Fig. 3 Semi-quantitative analysis of the density of collagen fibers within the central area of implanted dermal matrix. a Histologic views of MatriDerm and normal tissue. While few collagen fibers exist within the dermal matrix before implantation, normal human dermis contains abundant collagen fibers. b The density of collagen fibers was demonstrated over time (Masson’s trichrome stain, 9200). The
arrangement of collagen fibers becomes abundant and irregular as observed in healthy connective tissue. c Averaged OD according to the time period. The amount of collagen fibers increases with time. Furthermore, there was no significant difference between the two- and three-layer groups (P [ 0.05)
experimental groups was 1.27 ± 0.06 and 1.28 ± 0.03 mm after 3 days, and 0.81 ± 0.04 and 0.88 ± 0.07 mm after 40 days, respectively. The average thickness of a single layer did not reveal any significant differences between the threeand two-layer groups during the overall experimental period.
of the experimental group as much as the control group. The numbers of CD31-positive vessels in the control and experimental groups were 8.97 ± 1.26 and 9.3 ± 1.09, respectively, after 40 days. A comparative analysis of the capillary density revealed no significant differences between the control and experimental groups (Fig. 2b).
3.2 Capillary density of multilayered dermal matrix increases with time Over time, more CD31-positive vessels within central area of grafted matrix were observed after 40 days than after 3 days with statistical significance (P \ 0.01; Fig. 2a), suggesting that neovascularization arose in the middle layer
3.3 Collagen and elastin fibers significantly increase within implanted dermal matrix Few collagen and elastin fibers exist within the dermal matrix before implantation (Figs. 3, 4a). The collagen and elastin fibers within central area of grafted matrix became
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Fig. 4 Semi-quantitative analysis of the density of elastin fibers within the implanted dermal matrix. a Histologic views of MatriDerm and normal tissue. Few elastin fibers exist within the dermal matrix before implantation, but normal human dermis contains abundant elastin fibers. b The density of elastin fibers becomes abundant with time (Verhoeff van Gieson staining, 9200). c Averaged optical
density according to the time period. Time period is demonstrated on x-axis and averaged optical density on y-axis. There is a positive correlation between averaged optical density and amount of elastin fiber. There was no significant difference between the two- and threelayer groups (P [ 0.05)
more concentrated with time, and the original structure of Matriderm could not be maintained due to replacement with new collagen and elastin fibers after 40 days. The arrangement of collagen and elastin fibers was irregular as observed in healthy connective tissues (Figs. 3, 4b). Semi-quantitative analysis revealed the average density of collagen fibers of the experimental group was 24,354 ± 2,461 OD after 3 days and 60,103 ± 3,714 OD after 40 days. The average density of elastin fibers for the threelayer group was 34,948 ± 3,172 OD after 3 days and 69,237 ± 3,719 OD after 40 days. The amount of collagen
and elastin fibers increased over time with statistical significance (P \ 0.05). The average density within central area of the control group showed a similar appearance to the experimental group, and there were no significant differences between the control and experimental groups (Figs. 3, 4c).
