New Frontiers in Plastic and Cosmetic Surgery Melvin A Shiffman, Alberto Di Giuseppe
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Section 1
1Stem Cells
  • Tissue Engineering with Adipose-Derived Stem Cells (ADSCs) in Plastic and Reconstructive Surgery: Current and Future Applications2

Tissue Engineering with Adipose-Derived Stem Cells (ADSCs) in Plastic and Reconstructive Surgery: Current and Future Applications1

Aris Sterodimas
 
INTRODUCTION
Until very recently, most scientists and clinicians believed that damaged or diseased human tissue could only be replaced by donor transplants or with totally artificial parts. Tissue engineering (TE) promises a more advanced approach in which organs or tissues can be repaired, replaced, or regenerated for more targeted solutions. This approach also responds to clinical needs that cannot be met by organ donation alone.
Tissue engineering combines the principles of bioengineering, cell transplantation, and biomaterial engineering for the unique goal of generating bioartificial tissues and organs. Within just a few years, the possibility that the human body contains cells that can repair and regenerate damaged and diseased tissue has gone from an unlikely proposition to a virtual certainty.1 Tissue engineering aims to provide an alternative better means of treatment for tissue and organ damage through combining both biological and artificial components in such a way that a long-lasting repair is produced. In plastic and reconstructive surgical applications, adipose tissue has become central to an increasing number of translational efforts in TE.2 The growing interest in this area of research has resulted in the exploration of many novel research and clinical applications that utilize adipose-derived stem cell (ADSC) products obtained from this tissue source. Adipose-derived stem cells could also become the focus of an array of therapeutic solutions for many disease conditions, such as those affecting bone, cartilage, muscle, and neural expanding the possible indications and translational potential of tissue, cell-based, and regenerative medicine strategies.
After the introduction of liposuction, adipose tissue harvesting has become easier.3 ADSCs, because of their pluripotentiality and unlimited capacity for self-renewal, have allowed significant advances for distinct reconstructive procedures in the recent years. It contains a large number of multipotent cells, which is an essential prerequisite for stem-cell-based therapies. It has been described that stem and progenitor cells in the uncultured stroma-vascular fraction (SVF) from adipose tissue usually amount to up to 3% of the whole cells, and this is 2,500-fold more than the content of stem cells in bone marrow.4 ADSCs can easily be isolated by tissue digestion and centrifugation steps, followed by the outgrowth of the plastic adherent fraction from the SVF.5 Stromal-enriched lipograft (SEL), the new technique of TE using ADSCs has revolutionized various aspects in reconstructive and aesthetic surgery. Implied in the definition of TE is the use of stem cells, biomolecules, and biomaterials. The SEL is based on the use of ADSCs combined with a biomaterial that is the adipose tissue that has been processed to be used as a natural scaffold and biomolecules, cytokines, and growth factors, which are secreted by the stem cells and the adipose tissue.6
 
