**6. Tissue engineering**

promising results. [4] A recent study showed that the direct injection of ADSCs could restore blood flow in a mouse ischemic hindlimb model, as confirmed by clinical data. [22] The myogenic differentiation of ADSCs may be used in the treatment of muscular diseases such as Duchenne dystrophy and for regenerative cell therapy in heart failure. [23] Other novel potential clinical uses of ADSCs include the treatment of Alzheimer disease, of multiple sclerosis due to the anti-inflammatory effect of ADSCs, of neurogenic bladder and other neurologic disorders. A preliminary study showed that peri-urethral injection of autologous ADSCs acts positively in stress urinary after prostatectomy. Regarding current clinical applications of ADSCs, apart from a phase III trial on the treatment of Crohn's fistula, most clinical trials are in phase I. Beside the use in breast reconstruction, trials are in progress to treat acute myocardial infarction and chronic myocardial ischemia by intracoronary injection of SVF. Other trials are focused on the treatment of cirrhosis and of diabetes I or II. [4] Another trial adopted ADSCs (after purification and expansion) for the management of fistulas associated or not to Crohn's disease: results demonstrated an efficient control of inflammation and an improvement of healing process, most likely due to paracrine action that cells differ‐ entiation. Another trial investigated the restoration of volumes in hypotrophic scars after subcutaneous injection of ADSCs. Only two trials have studied the effect of ADSCs on chronic critical limb ischemia: the first adopting intra-muscular injection, the second by intravenous injection in diabetic patients. The literature regarding different clinical trials [Table 4.]demon‐ strates that ADSCs-based therapies are a concrete opportunity but despite these results, molecular, cellular e biological features of these cells are still uncertain and it is also unclear if regenerative therapy is related to their differentiation potential or paracrine activity: indeed,

more appropriate in vivo investigations are necessary.

Stress urinary after prostatectomy

194 Regenerative Medicine and Tissue Engineering

Chronic critical limb ischemia

in diabetic patients

**Pathology Operating methods Condition**

Cirrhosis intrahepatic arterial administration of autologous SVF Phase I Diabetes I intravenous injection of autologous SVF Phase I/II Diabetes II autologous SVF Phase I/II Hypotrophic scars subcutaneous injection of ADSCs Phase III Chronic critical limb ischemia intra-muscular injection of ADSCs Phase I

Myocardial infarction intracoronary injection of SVF Phase II/III Multiple sclerosis intravenous injection of autologous ADSCs Phase I/II Reumathoid arthritis intrarticular injection of autologous ADSCs Phase III