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4 Discussion The classical procedure for the coverage of full-thickness skin defects is autologous skin grafting. In functionally
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strained regions with high requirements for elasticity, pliability, and stability, such as joints, it is important to maintain the original tissue characteristics to preserve a full range of motion [13]. Dermal substitutes are an appropriate way to minimize scar contracture and optimize the quality of the grafted area [14]. Recently, the use of dermal matrix has been applied to soft tissue augmentation. The integration of host tissue into the implanted dermal matrix can facilitate volumetric expansion of soft tissue. There have been several reports for clinical application of acellular dermal matrix to augment soft tissues. Sclafani et al. [15]. found a 50 % decrease in the size of AlloDerm sheets within 6 months of implantation in a human study, after which the size stabilized. Gryskiewicz [16] reported longterm results of dorsal augmentation with AlloDerm, which showed that degradation occurs within 1 year and tends to be minimal. He recommended a multilayered technique rather than rolling the graft, and suggested that degradation rates were unrelated to the number of layers used. However, AlloDerm in the lip, as opposed to the nose, had a high degradation rate due to lip motion [17]. The purpose of this study was to compare the volumetric and histologic changes of multilayered Matriderm implanted subcutaneously in nude mice. Three-layered Matriderm was assigned as experimental group to clarify whether the middle layer that has not sufficient contact with host tissue can survive well. Therefore, the control group was set as two-layered Matriderm without the middle layer instead of single layered one. Comparison of average thickness in a single layer of each group did not show any significant differences between the groups as time passed, suggesting that the middle layer in the experimental group was preserved as much as the upper or lower layer. As previously mentioned, the middle layer would survive due to host endothelial cell and fibroblast ingrowth throughout the upper and lower layers. Endothelial ingrowth progresses significantly within the implanted dermal matrix of both two-layer and three-layer groups as seen by CD31 immunohistochemistry staining. A progressive increase in collagen fibers was observed, and dense collagen stroma was identified in both groups. Collagen fibers were distributed loosely and randomly, as opposed to the pathologic findings of a tissue with scar contracture, which has a parallel and regular distribution. The ingrowth of collagen and elastin fibers in the middle layer of Matriderm implies that host tissues replace the implanted dermal matrix. The integration with surrounding host tissues is a virtue of dermal matrix compared to synthetic materials such as silicone which is widely used for soft tissue augmentation clinically. Dermal matrix acts as regenerative scaffolds that undergo neovascularization and become remodeled into native host tissues [18, 19]. Above results showed that collagen and elastin fibers were newly
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formed in the grafted dermal matrix. Although dermal matrix degrades with time, it maintains the graft volume with newly formed tissues. Eventually, replaced host tissues remain and maintain volume retention. We anticipate that the thickness would be long lasting after host-derived collagen and elastin fibers replace the entire collagen-elastin matrix of Matriderm. The thickness decreased to 62.5 % by day 3 and to 42.5 % on day 40 in the three-layer group, and to 65 % on day 3 and to 40 % on day 40 in the two-layer group. The long-lasting effect may differ from the results of previous studies, because the type of implanted dermal matrix is different [8, 15]. The key point of this study is that the longlasting thickness is proportional to the degree of multiple layers. In other words, multilayered implantation with MatriDerm is considered appropriate for tissue ingrowth and can be used as a substitute for soft tissue augmentation. However, experimental results after a sufficient period of time (more than several months) are required prior to clinical application.
5 Conclusion We investigated whether tissue integration was possible in areas far from contact with the host tissue using multilayered implantation with MatriDerm. Three-layered Matriderm maintained its volume as much as two-layered Matriderm, although the thickness decreased after 30 days. Additionally, collagen and elastin fiber ingrowth from adjacent tissues was observed. These results suggest that implantation with MatriDerm could be used as a substitute for soft-tissue augmentation. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0022012, Lee WJ and No. 2013R1A1A1009764, Lee DW).
References 1. Sclafani AP, Romo T, Jacono AA, McCormick S, Cocker R, Parker A. Evaluation of acellular dermal graft in sheet (AlloDerm) and injectable (micronized AlloDerm) forms for soft tissue augmentation. Clinical observations and histological analysis. Arch Facial Plast Surg. 2000;2:130–6. 2. Tham C, Lai Y, Weng C, Chen Y. Silicone augmentation rhinoplasty in an Oriental population. Ann Plast Surg. 2005;54:1–5. 3. Sherris DA, Oriel BS. Human acellular dermal matrix grafts for rhinoplasty. Aesthet Surg J. 2011;31:95S–100S. 4. Achauer BM, VanderKam VM, Celikoz B, Jacobson DG. Augmentation of facial soft-tissue defects with Alloderm dermal graft. Ann Plast Surg. 1998;41:503–7. 5. Biesman BS, Wesley RE, Klippenstein KA, Termin P, Elson ML. Histopathologic evaluation of a new dermal allograft following explantation. Dermatol Surg. 2001;27:985–8.