TECHNIQUE
Marking of the areas to be liposuctioned are made while the patient is in standing position. Preoperative sedation in the surgical suite is administered. Anesthesia consists of an epidural block and intravenous sedation. The patient is placed in prone position. After the injection of normal saline wetting solution containing 1:500,000 of adrenaline by a small bore cannula and waiting 15 minutes, a 60-mL syringe attached to a 4-mm blunt cannula is inserted through small incisions in the abdominal area. Fat is aspirated by using the syringe method. The two thirds of the aspirated fat is used in order to isolate the SVF (Fig. 1.1). Digestion is done with 0.075% collagenase (Sigma, St Louis, MO, USA) in buffered saline and agitated for 30 minutes at 37°C in Celltibator (Medikan, Los Angeles, CA, USA). Separation of the SVF containing ADSCs is then done by 4using centrifugation at 1,200 × g for 5 minute.7
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Fig. 1.1: SVF isolation from adipose tissue by collagenase digestion and centrifugation. (SVF: Stroma-vascular fraction).
The Lipokit Centrifuge (Medikan, Los Angeles, CA, USA) is used. The SVF is located in the pellet derived from the centrifuged fat at the bottom of the lipoaspirate. When human adipose stem-cell expansion is needed, the SVF is cultured in a very low human serum medium in order to allow rapid expansion of autologous ADSCs.8 Biomaterials and biomolecules can then be used in order to guide the organization and differentiation of expanded ADSCs in the process of forming functional tissue.9 Growth factors, differentiation factors, angiogenic factors, and gene-modulated factors are the main components of the biomolecules and need to be strategically integrated in to the future tissue engineered constructs.
In SEL, freshly isolated SVF is attached to the aspirated fat, with the fat tissue acting as a living bioscaffold before transplantation.10 The remaining one third of the aspirated fat is treated in the following manner: with the syringe held vertically with the open end down, the fat and fluid are separated. Isotonic saline is added to the syringe, the fat and saline are separated and the exudate discarded. The procedure is repeated until the fat becomes yellow in color, free of blood, and other contaminants.11–14 Mixing of the SVF-containing ADSCs and the purified fat is then done (Fig. 1.2). This whole procedure is done inside the operating theatre, by two tissue engineers, manually, and the time required is about 90 minutes. The adipose tissue graft enriched with SVF is woven into the targeted tissues (face, breast, and body), injecting only a tiny amount with each pass as in order to obtain the most reliable clinical outcome. Tissue planes are created by using specific cannulas in different trajectories, always from the deeper aspect to more superficial areas. The fat is injected as the cannula is withdrawn in order to avoid intravascular fat injection. Antibiotics, analgesics, and anti-inflammatory medications are prescribed during the following seven postoperative days.
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Fig. 1.2: Stromal-enriched lipograft technique.
 
Current Human Applications of ADSCs in Plastic and Reconstructive Surgery
Traditional facial rejuvenation techniques address the face by lifting the soft tissues in two dimensions. The element not routinely addressed, is reduction and atrophy of the facial soft tissues, particularly the subcutaneous fat layer.15 This is the third dimension of facial aging. The SEL can achieve long-term volume replacement at the time of rhytidectomy and allows less aggressive surgical dissections in order to accomplish a more harmonious result (Figs. 1.3A to D). Successful applications of SEL in a series of patients for post-traumatic facial reconstruction have been published.7 Parry–Romberg syndrome patient has been treated by SEL.4
In addition to the well-established procedure for breast enlargement with silicone implants, the augmentation by fat grafting has become established in recent years. The use of SEL for breast augmentation has gained attention due to further improvements in the fat preparation and processing (Figs. 1.4A to D). There are though concerns about possible role of ADSCs in promoting of tumor formation or recurrence mediated by mechanisms, such as angiogenesis, and tumor stromal cells.5
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Figs. 1.3A to D: (A and B) Preoperative 72-year-old woman requesting facial rejuvenation. (C and D) Postoperative following facelifting assisted by stromal-enriched lipograft.
However, published clinical studies describing outcomes of fat grafting to the breast in >2,000 patients have not reported any increase in new or recurrent cancers.16 Recently the concept of composite breast augmentation has been introduced where combining breast silicone implant insertion and simultaneous SEL has been done in 20 patients with aesthetically favorable results (Figs. 1.5A to D).
The combination of circumferential liposuction, SEL of buttocks, and/or lower limbs in a single surgical procedure has been performed successfully in the last 8 years, emphasizing the low rate of complications and the high overall patient satisfaction (Figs. 1.6A to D).
Despite advances in wound closure techniques and devices, there is still a critical need for new methods of enhancing the healing process to achieve optimal outcomes. Nonhealing and radiation exposed wounds remain a significant challenge for plastic surgeons. Recent studies suggest that ADSCs secret angiogenic cytokines in vitro and in vivo, including VEGF, HGF, and FGF2, which increase neovascularization and enhance wound healing in injured tissues.176
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Figs. 1.4A to D: (A and B) Preoperative 22-year-old woman requesting breast augmentation. (C and D) Postoperative after breast augmentation by stromal-enriched lipograft.
Recently, autologou s ADSCs, together with angiogenic and mitogenic factor of basic fibroblast growth factor and an artificial dermis, were applied over the excised irradiated skin defect and tested for patients who were uneventfully healed with minimal donor-site morbidity, which has lasted for >1.5 years.18
Reconstruction of large bony defects after tumor resection involves harvesting of autologous bone causing donor site morbidity and risk of infection. Recently clinical case where ectopic bone was produced using ADSCs in microvascular surgery for novel maxillary bone reconstruction has been reported.19
 
