Elsevier

Acta Biomaterialia

Volume 10, Issue 5, May 2014, Pages 1907-1918
Acta Biomaterialia

The adipogenic potential of various extracellular matrices under the influence of an angiogenic growth factor combination in a mouse tissue engineering chamber

https://doi.org/10.1016/j.actbio.2013.11.019Get rights and content

Abstract

The extracellular matrix (ECM) Matrigel™ has frequently and successfully been used to generate new adipose tissue experimentally, but is unsuitable for human application. This study sought to compare the adipogenic potential of a number of alternative, biologically derived or synthetic ECMs with potential for human application, with and without growth factors and a small fat autograft. Eight groups, with six severe combined immunodeficient (SCID) mice per group, were created with bilateral chambers (silicone tubes) implanted around the epigastric vascular pedicle, with one chamber/animal containing a 5 mg fat autograft. Two animal groups were created for each of four ECMs (Matrigel™, Myogel, Cymetra® and PuraMatrix™) which filled the bilateral chambers. One group/ECM had no growth factors added to chambers whilst the other group had growth factors (GFs) (vascular endothelial growth factor-A (VEGF-A) plus fibroblast growth factor-2 (FGF-2) plus platelet-derived growth factor-BB (PDGF-BB)) added to both chambers. At 6 weeks, chamber tissue was morphometrically assessed for percent and absolute adipose tissue volume. Overall, the triple GF regime significantly increased percent and absolute# adipose tissue volume (p < 0.0005∗#) compared to chambers without triple GF treatment. The fat autograft also significantly increased percent (p < 0.0005) and absolute (p < 0.011) adipose tissue volume. Cymetra® (human collagen) constructs yielded the largest total tissue and absolute adipose tissue volume. We found that the pro-angiogenic FGF-2, VEGF-A and PDGF-BB combination in ECMs of synthetic and biological origin produced an overall significantly increased adipose tissue volume at 6 weeks and may have clinical application, particularly with Cymetra.

Introduction

Adipose tissue engineering has potential applications in breast reconstruction and in the treatment of soft-tissue defects. Although the transplantation of autologous adipose tissue grafts would be an ideal approach in breast and soft tissue reconstruction, in practice free fat grafts (without a vascular connection) are progressively absorbed [1]. The alternative – vascularized free fat flaps, where adipose tissue is transplanted with its supplying arterio-venous pedicle and anastomosed to a recipient artery and vein – is often unavailable [2] or leads to a significant donor site morbidity.

In adipose tissue engineering, an appropriate extracellular matrix (ECM) is required to support cell attachment, proliferation and differentiation until cells can secrete their own ECM. Cells with adipogenic potential must also be present. Adipogenesis is believed to occur in two stages: (1) commitment of mesenchymal stem cells (MSC) to a preadipocyte stage and (2) terminal differentiation of peradipocytes to mature adipocytes [3]. Adipose tissue engineering requires either MSCs or preadipocytes to be implanted directly into the ECM [4], [5], [6], or promotion of adipose progenitor cell recruitment from the body by the application of appropriate growth factors [7], [8]. This study exploits the second approach with a triple growth factor (GF) regime known to promote adipogenesis [9] with ECMs that are being used or potentially could be used in humans.

Vascularization of engineered tissue is a major hurdle in tissue engineering and there are several potential techniques that could be employed [10]. The O’Brien Institute has developed a novel intrinsic vascularization approach in vivo using a chamber model in which a vascular pedicle is isolated in a silicon tube that can be filled with a matrix, usually Matrigel™ [9], [11]. Within a week the pedicle spontaneously sprouts a capillary network into the ECM. Various cell populations derived from the circulating blood also invade the chamber construct, including macrophages, fibroblasts and adipocyte precursors [9], [12], [13].

There is a close relationship between angiogenesis and adipogenesis. Preadipocytes stimulate angiogenesis through VEGF secretion, and angiogenesis in turn drives preadipocyte differentiation [14]. Differentiation of preadipocytes into more mature cells does not necessarily diminish their ability to influence local angiogenesis [15].

In addition, adipose tissue endothelial cells (ECs) secrete various factors [16], [17], [18] that influence preadipocyte proliferation and differentiation. Further, by using fibroblast growth factor-2 (FGF-2) to stimulate neovascularization in subcutaneous Matrigel plugs in mice, Kawaguchi et al. [7] significantly enhanced adipogenesis at 5 weeks.

Preadipocyte and EC migration are also linked, as demonstrated by their concurrent expression of the integrin αvβ3 and plasminogen activator inhibitor-1 (PAI-1), a migratory regulator [19]. This connection may explain observations that preadipocyte migration follows angiogenesis in adipose tissue engineering models [7], [9].