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2676 6. Baxter RA. Intracapsular allogenic dermal grafts for breast implant-related problems. Plast Reconstr Surg. 2003;112:1692–6. 7. Cole PD, Stal D, Sharabi SE, Hicks J, Hollier LH. A comparative, long-term assessment of four soft tissue substitutes. Aesthet Surg J. 2011;31:674–81. 8. Hwang K, Hwang JH, Park JH, Kim DJ, Shin YH. Experimental study of autologous cartilage, acellular cadaveric dermis, lyophilized bovine pericardium, and irradiated bovine tendon: applicability to nasal tip plasty. J Craniofac Surg. 2007;18:551–8. 9. Wong AK, Schonmeyr B, Singh P, Carlson DL, Li S, Mehrara BJ. Histologic analysis of angiogenesis and lymphangiogenesis in acellular human dermis. Plast Reconstr Surg. 2008;121:1144–52. 10. Cervelli V, Brinci L, Spallone D, Tati E, Palla L, Lucarini L, De Angelis B. The use of MatriDermÒ and skin grafting in posttraumatic wounds. Int Wound J. 2011;8:400–5. 11. Garramone CE, Lam B. Use of AlloDerm in primary nipple reconstruction to improve long-term nipple projection. Plast Reconstr Surg. 2007;119:1663–8. 12. Nahabedian MY. Acellular dermal matrices in primary breast reconstruction: principles, concepts, and indications. Plast Reconstr Surg. 2012;130:44S–53S. 13. Haslik W, Kamolz LP, Manna F, Hladik M, Rath T, Frey M. Management of full-thickness skin defects in the hand and wrist
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14. 15.
16. 17. 18.
19.
region: first long-term experiences with the dermal matrix Matriderm. J Plast Reconstr Aesthet Surg. 2010;63:360–4. Yannas IV, Burke JF. Design of an artificial skin. I. Basic design principles. J Biomed Mater Res. 1980;14:65–81. Sclafani AP, Romo T, Jacono AA, McCormick SA, Cocker R, Parker A. Evaluation of acellular dermal graft (AlloDerm) sheet for soft tissue augmentation: a 1-year follow-up of clinical observations and histological findings. Arch Fac Plast Surg. 2001;3:101–3. Gryskiewicz JM. Dorsal augmentation with AlloDerm. Semin Plast Surg. 2008;22:90–103. Tobin HA, Karas ND. Lip augmentation using an alloderm graft. J Oral Maxillofac Surg. 1998;56:722–7. Lee JH, Park KR, Kim TG, Ha JH, Chung KJ, Kim YH, Lee SJ, Kang SH. A Comparative study of CG CryoDerm and AlloDerm in direct-to-implant immediate breast reconstruction. Arch Plast Surg. 2013;40:374–9. Carlson TL, Lee KW, Pierce LM. Effect of cross-linked and noncross-linked acellular dermal matrices on the expression of mediators involved in wound healing and matrix remodeling. Plast Reconstr Surg. 2013;131:697–705.
Multilayered implantation using acellular dermal matrix into nude mice Dong Won Lee • Myung Chul Lee Hyun Roh • Won Jai Lee
•
Received: 26 December 2013 / Accepted: 16 July 2014 / Published online: 24 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Soft tissue augmentation using acellular dermal matrix has gained popularity to overcome the shortcomings of autogenous and alloplastic materials. Sometimes it needs multilayered stacking to obtain enough volume. In this study, we investigated the efficacy of multilayered implantation using acellular dermal matrix (MatriDermÒ) for soft tissue augmentation. MatriDerm was implanted subdermally on each side of the dorsum of nude mice (n = 20), stacked two layers thick in the control group and three layers thick in the experimental group. Alterations of thickness, degree of angiogenesis, and collagen and elastin fiber syntheses were observed over 40 days. Three-layered implantation with MatriDerm maintained its volume similarly as in two-layered implantation, although the thickness decreased after 30 days in both groups. At the early stage of implantation, angiogenesis and collagen and elastin fiber syntheses occurred fluently on the central portion, which is the farthest away from the surface in contact with the host tissue. Collagen and elastin fibers became more concentrated over time, and the original structure of MatriDerm could not be maintained due to being replaced with newly formed collagen and elastin fibers 40 days after implantation. Multilayered implantation with MatriDerm is considered appropriate for tissue ingrowth and can be used as a substitute for soft tissue augmentation.