**Table 4.** Clinical trials using adipose-derived stem cells (ADSCs) or stromal vascular fraction (SVF).

Crohn's fistula injection into rectal mucosa of autologous of ADSCs with

fibrin glue

peri-urethral injection of autologous ADSCs Report of three

intravenous injection of ADSCs Phase I/II

initial cases

Phase III

#### **6.1. Adipose derived bio-products**

In the past decade, preclinical and translational efforts have established the future basis for the application of ADSCs from the bench to the bedside. Significantly, ADSCs have been widely used in tissue engineering, organ repair and gene therapy. These multipo‐ tent cells, have shown a remarkable plasticity and the ability to differentiate towards different cell lineages with similar yet enhanced properties (their multipotency and proliferative efficiency) in comparison to bone marrow-derived mesenchymal stem cells. [3,6-7,21-24,26] Moreover, ADSCs also show adjuvant angiogenic properties likely related to the secretion of vascular endothelial growth factor. [21] In vitro studies have rapid‐ ly increased during the last decade, resembling the need to optimize the variables of the differentiation process cells towards the desired lineage. The efficient use of biomateri‐ als, delivery vehicles and bioreactors has promoted the development of a large variety of novel tissue engineered products for repair and regeneration of various tissues and organs. The use of suitable animal models in an extensive preclinical literature has also established the basis for successful stem cell-based therapies that may implement current therapeutic solutions for several diseases. Thus, a focus of most interest for the scientif‐ ic community is posed today in the production of safe and reliable cell delivery vehicles/ scaffolds useful in applying ADSCs as a therapy as well as in the development of novel suitable in vivo animal models. A large variety of bioengineered products have been developed by means of selected differentiating cultures of ADSCs. [Table 5] Preclinical studies have experimentally reported the adoption of ADSCs in order to develop cells of mesodermal origin as well as cells of non-mesodermal lineage such as neural o neurallike cells for repair of neural traumatic injuries, fibroblast for reconstruction of soft tissue defects, tenocytes or regenerated tendon constructs for optimal musculoskeletal system reconstruction, osteoblasts for bone tissue replacement, chondrogenic lineages and cartilage substitutes for implantation, skeletal muscle cells and subsequent myotubelike formation depicting myogenic differentiation in vivo in muscular dystrophy model. Other reported lineages and engineered tissues that may be obtain through selective differentiation include hepatocytes, pancreatic endocrine cells, cardiomyocytes and vascular endothelial cells. [24] Most relevant transcription factors involved in differentia‐ tion into adipocytes, chondrocytes, myocytes and osteocytes are well-known. However, in addition to specific differentiation factors, tridimensional biomaterials are essential to address differentiation of ADSCs to the required cell type and to use them for tissueengineering purposes. Among investigated effective scaffolds and matrices we may include: type I collagen, hyaluronic, poly lactic-co-glycolic acid (PLGA) and silk fibroinchitosan. [26] Moreover, the combination with specific growth factors determines the overall outcome of the applied biopolymer.


**Table 5.** Synopsis of current approaches in ADSCs and tissue engineering.

**Figure 3.** Electron microscopy scanning of ADSCs cultured on a Hyaluronic acid-based biomaterial.

#### *6.1.1. Bio-engineered bone*

**Tissue Cell type Gene Scaffold Result**

Endothelia ADSCs - porous polycaprolactone

Tendon ADSCs - decellularized human

Nerve ADSCs - hyaluronan membrane

**Figure 3.** Electron microscopy scanning of ADSCs cultured on a Hyaluronic acid-based biomaterial.

**Table 5.** Synopsis of current approaches in ADSCs and tissue engineering.

Autologous ADSCs

196 Regenerative Medicine and Tissue Engineering

Bone human ADSCs BMP-2 - heal critical sized femoral


Cartilage ADSCs - polyglycolic acid scaffolds exhibit in vitro chondrogenic

ADSCs BMP-2 collagen sponge increase bone induction in SCID

Autologous SVF - bone graft treat calvarial defects in human

filled titanium scaffold

ADSCs - - improve outcome measures in

(PCL) scaffold

and fibrin meshes

tendon

defects in a nude mouse model

create neo-maxilla in human

mice

characteristics

recellularize

neuronal-like cells

osteoarthritis in dogs

endothelial differentiation

differentiate in glial-like and

There is still a clinical need to generate bone for the repair of large osseous defects, since current strategies are based on non-vascularized bone grafts, suitable only for small defects. As an alternative, progenitor cells might be implanted on biomaterials and differentiated in vivo supporting reconstruction of large bone losses. Osteo-inductive factors include vitamin D3, β-glicerophosphate, acid ascorbic and Bone Morphogenic Proteins (BMPs). [7] Treating ADSCs with recombinant BMP-2 has shown to stimulate osteogenic differentiation: [27] human ADSCs overexpressing BMP-2 could heal critical sized femoral defects in a nude mouse model. Similarly, ADSCs exposed to BMP-2 adenoviral transfection and seeded in collagen sponges increased bone induction in SCID mice. [27-28] These results suggest that transfected stem cells can replace the exogenous addition of growth factors when transplanted in a bio-engineered scaffold. The use of scaffolds is critical in repair of structural tissues such as bone. Deminer‐ alized bone matrix, collagen, PLGA, hydroxyapatite and β-tricalcium phosphate scaffolds were reported to be suitable for ADSC-derived osteochondral tissue engineering. Most of clinical trials of osteogenesis in ADSCs rely on murine studies and human trials are based on very limited reports. The first human case involved transplantation of SVF together with bone graft to treat calvarial defects [29] and in another case a neo-maxilla has been created using a β-tricalcium phosphate-filled titanium scaffold associated to cultured ADSCs. [30] Thus, ADSCs-based osteogenesis is possible, however, more adequate evidence is needed in the clinical setting.

