Self-synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber

Authors: Weiqing Zhan1,2, Qiang Chang1,2, Xiaolian Xiao2, Ziqing Dong2, Zhaowei Zeng2, Jianhua Gao2, MD, Feng Lu2,*

1. Weiqing Zhan and Qiang Chang contributed equally and should be considered cofirst author. 2. Department of Plastic and Cosmetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, P.R. China

*Corresponding author: Feng Lu Department of Plastic and Cosmetic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou North Road, 1838 Guangzhou, Guangdong, PR China Tel: +86 020 61641869 E-mail address: [email protected]

Keywords: adipose tissue regeneration; extracellular matrix; tissue engineering chamber

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1111/wrr.12292 This article is protected by copyright. All rights reserved.

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Funding statement: This work was supported by National Nature Science Foundation of China (81471881, 81372083, 81171834, 81201482), Key Clinical Specialty Discipline Construction Program, Health Collaborative Innovation major projects of Guangzhou (7414275040815).

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Abstract

The development of an engineered adipose tissue substitute capable of supporting reliable, predictable, and complete fat tissue regeneration would be of value in plastic and reconstructive surgery. For adipogenesis, a tissue engineering chamber (TEC) provides an optimized microenvironment that is both efficacious and reproducible; however, for reasons that remain unclear, tissues regenerated in a TEC consist mostly of connective rather than adipose tissue. Here we describe a chamber-based system for improving the yield of mature adipose tissue and discuss the potential mechanism of adipogenesis in tissue-chamber models. Adipose tissue flaps with independent vascular pedicles placed in chambers were implanted into rabbits. Adipose volume increased significantly during the observation period (week 1, 2, 3, 4, 16). Histomorphometry revealed mature adipose tissue with signs of adipose tissue remolding. The induced engineered constructs showed high-level expression of adipogenic (peroxisome proliferator-activated receptor γ), chemotactic (stromal cellderived factor 1a), and inflammatory (interleukin 1 and 6) genes. In our system, the extracellular matrix may have served as a scaffold for cell migration and proliferation, allowing mature adipose tissue to be obtained in a chamber microenvironment without the need for an exogenous scaffold. Our results provide new insights into key elements involved in the early development of adipose tissue regeneration.

Introduction

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Soft-tissue defects can be found in various pathologic cases [1,2]. Fat grafting, or lipotransfer, has become an integral part of a plastic surgeon's armamentarium for resolving both aesthetic and reconstructive problems. Lipotransfer is used in breast reconstruction after cancer treatment, and is also used to treat burn scars, congenital malformations, and post-traumatic malformations. It can also be used for achieving aesthetic treatment goals such as rejuvenation, and for treating other conditions [3,4,5]. Autologous fat is considered ideal for soft tissue augmentation because it is biocompatible, versatile, natural-looking, non-immunogenic, inexpensive, and readily obtainable with low donor site morbidity [6,7]. However, the graft survival rate remains unpredictable and often low, with resorption rates ranging from 25% to 80% [8]. The exacerbating factors include the poorly vascularized recipient bed and the high skin tension of the breast after cancer treatment. To improve graft survival, a new technique—a vacuum-based external breast expander-assisted autologous fat transfer—has been introduced. Although this procedure may be effective, completion of more than 3 operations per breast within 9 months was required on average [9].

Adipose tissue engineering may provide novel solutions for the regeneration of large-volume soft tissue defects. In 2007, researchers found significant adipose growth when a superficial inferior epigastric vascular pedicle-based adipose flap was placed into a hollow plastic chamber and then implanted subcutaneously in the groin of a rat [10]. This tissue engineering chamber (TEC) technique makes it possible to generate significant amounts of mature, vascularized and transferable adipose tissue, and hence, this technique introduces new possibilities in adipose tissue engineering. Large animal

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studies suggest that a stable, large volume adipose flap could be incubated by the TEC technique through only two operations (one for chamber implantation and one for removal), with a much shorter period of time (2 to 5 months) [11]. However, for adipose tissue engineering, an appropriate extracellular matrix (ECM) may be required to support cell attachment, proliferation, and differentiation until the adipocytes can secrete their own ECM [12]. An exogenous scaffold has been used in several studies, involving materials such as poly-lactic-co-glycolic acid (PLGA) and matrigel[11,13], which may be associated with additional health risks [14,15]. However, adipose tissue can also be generated without an exogenous scaffold [10]. In the present study, we hypothesized that the adipose tissue flap in the TEC could generate its own scaffold during the incubation period, and the self- synthesized ECM may play a similar role as an exogenous scaffold.

Materials and Methods Ethics statement All experiments were approved by the Nanfang Hospital Animal Ethics Committee Laboratory and were conducted according to the guidelines of the National Health and Medical Research Council (China). Adult New Zealand rabbits of 8 months for both sexes (1:1) weighing 2±0.5 kg were housed in specific pathogen-free conditions in the laboratory and received a stock diet and water ad libitum. The animals were maintained individually in rearing cages with appropriate temperature and light conditions.