D. W. Lee M. C. Lee H. Roh W. J. Lee (&) Department of Plastic and Reconstructive Surgery, Institute for Human Tissue Restoration, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752, Korea e-mail: [email protected]
1 Introduction Soft tissue augmentation is not to be overlooked in the field of plastic reconstructive surgery. Materials available for the soft tissue augmentation of contour defects continue to be developed. The ideal material should match the surrounding tissue in terms of texture, pliability, and color; neither transmit nor cause any disease in the recipient; and persist and ultimately be integrated into the host tissue [1]. Soft tissue augmentation using autogenous tissues such as fat, fascia, and dermis is preferable, since they do not trigger immunological reactions or cause inflammation [2]. However such tissues are often flawed due to donor site morbidity and the limited amount of extractable tissue. Alloplastic materials such as silicone are available but are associated with risks of infection, mobility, and extrusion [3]. On the other hand, soft tissue augmentation using allogenic or xenogenic materials stands out such that it helps to overcome the shortcomings of autogenous and alloplastic materials. Many different forms of allogenic or xenogenic materials have been introduced, and acellular dermal matrix has gained popularity because it minimizes rejection compared to other forms [4, 5]. The dermal matrix has another advantage in that integration with surrounding tissues is possible. A number of experiments using dermal matrix showed that active host cell proliferation and ingrowth of collagen fibers and vessels were observed during wound healing [1, 6–9]. Implanted dermal matrix is rapidly incorporated and maintains gross volume over a period of months [1]. Numerous dermal matrix products are available for clinical use. One of these is MatridermÒ (Dr. Suwelack Skin & Health care AG, Billerbeck, Germany), which is a highly porous collagen-elastin matrix consisting of a native bovine type I collagen fiber template coated with an
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a-elastin hydrolysate derived from bovine ligamentum nuchae [5]. The structurally intact native collagen serves as an essential component of new extracellular matrix for the migration of cells and vascularization [10]. As the healing process continues, the fibroblasts produce their own collagen matrix while Matriderm is degraded. Thick, acellular dermal matrix has been clinically used for breast reconstruction and soft tissue augmentation [3, 11, 12]. Sherris et al. [3] reported successful results of human acellular dermal matrix for augmentation rhinoplasty form from a series of 51 patients. Garramone et al. [11] demonstrated that acellular dermal matrix graft improves the long-term maintenance of projection in reconstructed nipples. However, thick dermal matrix sometimes needs multilayered stacking to obtain enough volume. Contact with the host tissue is sufficient only when the matrix is a bilayer, but multilayered matrix lacks contact. Therefore, it is doubtful whether multilayered matrix can survive as matrix with sufficient contact. There have been few experiments about the survival of matrix when implanted in multilayered stacks. In this study, we observed alterations of thickness, angiogenesis, and collagen and elastin fiber syntheses when Matriderm was implanted as two- or three-layer stacks and investigated whether multilayered Matriderm could be used for soft tissue augmentation.
2 Materials and methods 2.1 Nude mouse model and experimental protocols This study was approved by the Yonsei University Medical Center’s institutional animal care and use committee. The laboratory animals were housed in a room with regulated airflow, where temperature, humidity, and light were controlled. Four- to five-week-old nude mice (BALB/cAnNCrjBgi-nu/nu, n = 20) were anesthetized by injecting ZolazepamÒ intraperitoneally. The left sides of their dorsal skin composed the control group, and the right sides composed the experimental group. After anesthesia was administered, 15-mm incisions were made on both sides, 5 mm parallel to the central line on the dorsal skin. Adequate space was secured for implantation by dissecting a 15 9 15-mm2 region subdermally. A 2-mm thickness of Matriderm was cut into a square, 10 mm on each side. Two layers of Matriderm were inserted on the left side (control group) and three layers were implanted on the right (experimental group). After insertion, the incised skin was repaired with nylon #5–0 (Johnson & Johnson, New Brunswick, US). After the insertion of two- or three-layered Matriderm, the dorsal skin with implanted Matriderm and the