Various growth factors including insulin-like growth factor [20] and FGFs [7], [8] have previously been used to stimulate adipogenesis. Our group has established that a triple combination of angiogenic growth factors (FGF-2, vascular endothelial growth factor (VEGF-A) and platelet derived growth factor-BB (PDGF-BB)) can significantly stimulate early angiogenic development and several weeks later adipose tissue growth in Matrigel [9]. Matrigel™ has frequently and successfully been used to generate new adipose tissue experimentally, but is synthesized from a mouse sarcoma and therefore has no human clinical application. To make the transition from experimental adipose tissue engineering to human applications, more suitable ECMs for human use must be investigated.

The purpose of the this study was to use the triple angiogenic growth factor regime, with alternative ECMs (Myogel, an ECM derived from muscle; Cymetra®, human collagen; and PuraMatrix™, a synthetic ECM) that may support adipogenesis in humans.

Section snippets

Materials and methods

Experiments were approved by St Vincent’s Hospital, Melbourne Animal Ethics Committee under National Health and Medical Research Council of Australia guidelines.

Results

During chamber creation injection of all matrices was successfully achieved; however, observations on Myogel chambers indicated that this ECM may have not set prior to wound closure.

Two animals died, both in the Cymetra plus triple GF group due to anaesthetic death, leaving only four animals in that group. Six chambers were examined in all other groups.

Discussion

This study demonstrated significant increases in total construct tissue volume (p < 0.001), percent adipose tissue (p < 0.0005) and absolute adipose volume (p < 0.0005) formed at 6 weeks when the triple GF combination of FGF-2, VEGF-A and PDFG-BB were applied to four different ECMs within an enclosed chamber surrounding the epigastric pedicle. Implantation of a fat autograft with or without triple GF did not influence construct weight or volume, but significantly increased percent adipose tissue

Conclusion

This study supports the widespread use in a variety of ECMs of the triple GF combination of 100 ng ml−1 FGF-2, 100 ng ml−1 VEGF-A120 and 100 ng ml−1 PDGF-BB to significantly stimulate adipose tissue growth. It is likely that this growth factor combination mediates its effects by encouraging the early establishment of a stable vasculature and by improving the recruitment, proliferation and differentiation of adipogenic MSC.

This study demonstrates that the extracellular matrix Cymetra (an acellular

Author disclosure statement

No competing financial interests exist for any author of this paper.

Acknowledgements

The authors would like to thank the Experimental and Surgical Research Unit staff at St Vincent’s Hospital, Melbourne: Sue McKay, Liliana Pepe, Anna Deftereos and Amanda Rixon for their assistance in animal surgery and housing. We would also like to thank Assoc. Prof. Keren Abberton and Dr Alan Woods for the Myogel, Dr Gregery PL Thomas for surgical advice and Ms Effie Keramidaris for technical assistance.

The authors acknowledge the assistance of the Victorian State Government’s Department of

References (40)

  • C.W. Patrick et al.

    Long-term implantation of preadipocyte-seeded PLGA scaffolds

    Tissue Eng

    (2002)
  • N. Kawaguchi et al.

    De novo adipogenesis in mice at the site of injection of basement membrane and basic fibroblast growth factor

    Proc Natl Acad Sci USA

    (1998)
  • Y. Tabata et al.

    De novo formation of adipose tissue by controlled release of basic fibroblast growth factor

    Tissue Eng

    (2000)
  • Z. Lokmic et al.

    Engineering the microcirculation

    Tissue Eng

    (2008)
  • K.J. Cronin et al.

    New murine model of spontaneous autologous tissue engineering, combining an arteriovenous pedicle with matrix materials

    Plast Reconstr Surg

    (2004)
  • F. Stillaert et al.

    Host rather than graft origin of Matrigel-induced adipose tissue in the murine tissue-engineering chamber

    Tissue Eng

    (2007)
  • H.E. Lilja et al.

    An adipoinductive role of inflammation in adipose tissue engineering: key factors in the early development of engineered soft tissues

    Stem Cells Dev

    (2013)
  • D.A. Fukumura et al.

    Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis

    Circ Res

    (2003)
  • F. Verseijden et al.

    Angiogenic capacity of human adipose-derived stromal cells during adipogenic differentiation: an in vitro study

    Tissue Eng

    (2009)
  • L.J. Hutley et al.

    Human adipose tissue endothelial cells promote preadipocyte proliferation

    Am J Physiol Endocrinol Metab

    (2001)
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    Present address: Royal Children’s Hospital, 50 Flemington Rd, Parkville, Victoria 3052, Australia.

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