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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online July 12, 2005 as doi:10.1096/fj.04-3241fje. |
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,1
* Bernard O’Brien Institute of Microsurgery, St. Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia; and
Department of Surgery, University of Melbourne, Parkville, Victoria, Australia
1 Correspondence: Bernard O’Brien Institute of Microsurgery, 42 Fitzroy St., Fitzroy 3065, Victoria, Australia. E-mail: messinaa{at}svhm.org.au
SPECIFIC AIMS
We have shown that when a functional arterio-venous (AV) loop is constructed in the rat groin and placed inside a semi-sealed chamber, an extensive vasculature with its supporting matrix is spontaneously generated. Our aims were to determine: 1) whether we could grow specific new tissue in the AV loop chamber, by using skeletal muscle as the inductive source; and 2) whether the neovasculature that is generated within the chamber would develop, integrate with, and support differentiation and growth of this tissue. These are critical requirements for any model intended to grow neo-organs.
PRINCIPAL FINDINGS
1. By implanting chambers with skeletal muscle tissue, we can grow a complex 3D neo-organ that is comprised of specific tissue, is well integrated with the chamber neovasculature, and has its own vascular supply
To determine whether we could grow specific new tissue we implanted AV loop chambers with minced skeletal muscle tissue as the inductive source, because of its high capacity for regeneration, and compared the outcome with control chambers. In some chambers we included the cut proximal end of the epigastric and femoral nerves as a source of growth factors and to potentially innervate the new tissue formed. All chambers were explored after 6 wk incubation. In the first experiment we implanted a small amount (50 mg) of rat skeletal muscle that had been preincubated in vitro for 24 h. In later experiments we implanted 400 mg fresh muscle, to increase the quality of muscle, the quantity of precursor cells, and to reproduce studies where muscle tissue gives rise to myogenesis. Gross examination of the newly generated tissue revealed an intact, soft, encapsulated, pulsating mass of tissue that filled 
40%–70% of the available volume (0.5 mL; Fig. 1
A, B). Implanting muscle tissue in the chambers resulted in the unexpected generation of adipose tissue (Fig. 1)
, which was uniform, well vascularized, and devoid of inflammatory cells, fibroblastic cells, and collagen. More tissue grew in chambers implanted with 400 mg fresh muscle (compared with 50 mg) and the proportion of adipose tissue in these chambers was doubled (P<0.01). New desmin-positive, small diameter striated skeletal muscle fibers were also grown in these chambers. They were 10-fold more numerous in chambers that included a nerve (P<0.05). We show that a small amount of preincubated skeletal muscle is adequate to induce both adipogenesis and myogenesis in our chamber and that this tissue is incorporated and vascularized on the AV loop. This specific tissue growth occurred more consistently in chambers inclusive of a nerve (Table 1
).
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2. Implanting human muscle into rat chambers induces both adipogenesis and myogenesis
Human muscle (200 mg preincubated in vitro) was implanted into nude rat chambers to determine whether our observations with rat muscle hold true for this species and to indicate whether there is a potential clinical application. Implanting human muscle tissue into the chamber resulted in both adipogenesis and myogenesis, comparable to that observed with rat muscle tissue, confirming that the phenomenon is not restricted to rat muscle tissue.
3. Skeletal muscle grown in chambers implanted with muscle tissue or myoblasts was derived from donor precursors; adipose tissue grown in chambers implanted with muscle tissue was derived from recipient precursors; a nonviable component of skeletal muscle and an acellular muscle matrix induce adipogenesis in the rat chamber model
Immunohistochemical labeling using a human mitochondrial specific antibody was carried out to determine the precursor cell origin of the new skeletal muscle and or the adipose tissue grown in chambers implanted with human muscle tissue. This antibody labeled adipose and muscle tissue in control human specimens. In chambers implanted with human muscle, all skeletal muscle fibers observed were positively labeled, indicating that they were derived from the donor human muscle cell source. A small number of adipocytes, perivascular cells, and interstitial cells were also labeled, indicating that recipient precursors gave rise to these components. However, the majority of these cells were not labeled and most likely to be derived from recipient cells. To confirm this observation we implanted 400 mg of rat muscle tissue that had been rendered nonviable by repeated freeze-thawing into the chamber. This resulted in considerable in vivo adipose tissue generation but no skeletal muscle fiber generation, confirming our observation that the majority of adipose tissue was derived from a recipient cell source. This observation indicates that a nonviable component or extracellular matrix of the implanted muscle tissue is the adipo-inductive factor.