Future Human Applications of ADSCs in Plastic and Reconstructive Surgery
Facial volume enhancement for aesthetic and reconstructive purposes is probably going to be one of the near future applications of TE with ADSCs. Injectable microcarrier beads combined with ADSCs in order to form a minimally 7invasive implant that will stimulate regeneration of host adipose cells and fill a soft-tissue void upon injection in vivo.20
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Figs. 1.5A to D: (A and B) Preoperative 29-year-old woman with pectus excavatum requesting breast augmentation revision and treatment of pectus. (C and D) Postoperative following composite breast augmentation combining silicone implant insertion and stromal-enriched lipograft.
The system is comprised of cells seeded on hyaluronic acid biodegradable beads of an injectable size, resulting in a composite that may be injected into a patient through a syringe at the defect site. Stromal-enriched lipograft has been recently used as a nonsurgical alternative to the modeling of nasal shape and profile in secondary cases of rhinoplasty patients.10
Total auricular reconstruction represents one of the greatest challenges for the plastic and reconstructive surgery field. The ability to construct a fully satisfactory complete external ear has for centuries been an elusive goal. Tissue engineering using ADSCs could have the potential to provide ear-shaped cartilaginous constructs in the near future. Adipose-derived stem cells obtained by liposuction have been differentiated to chondrocytes, expanded in vitro and seeded onto biodegradable alginate and silk polymer ear-shaped scaffolds. Microporous three-dimensional (3D) scaffolds were fabricated by solvent casting technique using molds made through rapid prototyping from a normal ear CT image.21 A recent published study has confirmed that the association of silk, alginate, and ADSCs is a reliable method to produce an engineered 3D auricular cartilage construct (Fig. 1.7).22
Breast augmentation or reconstruction based on ADSCs cultured on absorbable tailor-made breast scaffolds that are then implanted in vivo has been the target in the past years. Achieving simultaneous cellular proliferation and scaffold resorption can result in mature adipose 8tissue and possibly is superior to traditional fat grafting to the breast.23
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Figs. 1.6A to D: (A and B) Preoperative 31-year-old woman requesting body contouring. (C and D) Postoperative following body contouring by stromal-enriched lipograft.
The future research is focusing on ADSCs-scaffold implantation into the patient in order to ideally restore the aesthetic function of the tissue by imparting a soft, smooth feel closely resembling that of natural breast (Fig. 1.8).
Use of a skin flap has been a common technique in reconstructive surgery for more than five decades. However, partial necrosis of its distal end is still a serious postoperative complication. Advances in the treatment of ischemia-reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and 9implanted tissues.
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Fig. 1.7: Ear construct manufactured by auricular tissue engineering ready for implantation.
A recent study reports that ADSCs treatment significantly enhances skin flap survival in the aftermath of ischemia to an extent that almost equals surgical results without ischemia. This effect is accompanied with a pronounced and significant angiogenic response and an improved blood perfusion to the flap.24,25
 
