A current challenge in the field of tissue engineering is soft tissue replacement. Techniques for soft tissue reconstruction include use of autologous fat transplantation, alloplastic implants and autologous tissue flaps. However, these approaches have disadvantages, including donor-site morbidity, implant migration, foreign body reaction and immune system rejection. The use of autologous stem cells expanded
The presence of self-renewing cells within the bone marrow of mice was reported in 1963 which was later known as hematopoietic stem cells (HSCs) (Becker, et al.;1963, Zhang, et al.;1999). Several years later, HSCs were identified in umbilical cord blood by other investigators (Aust, et al.; 2004, Dellavalle, et al.;2007). Furthermore, several other adult stem cell types such as neural stem cells (Dellavalle, et al.; 2007, Guilak, et al.;2010), were isolated and identified. Moreover, a population of plastic adherent cells were isolated from collagenase digests of adipose tissue.
Adipose tissue derived stem cells were termed as: adipose derived stem/stromal cells (ASCs), adipose-derived adult stem (ADAS) cells, adipose-derived adult stromal cells, adipose-derived stromal cells (ADSCs), adipose stromal cells (ASCs), adipose mesenchymal stem cells (AdMSCs), lipoblast, pericyte, preadipocyte, and processed lipoaspirate (PLA) cells. To prevent the confusion in the literature, the International Fat Applied Technology Society reached a consensus to adopt the term “adipose-derived stem cells” (ASCs) to identify the isolated, plastic-adherent, multipotent cell population (Gimble, et al.;2007). Adipose tissue derives from the mesodermal layer of the embryo and develops both pre- and postnatally (Gonda, et al.;2008, Kakudo, et al.;2008). Researchers studying the development of adipose tissue have long worked with a fibroblastic cell line, known as preadipocytes, isolated from adipose tissue (Cowherd, et al.;1999, Gregoire, et al.;1998, Kirkland and Hollenberg;1998, Nam and Lobie;2000, Sorisky;1999).
The reason why adipose tissue would contain a stem cell population is not still clear. There is some discussion whether the cells are subpopulation of fibroblasts reside within the fat tissue or are perhaps mesenchymal or peripheral blood stem cells passing through the fat tissue (Crisan, et al.; 2008, Dellavalle, et al.; 2007).
The ASCs represent a readily available source for isolation of potentially useful stem cells (Sterodimas, et al.; 2010). In culture, they have shown to have an impressive developmental plasticity, including the ability to undergo multilineage differentiation and self-renewal (Liu, et al.; 2009). When ASCs are compared with BM-MSCs, further similarities have been demonstrated in regards to their growth kinetics, cell senescence, gene transduction efficiency (De Ugarte, et al.; 2003), as well as CD surface marker expression (Gronthos, et al.; 2001, Katz, et al.; 2005, Zuk, et al.; 2002) and gene transcription (Katz, et al.; 2005). Compared to bone marrow MSCs, ASCs have potential advantages for tissue engineering application, because of the tissue accessibility, multipotency and ease of isolation without painful procedures or donor site injury.
In this chapter we will discuss the potential use of adipose-derived stem cells in the field of tissue engineering.
2. Isolation and expansion of ASCs
The initial methods to isolate cells from adipose tissue were pioneered by Rodbell and colleagues in the 1960s (Rodbell; 1966, Rodbell; 1966, Rodbell and Jones; 1966) using rat fat tissue. These methods were further adapted for human tissues by several other groups (Deslex, et al.; 1987, Engfeldt, et al.; 1980, Ho, et al.; 2010). The current methods for isolating ASCs rely on a collagenase digestion followed by centrifugal separation to isolate the stromal/vascular cells from primary adipocytes. The pellet is resuspended with a basal medium containing 10% foetal bovine serum (Estes, et al.; 2008). The cell suspension is filtered through 100 µm cell strainer and the cells are plated and incubated at 37ºC in the presence of 5% CO2. The medium is changed every second day until the cells reach 80-90% confluence. A large number of ASCs can be harvested in this manner, with yields of approximately 250,000 cells per gram of tissue (Aust, et al.; 2004, Guilak, et al.; 2010). In order to remove the use of animal products in human ASC cultures, a very low human serum expansion medium and a completely serum-free medium have been recently reported (Parker, et al.; 2007). Furthermore it was reported that use of platelet- rich plasma can enhance the proliferation of human ASCs. These results can support the clinical application of platelet-rich plasma for cell based, soft-tissue engineering and wound healing (Kakudo, et al.; 2008).