4. Skeletal muscle tissue has both adipogenic and myogenic potential in culture
Rat skeletal muscle tissue was placed in DMEM supplemented with 10% fetal calf serum and incubated in vitro for up to 6 wk. A fusiform cell outgrowth was observed surrounding small attached pieces of muscle tissue and at random throughout the tissue culture plates. Phase bright adipocytes appeared after 5-day culture and increased in number thereafter, reaching confluence by 4 wk. If the serum level was decreased to 2%, myotubes also formed in these cultures, confirming both adipogenic and myogenic potential of skeletal muscle tissue.
DISCUSSION
Tissue engineering holds the promise of creating neo-organs from stem cells, but first the issue of how to vascularize these neo-organs must be resolved. We have taken a novel in vivo approach to address this issue by developing a model in which a functional vasculature is spontaneously generated on an arterio-venous loop that is constructed surgically and placed inside a polycarbonate chamber in the rat groin (Fig. 2
). This differs markedly from other approaches in which attempts are being made to grow a vasculature in vitro or attract a vasculature to cell seeded matrices in vivo by using proangiogenic factors.
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In this study, we induced the growth of a complex vascularized neo-organ by placing donor skeletal muscle tissue as the inductive source into this unusually angiogenic chamber environment. We show proof of the concept that the angiogenesis generated by our in vivo chamber model is able to develop and integrate with a specific new tissue, in this case muscle and adipose tissue that was derived from donor and recipient precursor cells. This new tissue forms a relatively large, complex, vascularized "neo-organ." It is encapsulated and well demarcated from other tissues and is thus easily retrievable. It resembles an organ by virtue of the fact that it is generated on its own vascular pedicle and is also transplantable.
The induction of adipogenesis by adult rat and human skeletal muscle tissue was unexpected. In vivo, adipose tissue replacement of skeletal muscle in "nerveless" limbs implanted into chick embryos and of granulation tissue that forms within hollow nylon mesh tubes inserted intramuscularly has been described. On the other hand, when a similar preparation of muscle tissue is reimplanted into its original bed, myogenesis (not adipogenesis) occurs. These data differ from ours in several aspects. In our model: 1) adipogenesis occurs in chambers containing implanted muscle irrespective of the inclusion of a nerve; 2) the granulation tissue formed in our chamber is not replaced by adipose tissue unless muscle tissue is included; and 3) the implanted muscle induces adipogenesis as well as myogenesis. We show that the adipogenic properties of the muscle tissue lie in the extracellular or nonviable cell component whereas the myogenic properties derive from the cell component and that these phenomena are also observed in human muscle tissue. In general, the observations point to a role for site (chamber vs. muscle bed or groin vs. intramuscular); environmental, extracellular, and cellular cues; and the type of inductive implant, in determining the ultimate type of tissue grown. All these factors should be taken into consideration when deciding how and which type of tissue to grow. One could postulate that by refining the composition of the inductive tissue or matrix implanted around the AV loop, the type of tissue grown in the chamber would be better controlled.
The generation of a vasculature which is able to develop and integrate with newly growing tissue has a significant application in the field of organogenesis and tissue engineering as a source of vascularization for the creation of complex neo-organs such as fat and muscle (as demonstrated in this study) and has the potential to enable creation of complex neo-organs such as liver. It is a conceptual advance in the field of tissue replacement and represents an important and practical step in the evolution of tissue regeneration that merges surgical technique to cell biology.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3241fje;
This article has been cited by other articles:
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  A. N. Morritt, S. K. Bortolotto, R. J. Dilley, X. Han, A. R. Kompa, D. McCombe, C. E. Wright, S. Itescu, J. A. Angus, and W. A. Morrison Cardiac Tissue Engineering in an In Vivo Vascularized Chamber Circulation, January 23, 2007; 115(3): 353 - 360. [Abstract] [Full Text] [PDF]
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