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surrounding tissues were harvested at 3, 10, 20, 30, and 40 days (n = 4, all time periods). With the specimens acquired from each time period, paraffin slices, including the skin, dermal substitute, and subcutaneous tissue, were made. Specific staining methods were carried out to analyze each item: (1) hematoxylin and eosin (H&E) stain for alteration of thickness; (2) CD31 immunohistochemistry stain for angiogenesis; (3) Masson’s trichrome stain for collagen fiber synthesis; and (4) Verhoeff van Gieson (VVG) stain for elastin fiber synthesis. 2.2 Thickness of implanted multilayed dermal matrix The tissue samples were stained with H&E for histological examination. The thickness of the multilayered dermal matrix (Matriderm) was defined as the vertical distance from the innermost to the outermost surfaces of the Matriderm, and was measured at five points in each specimen and averaged. Thickness was presented according to the time period (3, 10, 20, 30, and 40 days after implantation) for the control and experimental groups, respectively. Furthermore, measured thickness was bisected for the control group and trisected for the experimental group to calculate average thickness of a single layer in each group, and the measurements were presented and compared to identify the rate of maintenance. 2.3 Neovascularization assessment within the dermal matrix To measure the extent of angiogenesis within the dermal matrix, CD31 immunohistochemistry staining was performed. Tissue sections were pretreated with a 3 % hydrogen peroxide solution for 10 min to block endogenous peroxidase and treated with protein block serum-free reagent (X0909; DAKO, Carpinteria, CA) for 30 min to prevent non-specific reactions. Sections were incubated at 4 °C overnight with primary antibodies [rabbit anti-vascular endothelial growth factor, RB-222-P, Laboratory Vision, Fremont, CA; anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM/CD31) polyclonal antibody, M20, Santa Cruz Biotechnology, Santa Cruz, CA, USA] and incubated at room temperature for 20 min with DAKO Envision Kit (DAKO) secondary antibodies. To calculate the amount of neovascularization, the CD31 positively stained vessels, which were covered with a single layer of endothelial cells and without a muscular layer, were counted, and a comparative analysis of the number of vessels at each high-power field (9200) was performed. For each slide, the number of blood vessels was measured on five different slides in the central area, which is the farthest away from the surface of the dermal matrix (in the three-layer group, the middle layer was included),
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Fig. 1 Thickness of implanted dermal matrix. a Comparison of microscopic views between the two- and three-layer groups (H&E stain, 940). b Averaged thickness according to the time period. The thickness of the three-layer group was maintained better than the two-layer group overall
and the average vascular density (the number of vessels/ high-power field) was calculated. The vascular density was presented according to the time period in each group. 2.4 Collagen and elastin fiber syntheses within the dermal matrix To analyze the elastin and collagen fibers, Masson’s trichrome and VVG staining were performed. The staining solution for Masson’s trichrome stain was prepared with Bouin’s solution (picric acid solution, 75 ml; 37 % formalin, 25 ml; glacial acetic acid, 5 ml), Weigert’s iron hematoxylin solution (hematoxylin, 1 g; 95 % ethanol, 100 ml; ferric chloride, 2 g; concentrated HCl, 1 ml; distilled water, 95 ml), Biebrich scarlet-acid fuchsin solution (1 % Biebrich scarlet, 90 ml; 1 % acid fuchsin, 10 ml; Glacial acetic acid, 1 ml), phosphomolybdic–phosphotungstic acid (phosphomolybdic acid, 2.5 g; phosphotungstic acid, 2.5 g) and an aniline blue
solution (aniline blue, 2.5 g; distilled water, 100 ml; glacial acetic acid, 2 ml); the staining solution for VVG stain was prepared with Weigert’s iron hematoxylin solution and van Gieson solution (picric acid solution, 100 ml; 1 % acid fuchsin, 10 ml). Both the arrangement and presence of collagen and elastin fibers were analyzed in the central area (in the threelayer group, the middle layer was included). A semi-quantitative analysis of the density of the collagen and elastin fibers was executed using MetaMorphÒ image analysis software (Universal Image Corporation, Buckinghamshire, UK). Results are expressed as the average optical density (OD) for five different digital images. OD quantifies the opacity of objects when exposed to transmitted light, and it can be thought of as analogous to the inverse of the grayscale values, which pertain to the amount of spectral, or reflected, light. Matriderm sheets that were not implanted were also stained to observe the arrangement and density of collagen and elastin fibers within the matrix alone.