ASCs should be harvested at 80% confluence for freezing. Cryopreservation medium consists of 80% fetal bovine serum, 10% dimethylsulfoxide (DMSO) and 10% DMEM/Ham’s F-12. The cells should be stored in a final concentration of 1–2 million viable cells per milliliter of cryopreservation medium. Aliqouted vials are first frozen in an alcohol freezing container and store and are stored at -80ºC overnight. On the next day, the frozen vials can be transferred to a liquid nitrogen container for long-term storage. Successful storage of ASCs more than 6 months has been shown. This ensures the availability of autologous banked ASCs for clinical applications in the future (De Rosa, et al.;2009, Gonda, et al.;2008).
2.1. Characterization of ASCs
In order to characterize the undifferentiated animal or human ASC cells cultured in vitro flow cytometric and immunohistochemical methods are widely used (Gronthos, et al.; 2001, Zuk, et al.; 2001). The cell surface phenotype of ASC is quite similar to MSCs (mesenchymal stem cells). Both ASC and MSC cells express CD29, CD44, CD71, CD90, and CD105 (Zuk, et al.; 2002). In contrast, no expression of the hematopoietic lineage markers CD31, CD34 and CD45 was observed in either of the cultures. In addition the ASC cells express the neutral endopeptidase (CD10 or common acute lymphocytic leukemia antigen CALLA), aminopeptidase (CD13), and ecto nucleotidase (CD73). Furthermore, ASC cells produce Type I and Type III collagens, osteopontin, ostenectin, Thy-1 (CD90), and MUC-18 (CD146) (Gimble and Guilak; 2003).
Different investigator have reported different pattern of expression. For example, while Gronthos et al. (Gronthos, et al.;2001) detected CD34 and VCAM (CD106) on ASC cells, Zuk et al. (Zuk, et al.;2002) did not. Likewise, while Zuk et al. (Zuk, et al.;2002) detected Stro-1, Gronthos et al. did not. These discrepancies could be due to the differences in cell isolation methods, how long the cells were cultured prior to analysis and sensitivity differences between immunohistochemical and flow cytometric detection methods (Zuk, et al.;2002, Zuk, et al.;2001).
3. ASCs applications
Adipose tissue has proven to serve as an abundant source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. ASCs can be differentiated into variety of cell types. Differentiation is commonly induced by insulin, dexamethasone, cyclic AMP agonist, β-glycerophosphate, heparin, ascorbate and different cytokines depending on the lineage type.
ASCs like BM-MSCs, differentiate
3.1. ASCs differentiation
ASCs in response to inductive compounds including glucocorticoid receptor ligands (dexamethasone), insulin, cyclic AMP agonist (forskolin) and peroxisome proliferator-activated receptor gamma (PPARγ) undergo adipogenic differentiation (Farmer; 2006, Hauner, et al.; 1989, Lazar; 2005, Zuk, et al.; 2001). During the differentiation process ASCs reduce their proliferation rate and undergo morphological changes. ASCs are induced in the adipocyte differentiation medium containing biotin, d-pantothenate, dexamethasone, methylisobutylxanthine, insulin and equivalent PPARγ agonist. After induction for 2 weeks in adipogenic medium the human ASC contain vacuoles filled with neutral lipid cells which can be further stained for intracellular lipid droplets accumulation using an Oil Red O stain (Preece;1972).
In addition, these cells secrete increased amounts of the adipocyte protein leptin, and transcribe adipogenic mRNAs such as the fatty acid binding protein, aP2 and lipoprotein lipase (Halvorsen, et al.; 2001, Hauner, et al.; 1989, Sen, et al.; 2001). Some of these parameters such as leptin, aP2 mRNA levels were quantified and found to be increased by several hundred-fold during the differentiation process (Halvorsen, et al.; 2001, Sen, et al.; 2001). It is reported that ASCs harvested from female mice differentiate more efficiently into adipocytes than those from male mice (Ogawa, et al.; 2004).
One of the most important uses of ASCs is for the replacement of adipose tissue itself. Large soft tissue defects are a common problem following trauma, burns and oncological resections. Several studies demonstrated the
3.1.2. Smooth muscle
ASCs can be differentiated to smooth muscle cells (SMCs) and might offer a cell source for hollow organ engineering. For myogenic differentiation ASCs at passages 3 through 5 are cultured in smooth muscle inductive medium consisting of MCDB131 supplemented with 1% FBS and 100u/ml of heparin for up to 6 weeks at 37ºC with 5% CO2. The media is changed every 3 days and cell splitting is not required (Jack, et al.; 2009).