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Tissue engineering chambers Silicon chambers were manufactured by the clinical laboratory of Nanfang Hospital (Guangzhou, China). The perforated chamber has an internal diameter of 30 mm; the flat base and dome-shaped lid clip together to form a 6-ml chamber with a dome height of 15 mm (Figure 2). The base has a 2-mm–wide silicone plate that allows the chamber to be anchored to the surrounding tissues. An opening in the wall allows for both the entry of blood vessels and exchange between the inside and outside of the chamber. A side well designed to a semicircular hole of 2 mm in diameter. Experimental procedures Animals were anesthetized by an intramuscular injection of 2 mg/kg sodium pentobarbital. The dorsal area was shaved and scrubbed with ethanol. The superficial dorsal vessels on both sides were exposed. The adipose tissue flap derived from these vessels was dissected in the dorsum along the dorsomedian line of the animal. The two flaps were then placed into the TECs and the chambers were secured beneath the dorsal skin with 6-0 Prolene (Ethicon, Inc, Somerville, NJ) holding sutures. The wounds were closed with 4-0 silk sutures. (Figure 1) Tissue harvesting and long-term stability At days 7, 14, 21, and 28 after implantation, the rabbits were anesthetized (n=7, 14 chambers per time-point) and the implanted regions were exposed. Nine other rabbits were observed for a further 12 weeks to evaluate the long-term stability of the system. The chambers were removed and the chamber contents were isolated carefully. The harvested chamber constructs were evaluated with respect to their

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volume and morphology. The anesthetized animals were killed by a sodium pentobarbital injection (15 ml of a 30 mg/ml solution) into the auricular vein. Tissue volume analysis and histology The volume of the harvested vascularized adipose tissue was determined based on the volume of fluid displacement, achieved as follows: The harvested tissue was suspended on a fine cotton suture thread and immersed entirely in a beaker of saline that had been placed on a balance. The volume was calculated by assuming a density of 1 g/ml for the displaced saline. The specimens were then cut serially into coronal sections 1 mm thick, fixed in 4% paraformaldehyde, dehydrated, and paraffinembedded with buffered formalin. A Nikon Coolpix 995 digital camera (Nikon, Tokyo) was used to capture images of the constructs. The paraffin blocks were cut into 5-µm-thick sections, which were stained with hematoxylin and eosin and assessed under an Olympus IX71 microscope (Tokyo, Japan) using the Olympus DP Controller software. Enzyme-linked immunosorbent assays (ELISAs) The levels of stromal cell-derived factor 1a (SDF-1a) and vascular endothelial growth factor A (VEGF-A) were determined using Quantikine high sensitivity ELISA kits (Yanji Biotechnology, Shanghai, China). Chamber fluid was extracted through a syringe at different times and stored at −80°C until needed. The fluid was thawed by incubation in 1 ml of phosphate-buffered saline at 4°C and then centrifuged for 20 min. The supernatant was stored at −80°C and then transferred to 4°C for use in the assay. Plates were read within 15 min on a Multiskan microplate reader (Pathtech) at 450 nm absorbance and analyzed using Genesis 2 v3.04 (Lifesciences, Cambridge, UK). Values were calculated as nanograms per liter of the original sample volume.

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Quantitative real-time polymerase chain reaction (PCR)

Total RNA was extracted from the chamber contents using Trizol and SYBR Green qPCR SuperMix (Invitrogen). The samples were treated with RNase-free DNase (Qiagen, Germany) to avoid genomic DNA contamination. A control without template was used in each experiment. cDNA was synthesized using Multiscribe reverse transcriptase (RT, Applied Biosystems, CA, USA). Quantitative RT-PCR was carried out according to the TaqMan method (50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min) with previously designed primers (Applied Biosystems) and using the Ct method for the analysis. Relative expression was normalized to that of GAPDH. Whole-mount staining of the chamber contents The engineered soft tissue was visualized as described [16]. Briefly, adipose tissue was cut into 0.5- to 1-mm pieces and incubated with the following antibodies at 4°C overnight: perilipin (H-300; sc-67164) and CD34 (sc-7045; both from Santa Cruz Biotechnology, Santa Cruz, CA), and Alexa Fluor 488-conjugated isolectin GS-IB4 (Molecular Probes). The latter was used to stain endothelial cells, which were then incubated with Alexa-Fluor-547-conjugated AffiniPure donkey anti-rabbit IgG (H+L) and donkey anti-goat IgG (H+L)-DyLight594 for 2 h and washed three times. Hoechst 33342 (Sigma, St. Louis, MO, USA) was added for 30 min to stain nuclei. Samples were washed and observed by confocal microscopy (Leica TCS SP2; Leica Microsystems, Wetzlar, Germany). The adipose progenitor cell lineage was identified by double-positive staining for CD34 and perilipin. Scanning electron microscopy of self-synthesized extracellular matrix gel

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The transparent, newly generated tissue components harvested from the chambers were immersed in 2% glutaraldehyde solution for 2 days, then fixed with osmium tetroxide, dried, and sputter-coated. Samples were viewed under a Hitachi S3000N scanning electron microscope (Japan). Statistical analysis Data are presented as the mean ± standard error of the mean (SEM). A one-way analysis of variance (ANOVA) was used to analyze the results obtained at different time points, followed by the SNK-q test. P

Self-synthesized extracellular matrix contributes to mature adipose tissue regeneration in a tissue engineering chamber.

The development of an engineered adipose tissue substitute capable of supporting reliable, predictable, and complete fat tissue regeneration would be ...
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