#### *6.1.2. Bio-engineered cartilage*

ADSCs might be used to generate cartilage for clinical use in the treatment of degenerative joints. The list of potentially useful growth factors for cartilage repair comprises TGFβ, IGF-1, FGFs, EGF and BMPs, transcription factors as SOX9 and signal transduction molecules such as SMADs. Several in vitro studies have shown the chondrogenic differentiation of ADSCs and this feature is confirmed by their ability to generate cartilage in a variety of experimental models. ADSCs seeded into polyglycolic acid (PGA) scaffolds exhibited in vitro chondrogenic characteristics and they could synthesized cartilage extracellular matrix. [23] The great potential of ADSCs in cartilage tissue engineering was also demonstrated in different studies in vivo. Moreover ADSCs have been used recently for treatment of osteoarthritis in dogs [32] and rheumatoid arthritis in human. [33] However, given the lack of evidence, it seems likely that the symptomatic benefits seen in these trials may relate to the anti-inflammatory proper‐ ties of ADSCs rather than to a real chondrogenic differentiation.

#### *6.1.3. ADSCs and vascular/endothelial tissue engineering*

The vascularization of regenerated tissues is an important field of research since it allow the survival of tissue and the differentiated cells. [24] It has been reported that human ADSCs have the potential for endothelial differentiation and they can participate in blood vessel formation by means of the secretion of several pro-angiogenic factors, like vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). [23] This feature makes these cells suitable for regenerative cell therapy, treatment of ischemic disorders and construction of vascularized grafts in one-step procedure, as it has already been performed in many experi‐ ments on animal models. [22] Furthermore, as reminded, the angiogenetic properties of ADSCs have been already investigated in several clinical trials to treat various diseases.

#### *6.1.4. Bio-engineered tendon*

Tendon tissue engineering is relatively unexplored due to the difficulty to maintain in vitro preservation of tenocyte phenotype: only recently research has demonstrated the fundamental role of in vitro mechanical stimuli in maintaining the phenotype of tendinous tissues. [34] The main growth factors inducing tendon differentiation include fibroblast growth factor (FGF), platelet-derived growth factor-BB (PDGF-BB), epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and members of the transforming growth factor-β (TGF-β)/bone morphogenetic proteins (BMPs) family. Several in vivo and in vitro studies have showed the ability of ADSCs to differentiate in tenocytes under specific stimuli and under biomechanical force. [34] Furthermore, recent experiments have focused on the possibility of re-cellularize by means of seeded ADSCs a decellularized human tendon. [35] Thus, an integration of ADSCs, growth factors, mechanical stimuli and biopolymers may provide a solution for the treatment of difficult tendon injuries

#### *6.1.5. ADSCs and neuronal tissue-engineering*

Incubation of ADSCs under neuro-inductive conditions (culture medium containing EGF, FGF, NGF and BDNF) has shown the potential to form neurospheres expressing neurospecific markers, including nestin¸ βIII tubulin, S100 and glial fibrillar acidic protein (GFAP). [36] Moreover, seeding of these neurospheres in different scaffolds (hyaluronan based membranes and fibrin glue meshes) demonstrated further differentiation in glial-like and neuronal-like cells. [37] Although these are only preliminary researches, these promising results are of significant clinical interest. ADSCs-induced neural cells may provide beneficial therapeutic effects in treatment of injuries occurring to both the peripheral and central nervous systems such as in the treatment of neurodegenerative states, including Parkinson's disease, Hungtin‐ ton's disease, multiple sclerosis and Alzheimer's disease.