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Fig. 2 Capillary density of implanted dermal matrix. a Comparison of microscopic views in the central area implanted dermal matrix between the two- and three-layer groups (CD31 immunohistochemistry stain, 9200). b Averaged vascular density according to the time
period. There was no significant difference between the two- and three-layer groups (P [ 0.05), meaning adequate angiogenesis of the all layers in both groups
2.5 Statistical analysis
three layers of Matriderm were implanted under the assumption that the middle layer would survive with vascular and connective tissue ingrowth throughout the upper and lower layers, which have homogeneous characteristics. The original structure of Matriderm was stable 10 days after implantation, but could not be maintained for 40 days (Fig. 1a). The average thickness of the control (two-layer group) and experimental (three-layer group) groups were 2.55 ± 0.12 and 3.86 ± 0.11 mm, respectively, after 3 days. After 40 days, the average thickness of the control and experimental groups were 1.76 ± 0.14 and 2.45 ± 0.14 mm, respectively. While three-layer grafts were thicker than two-layer grafts significantly overall (P \ 0.05), thickness decreased with time in both groups, and the difference between days 20 and 30 was statistically significant (P \ 0.05). However, there were no significant differences between thicknesses on days 30 and 40 (Fig. 1b). To compare the average thickness of a single layer in each group, measured thickness was bisected in the control group and trisected in the experimental group. As a result, the average thickness of a single layer in the control and
Paired t test was used to compare the thickness, number of new blood vessels, and quantity of collagen and elastin fibers between the two- and three-layer groups. One-way analysis of variance was also used to test whether there was any difference between time periods on those variables in each group. Significant analysis of variance results were followed by the post hoc test for pairwise comparisons, adjusted by Bonferroni correction. The statistical significance was evaluated with a 95 % confidence interval.
3 Results 3.1 Thickness of implanted dermal matrix decreases with time, but was maintained for 30 days We assumed that two layers of Matriderm could be maintained, because each layer was supplemented from the adjacent tissues above and below the implanted matrix. However,
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Fig. 3 Semi-quantitative analysis of the density of collagen fibers within the central area of implanted dermal matrix. a Histologic views of MatriDerm and normal tissue. While few collagen fibers exist within the dermal matrix before implantation, normal human dermis contains abundant collagen fibers. b The density of collagen fibers was demonstrated over time (Masson’s trichrome stain, 9200). The
arrangement of collagen fibers becomes abundant and irregular as observed in healthy connective tissue. c Averaged OD according to the time period. The amount of collagen fibers increases with time. Furthermore, there was no significant difference between the two- and three-layer groups (P [ 0.05)
experimental groups was 1.27 ± 0.06 and 1.28 ± 0.03 mm after 3 days, and 0.81 ± 0.04 and 0.88 ± 0.07 mm after 40 days, respectively. The average thickness of a single layer did not reveal any significant differences between the threeand two-layer groups during the overall experimental period.
of the experimental group as much as the control group. The numbers of CD31-positive vessels in the control and experimental groups were 8.97 ± 1.26 and 9.3 ± 1.09, respectively, after 40 days. A comparative analysis of the capillary density revealed no significant differences between the control and experimental groups (Fig. 2b).
3.2 Capillary density of multilayered dermal matrix increases with time Over time, more CD31-positive vessels within central area of grafted matrix were observed after 40 days than after 3 days with statistical significance (P \ 0.01; Fig. 2a), suggesting that neovascularization arose in the middle layer
3.3 Collagen and elastin fibers significantly increase within implanted dermal matrix Few collagen and elastin fibers exist within the dermal matrix before implantation (Figs. 3, 4a). The collagen and elastin fibers within central area of grafted matrix became
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Fig. 4 Semi-quantitative analysis of the density of elastin fibers within the implanted dermal matrix. a Histologic views of MatriDerm and normal tissue. Few elastin fibers exist within the dermal matrix before implantation, but normal human dermis contains abundant elastin fibers. b The density of elastin fibers becomes abundant with time (Verhoeff van Gieson staining, 9200). c Averaged optical
density according to the time period. Time period is demonstrated on x-axis and averaged optical density on y-axis. There is a positive correlation between averaged optical density and amount of elastin fiber. There was no significant difference between the two- and threelayer groups (P [ 0.05)
more concentrated with time, and the original structure of Matriderm could not be maintained due to replacement with new collagen and elastin fibers after 40 days. The arrangement of collagen and elastin fibers was irregular as observed in healthy connective tissues (Figs. 3, 4b). Semi-quantitative analysis revealed the average density of collagen fibers of the experimental group was 24,354 ± 2,461 OD after 3 days and 60,103 ± 3,714 OD after 40 days. The average density of elastin fibers for the threelayer group was 34,948 ± 3,172 OD after 3 days and 69,237 ± 3,719 OD after 40 days. The amount of collagen
and elastin fibers increased over time with statistical significance (P \ 0.05). The average density within central area of the control group showed a similar appearance to the experimental group, and there were no significant differences between the control and experimental groups (Figs. 3, 4c).
