Sustained volume retention in vivo with adipocyte and lipoaspirate seeded silk scaffolds

Abstract

Current approaches to soft tissue regeneration include the use of fat grafts, natural or synthetic biomaterials as filler materials. Fat grafts and natural biomaterials resorb too quickly to maintain tissue regeneration, while synthetic materials do not degrade or regenerate tissue. Here, we present a simple approach to volume stable filling of soft tissue defects. In this study, we combined lipoaspirate with a silk protein matrix in a subcutaneous rat model. Silk biomaterials can be tailored to fit a variety of needs, and here were processed silk biomaterials into a porous sponge format to allow for tissue ingrowth while remaining mechanically robust. Over an 18 month period, the lipoaspirate seeded silk protein matrix regenerated subcutaneous adipose tissue while maintaining the original implanted volume. A silk protein matrix alone was not sufficient to regenerate adipose tissue, but yielded a fibrous tissue, although implanted volume was maintained. This work presents a significant improvement to the standard approaches to filling soft tissue defects by matching biomaterial degradation and tissue regeneration profiles.

Copyright © 2013 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Clinically translatable process for soft tissue regeneration. (a) Porous silk sponges were prepared as described in Methods (i). The aqueous solution was lyophilized, re-dissolved in an organic solvent, and cast into a porous sponge format using a salt-leaching method (ii), cut to the desired dimensions and autoclaved. No further processing was required for unseeded groups. The seeded groups were prepared by first obtaining lipoaspirate, and clearing the fat of free oils and blood (iii). For the ASC-seeded group, ASCs were isolated by a collagenase digestion followed by an adherence selection process. ASCs were seeded onto the silk sponges and cultured for 1 month under adipogenic conditions (iv) prior to implantation. For the lipo-seeded group, the silk sponges were soaked in the processed lipoaspirate for 1 h (iv) prior to implantation. Macroscopic images of unseeded (left), ASC-seeded (middle) and lipo-seeded (right) constructs prior to implantation (v). The constructs were implanted subcutaneously in the dorsal region of a nude rat for 3, 6, 12 and 18 months. (b) Macroscopic images of constructs after 3 (top row), 6 (second row), 12 (third row) and 18 (bottom row) months in vivo, prior to being explanted. Integration with the surrounding host tissue increased when the silk sponges were pre-seeded. By 12 months, all groups were similarly integrated. (c) Functional blood vessels support regenerating tissue. H&E image of unseeded group at 6 months show functional blood vessels containing red blood cells (white arrows). The vasculature is a result of ingrowth from the host. Scale bar – 100 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Tissue regeneration occurs with silk sponge degradation. (a) The silk sponge volume was calculated by measuring the silk sponge thickness and diameter upon explantation. The volume was maintained, i.e. no biomaterial degradation, through 6 months. At 12 and 18 months, the seeded groups degraded more quickly than the unseeded group. Tissue thickness had significantly increased at 6, 12 and 18 months when compared to 3 months (* indicates p < 0.0001). Sponge size had significantly decreased at 12 months for the lipo-seeded group, and at 18 months for all groups (§ indicates p < 0.05). (b) Cross-sections of 12 (top) and 18 (bottom) month explants are shown for unseeded (left), ASC-seeded (middle) and lipo-seeded (right) groups. The region in the white dotted line outlines the silk sponge, while the black dotted line demarcates the skin from the subcutaneous tissue. The arrows point to regions of subcutaneous fat. The subcutaneous fat formation was greatest in the lipo-seeded group (right column) and least in the unseeded group (left column). Scale bar – 2 mm. (c) Approximated thickness of the newly formed tissue was determined by measuring the thickness of the fat and subtracting the thickness of the silk sponge by image analysis in ImageJ. There were no statistically significant changes in total % retention.

Fig. 3

Silk sponge pore wall thickness did not change over time. Silk sponge pores were observed by SEM. No differences in thickness were seen over time which confirmed our observation that degradation occurred from outer part of the sponge inwards. The ranges of pore wall thickness were determined by image analysis in SmartTiff (Carl Zeiss Microscopy). Average pore wall thicknesses ranged from ~ 10 to 40 µm. The overall sponge structure remained intact with open pores and tissue infiltration. Each pore wall was measured 3 times, and each sample was measured in 8 different locations per image. Scale bar – 100 µm.

Fig. 4

Histological tracking of regenerated tissue show an active tissue remodeling process. (a) H&E images at 3 (top) and 6 months (bottom) show a decrease in macrophages within the sponge from 3 to 6 months. Macrophages are seen surrounding the silk pore wall in the unseeded group (left) at 3 and 6 months. The intact silk sponge stains a dark purple and is visible in all groups. Hemosiderin deposits (dark or black deposits) were present is some sections as with the later timepoints (see Supplementary Fig. 2). Scale bar – 100 µm, inset – 200 µm. (b) Masson’s Trichrome staining for tissue organization at 3 (top) and 6 months (bottom). The intact silk sponge stains red and is visible in all groups. At 3 months, the unseeded study group (left) has a collagenous matrix (blue) that is poorly organized in comparison to the seeded groups. By 6 months (bottom) more matrix (blue) is seen. Scale bar – 100 µm, inset – 200 µm. (c) Oil Red O (ORO) for staining mature adipocytes at 3 (top) and 6 months (bottom). The intact silk sponge (asterisk) stains a light purple and is visible in all groups. Areas of ORO positive staining are pointed to with a red arrow. At 3 months (top), only the seeded groups (middle, right) were positive for ORO, the unseeded group (left) was positive for ORO only near the underlying muscle as shown. The lipo-seeded group (right) stained the most densely for ORO. Scale bar – 200 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Histological tracking of regenerated tissue shows mature adipocytes/fat formation in seeded groups at 12 and 18 months. (a) H&E images at 12 (top) and 18 months (bottom) show that fat formation was present in seeded groups. The silk sponge stains a dark purple and is visible in all groups. At 12 months, the silk sponge walls began to fracture as the sponge degraded. In some sections, dark, or black deposits of hemosiderin were seen (see Supplementary Fig. 2). Scale bar – 100 µm, inset – 200 µm. (b) Masson’s Trichrome staining for tissue organization at 12 (top) and 18 months (bottom). The silk sponge stains red and is visible in all groups. The cellularity (nuclei stain dark or black), decreased from 12 to 18 months in all groups, and then is replaced with a well-organized collagenous matrix (blue). Scale bar – 100 µm, inset – 200 µm. (c) Oil Red O (ORO) for staining mature adipocytes at 12 (top) and 18 months (bottom). The silk sponge (asterisk) stains a light purple and is visible in all groups. Areas of ORO positive staining are pointed to with a red arrow. The lipo-seeded groups (right) stained the most densely for ORO. Scale bar – 200 µm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

At 12 and 18 months in the lipo-seeded group, a large vessel was found feeding into the lipo-seeded construct from the underlying muscle. This was not seen at earlier timepoints or in other groups. Scale bar – 1 mm.