DISCUSSION
Tissue engineering using ADSC is a fairly young field of research that has shown huge potential so far. It is imperative to be humble enough to realize that TE combines several different fields of research, in themselves very complicated, as biology, medicine, chemistry, material science, and more.
The presence of ADSC cells in adipose tissue transplantation may contribute to neoangiogenesis in the acute phase by acting as endothelial progenitor cells or angiogenic-factor-releasing cells.26 In vivo, ADSCs demonstrate the capacity to proliferate in response to a hypoxic insult remote from their resident niche, and this has been supported by in vitro studies showing increasing ADSCs proliferation with greater degrees of hypoxia. The number of functional ADSCs is likely to be important for tissue repair and remodeling and ADSCs differentiate into vascular endothelial cells that contribute to neoangiogenesis in the acute phase of transplantation. Adipose-derived stem cells also upregulate their proneovascular activity in response to hypoxia, and may harbor the capacity to home to ischemic tissue and function cooperatively with existing vasculature to promote angiogenesis.4
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Fig. 1.8: Future breast tissue engineering based on stromal-enriched lipograft.
In 2011, the first study comparing the autologous fat grafting to SEL was published.7 A group of 20 patients with congenital or acquired facial tissue defects were included in this study and randomly divided into two groups. Ten patients were treated with autologous fat transplantation, and the remaining 10 were treated with ADSC-enriched lipografts. Stromal-enriched lipografts produced superior results without the need for repeat treatment sessions, which were necessary with autologous fat transplantation. Even when large areas need reconstruction, like in head and neck cancers; it is possible to use the SEL technique as a TE method of reconstruction. The SEL technique also offer the possibility of prefabrication of flaps, reconstruction of auricular framework defects and fabrication of new skin for massive burns when SVF is used either alone or combined with other natural or synthetic biomaterials. In aesthetic plastic surgery, the SEL technique provides a safer alternative to allogenic implant use, resulting in the creation of a functional tissue that has a more natural look and carries fewer risks than currently available augmentation options.
One of the main obstacles in the progress of TE using ADSCs today is the vasculature system. Any biological structure of sizes larger than a couple of hundred microns in diameter needs a circulatory system for 10nutritional reasons. This is mirrored in the fact that the tissues that have found their ways to the clinical setting are all tissues and organs with the least complex blood circulation: fat, skin, cartilage, and bone. In order to succeed also with more complex organs and tissues, this problem needs to be resolved. Recent advances in nanotechnology may allow the development of nanostructured scaffolds with a cellular environment that maximally enhances not only cell expansion but also the neovascularization that is crucial for long-term maintenance of cell volume. Another obstacle is the limited materials that are currently available as carriers or scaffolds in the field of plastic and reconstructive surgery. Currently, fat tissue appears to be the best available natural biomaterial. Innovative synthetic materials, such as polypeptides or novel biodegradable polymers, need to be introduced. Advances in materials design may generate “smart” scaffolds that will control tissue topology and have surface modifications to stimulate cell attachment, differentiation, and growth.27
In 2009, the use of ADSCs to create induced pluripotent cells (iPS cells) was reported.28 In their derivation of iPS cells from ADSCs, researchers cite the enormous clinical utility for an embryonic-like stem-cell population that can be easily derived and used without the ethical issues of an embryonic stem cell. Whether ADSC-derived iPS cells will be ultimately be used clinically will depend on a couple of important factors, namely, how easy is it to develop iPS lines and their safety.29
The apparent commercial and industrial interest on TE using ADSCs that has emerged lately should encourage the scientific community in adopting a disciplined strategy in pursuing this field and clinical trials can lead to optimization of it.30 An interdisciplinary effort at and the government front will bring successful realization of this therapy in to the field of regenerative medicine.
 
CONCLUSION
There is a wealth of published clinical data showing safety, feasibility, and efficacy of the SEL technique. Adipose tissue stem-cell-based regenerative strategies hold tremendous promise, and this great potential must be balanced against stringent standards of scientific and clinical investigation, before developing “off-the-shelf” TE products. Further investigations should be encouraged toward bench side and bedside to resolve various issues.
 
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