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4 Discussion The classical procedure for the coverage of full-thickness skin defects is autologous skin grafting. In functionally
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strained regions with high requirements for elasticity, pliability, and stability, such as joints, it is important to maintain the original tissue characteristics to preserve a full range of motion [13]. Dermal substitutes are an appropriate way to minimize scar contracture and optimize the quality of the grafted area [14]. Recently, the use of dermal matrix has been applied to soft tissue augmentation. The integration of host tissue into the implanted dermal matrix can facilitate volumetric expansion of soft tissue. There have been several reports for clinical application of acellular dermal matrix to augment soft tissues. Sclafani et al. [15]. found a 50 % decrease in the size of AlloDerm sheets within 6 months of implantation in a human study, after which the size stabilized. Gryskiewicz [16] reported longterm results of dorsal augmentation with AlloDerm, which showed that degradation occurs within 1 year and tends to be minimal. He recommended a multilayered technique rather than rolling the graft, and suggested that degradation rates were unrelated to the number of layers used. However, AlloDerm in the lip, as opposed to the nose, had a high degradation rate due to lip motion [17]. The purpose of this study was to compare the volumetric and histologic changes of multilayered Matriderm implanted subcutaneously in nude mice. Three-layered Matriderm was assigned as experimental group to clarify whether the middle layer that has not sufficient contact with host tissue can survive well. Therefore, the control group was set as two-layered Matriderm without the middle layer instead of single layered one. Comparison of average thickness in a single layer of each group did not show any significant differences between the groups as time passed, suggesting that the middle layer in the experimental group was preserved as much as the upper or lower layer. As previously mentioned, the middle layer would survive due to host endothelial cell and fibroblast ingrowth throughout the upper and lower layers. Endothelial ingrowth progresses significantly within the implanted dermal matrix of both two-layer and three-layer groups as seen by CD31 immunohistochemistry staining. A progressive increase in collagen fibers was observed, and dense collagen stroma was identified in both groups. Collagen fibers were distributed loosely and randomly, as opposed to the pathologic findings of a tissue with scar contracture, which has a parallel and regular distribution. The ingrowth of collagen and elastin fibers in the middle layer of Matriderm implies that host tissues replace the implanted dermal matrix. The integration with surrounding host tissues is a virtue of dermal matrix compared to synthetic materials such as silicone which is widely used for soft tissue augmentation clinically. Dermal matrix acts as regenerative scaffolds that undergo neovascularization and become remodeled into native host tissues [18, 19]. Above results showed that collagen and elastin fibers were newly
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formed in the grafted dermal matrix. Although dermal matrix degrades with time, it maintains the graft volume with newly formed tissues. Eventually, replaced host tissues remain and maintain volume retention. We anticipate that the thickness would be long lasting after host-derived collagen and elastin fibers replace the entire collagen-elastin matrix of Matriderm. The thickness decreased to 62.5 % by day 3 and to 42.5 % on day 40 in the three-layer group, and to 65 % on day 3 and to 40 % on day 40 in the two-layer group. The long-lasting effect may differ from the results of previous studies, because the type of implanted dermal matrix is different [8, 15]. The key point of this study is that the longlasting thickness is proportional to the degree of multiple layers. In other words, multilayered implantation with MatriDerm is considered appropriate for tissue ingrowth and can be used as a substitute for soft tissue augmentation. However, experimental results after a sufficient period of time (more than several months) are required prior to clinical application.
5 Conclusion We investigated whether tissue integration was possible in areas far from contact with the host tissue using multilayered implantation with MatriDerm. Three-layered Matriderm maintained its volume as much as two-layered Matriderm, although the thickness decreased after 30 days. Additionally, collagen and elastin fiber ingrowth from adjacent tissues was observed. These results suggest that implantation with MatriDerm could be used as a substitute for soft-tissue augmentation. Acknowledgments This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0022012, Lee WJ and No. 2013R1A1A1009764, Lee DW).
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