The cellular changes after differentiation can be investigated by real- time PCR at mRNA level. As reported the expression of muscle actin (SMA), calponin and myosin heavy chain showed an increase after growth in differentiation medium (Jack, et al.; 2009). The same was observed at protein levels, induction media induced differentiation of the ASCs into a smooth muscle phenotype in which the expression of smooth muscle specific proteins SMA, caldesmon, and myosin heavy chain (MHC) was increased (Jack, et al.; 2009).
Differentiation is a complex process and has a dramatical effect on cell size, shape, membrane potential, metabolic activity and responsiveness to external signals. One of the main characteristic of SMCs is their contractility which plays important roles in angiogenesis, blood vessel maintenance, and mechanical regulation of hollow organs such as bladder.
Differentiated SMCs can show two specific phenotypes, which vary from synthetic and proliferative to contractile and quiescent (Beamish, et al.; 2010). SMCs exhibit a contractile phenotype characterized by high expression of specific contractile proteins including SMA, calponin, SM22, smoothelin,
Since differentiated human ASCs express smooth muscle specific proteins they may prove to be of value in the repair of smooth muscle defects in the gastrointestinal and urinary tracts. Juan et al. reported that the ASCs from different sites show different myogenic differentiation abilities
3.1.3. Osteogenesis. Bone defect repair
In the past decade, several groups isolated cells from the adipose tissue of humans and other species capable of differentiating into osteoblasts
For osteogenic differentiation confluent ASCs cells are incubated for 3 weeks in DMEM containing 10% FBS, 100 nM dexamethasone, 10 mM β-glycerophosphate and 50 μM L-ascorbic acid-2-phosphate. After fixation cells are incubated at 37ºC for 1 hour with 0.16% naphthol AS-TR phosphate and 0.8% Fast Blue BB dissolved in 0.1 M tris buffer (pH 9.0). For osteogenic differentiation cells were also incubated in 1% alizarin red S for 3 minutes to detect calcium deposition (Sakuma, et al.; 2009).
Over a 2- 4 week period
3.1.4. Myogenesis: skeletal muscle repair
There is several line of evidence that ASC cells can differentiate along each of the myocyte lineage pathways when cultured in myogenic induction medium containing 0.1
Skeletal myogenesis is characterized by a period of myoblast proliferation, followed by the expression of muscle-specific proteins and fusion to form multinucleated myotubules. Early myogenic differentiation is characterized by the expression of several myogenic regulatory factors including myogenic determination factor MyoD1 (Weintraub, et al.; 1991). Terminally differentiated myoblasts can be characterized by the expression of myosin and the presence of multiple nuclei (Silberstein, et al.; 1986).
In the first
ASCs display chondrogenic characteristics following induction with ascorbate, dexamethasone and transforming growth factor-β (Awad, et al.; 2003, Huang, et al.; 2004, Zuk, et al.; 2001). Under inductive conditions ASCs express aggrecan, chondroitin sulphate, collagen type II and IV and proteoglycans associated with chondrogenic phenotype.(Awad, et al.; 2004, Erickson, et al.; 2002, Wickham, et al.; 2003, Zuk, et al.; 2001)
For chondrogenic differentiation, ASCs cells are grown to confluency in 30-mm dishes and incubated for 3 weeks in DMEM containing 1% FBS, 50 mM L-ascorbic acid-2-phosphate, 40 mg/ml proline, 100 mg/ml pyruvate, 10 ng/ml transforming growth factor (Harriman, et al.)-b3, and 1x ITS. Induction medium is replaced every 3 days. At the indicated time points, differentiated cells are fixed for 1h with 4% paraformaldehyde and rinsed with PBS. Accumulation of chondrocyte matrix is detected with alcian blue staining (pH 2.5, Wako) (Matsumoto, et al.; 2008).
3.1.6. Neuronal differentiation
There is preliminary evidence suggesting that human ASCs can display neuronal and/or oligodendrocytic markers. ADSC at passages 2–5 are seeded in six-well plates at 40%–60%
confluence. After three washes with PBS, the cells are induced with NIM (DMEM supplemented with 500 mM IBMX, 200 mM INDO, and 5 mg/ml insulin) for 1 hr. The cells are then examined for the expression of neuronal markers S100, NF70, and nestin followed by hematoxylin-eosin (HE) staining (Ning, et al.; 2006).
4. Biomaterials in tissue engineering with ASCs
Currently, autologous and allogenic adipose tissues represent a ubiquitous source of material for fat reconstructive therapies. However, these approaches are limited, and often accompanied by a 40–60% reduction in graft volume following transplantation. A number of factors including a stable scaffold support structure and vascularisation is necessary to support
For functional tissue replacement such as bone, Silk-based biomaterials have previously been demonstrated to offer exceptional benefits over conventional synthetic (e.g. poly-glycolic and lactic acid copolymers) and natural (e.g. collagen type I) biomaterials (Meinel, et al.; 2004). The slow degradation and mechanical integrity of silk scaffolds in comparison with other conventional biomaterials such as collagen and PLA, above all for long-term
Tissue-specific scaffolds and signalling systems are essential to differentiate stem cells into the required cells and use them effectively to construct three-dimensional (3D) tissues (Sterodimas, et al.; 2010). It has also been proved that adipose tissues engineered with ASCs and type I collagen scaffolds can serve
Porous collagenous microbeads can be useful as injectable cell delivery vehicles for adipose-derived stem cells, allowing
Placental decellular matrix (PDM) holds potential as a scaffold for adipose tissue engineering applications. The placenta is a rich source of human extracellular matrix (ECM) components that can be harvested without harm to the donor. Constructs derived from the ECM may mimic the native environment of the body, promoting normal cellular organization and behavior. Natural materials also have advantages in terms of ease of processing, biodegradability and biocompatibility (Schmidt and Baier; 2000). Cell-adhesive placental decellular matrix scaffolds facilitate proliferation and viability, while differentiation is augmented when the cells are encapsulated in non-adhesive cross-linked hyaluronan (XLHA) scaffolds (Flynn, et al.; 2008). Incorporation of XLHA into the PDM scaffolds may improve the construct bulking properties and may influence cellular infiltration, differentiation and wound healing (Shu XZ; 2004).
Other candidate for suitable scaffolds is non-woven polyglycolic acid (PGA) and hyaluronic acid gel. In a recent study it has been shown that more adipose-tissue-like construct is regenerated when using type I collagen sponge than when the non-woven polyglycolic acid or hyaluronic acid gel are used (Itoi, et al.; 2010). In addition, significant evidence has been shown that ASCs and PLGA spheres can be used in a clinical setting to generate adipose tissue as a noninvasive soft tissue filler (Choi, et al.; 2006).
Altman et al. could show that human adipose-derived stem cells seeded on a silk fibroin-chitosan scaffold enhance wound healing and show differentiation into fibrovascular, endothelial, and epithelial components of restored tissue (Altman, et al.; 2009). In addition, it has been shown that transfection of human ASCs with liposome- enveloped xenogenic protein from a neonatal rat tissue preparation can induce differentiation of stem cells along the directed lineage (Gaustad, et al.; 2004). These observations support the hypothesis that the inductive biochemical and structural cues of the microenvironment are conserved across species and that a silk fibroin-chitosan delivery vehicle can provide a beneficial niche in supporting migration, proliferation, and differentiation of the applied cells (Altman, et al.; 2009).
Another complex biomolecule which has sparked great interest for tissue engineering is Hyaluronic acid which has been stated to support the growth and development of progenitor cells (Brun, et al.; 1999, Solchaga, et al.; 1999). The material has a progressive rate of biodegradation, lacks cytotoxicity and does not induce a systemic immune response or chronic inflammation in a human
Long-standing, 3D predefined-shape adipose tissue from hAD-MSCs of human adipose tissue remains a challenge. Lin et al. cultured scaffolds (Gelatin sponges, monofilament polypropylene and polyglycolic acid meshes) with hAD-MSCs in adipogenic medium for 2 weeks before implantation, and implanted scaffolds were harvested after 2, 4, and 6 months
There are numerous adipose tissue engineering culture strategies in which the core tissue engineering principles comprising appropriate cells, scaffold, and microenvironment are optimized (Patrick; 2001). Specifically, there are static versus dynamic culture, co-cultivation, and addition of growth factors, vascularization, and long-term sustainability of engineered constructs. The advantage of dynamic culture includes increased nutrient and oxygen delivery to cells within a 3D construct (Frye and Patrick; 2006). Improved bioreactor designs to address direct perfusion conditions are necessary to advance dynamic culture techniques (Choi, et al.; 2010).
It has been shown that hASCs express pericyte lineage markers
ASCs provide an abundant and readily accessible source of multipotent stem cells. The use of autologous stem cells expanded
However, further studies are needed before ASCs can be used clinically. In particular, investigators need to demonstrate the safety and efficacy of ASCs cells in animal models, either alone or in combination with novel biomaterial scaffolds.
Both the authors Dr. Mathias Tremp and Dr. Souzan Salemi contributed equivalently in writing this chapter.