Biomaterials as cell carriers for augmentation of adipose tissue-derived stromal cell transplantation

Abstract

Adipose tissue-derived stromal cells (ADSCs) contain lineage-committed progenitor cells that have the ability to differentiate into various cell types that may be useful for autologous cell transplantation to correct defects of skin, adipose, cartilage, bone, tendon, and blood vessels. The multipotent characteristics of ADSCs, as well as their abundance in the human body, make them an attractive potential resource for wound repair and applications to tissue engineering. ADSC transplantation has been used in combination with biomaterials, including cell sheets, hydrogel, and three-dimensional (3D) scaffolds based on chitosan, fibrin, atelocollagen, and decellularized porcine dermis, etc. Furthermore, low molecular weight heparin/protamine nanoparticles (LH/P NPs) have been used as an inducer of ADSC aggregation. The tissue engineering potential of these biomaterials as cell carriers is increased by the synergistic relationship between ADSCs and the biomaterials, resulting in the release of angiogenic cytokines and growth factors. In this review article, we describe the advantages of ADSC transplantation for tissue engineering, focusing on biomaterials as cell carriers which we have studied.

Keywords

Adipose tissue-derived stem cells cell transplantation scaffold cell aggregation angiogenesis wound healing

1. Introduction

Regenerative medicine has evolved with recent advances in stem cell research. Exciting potential of stem cells for tissue regeneration, as well as in the restoration of diseased organs and systems, has been shown in the last couple of decades. Despite these advances, the availability of stem cells remains a challenge for both scientists and clinicians with an interest in regenerative medicine. In general, a stem cell is characterized by its ability to self-renew and differentiate into multiple lineages. With regard to regenerative medicine, the ideal stem cell population should be present and accessible in quantity, harvestable by a relatively noninvasive procedure, able to differentiate into a variety of cell lineages, and easy to transplant to an autologous or allogeneic host [1–3]. The two major types of stem cells are embryonic stem (ES) cells and adult stem cells, also known as somatic stem cells. Other stem cell types, including induced pluripotent stem cells (iPSCs), are produced in the lab by reprogramming adult somatic cells to express ES characteristics. ES cells are obtained by extracting cells from the inner cell mass of embryos at the blastocyst stage, and subsequently expanding them in culture. However, sourcing ES cells is difficult and fraught with ethical concerns [4,5].

In contrast to ES cells, adult stem cells can differentiate into a limited set of specialized cells concomitant with the tissue of origin. In adults, tissue-specific stem cells are located throughout the body. The hematopoietic stem cells found in bone marrow and umbilical cord blood can differentiate into all hematopoietic cell types. These cells have been used in bone marrow transplant therapy for diseases, such as leukemia [6]. Other types of tissue-specific stem cells are usually found in specific niches deep within tissues, and are thus more difficult to obtain and to study, especially in humans [7]. An addition to the list of known stem cell sources is the pool of adipose tissue-derived stromal cells (ADSCs) found in the perivascular region of white adipose tissue, which includes subcutaneous fat deposits [8]. Because of their abundance, ease of isolation, and non-controversial nature, ADSCs have been considered for applications in regenerative medicine.

Although various tissues are known to contain adult stem cells can differentiate into a limited set of specialized cells concomitant with the tissue of origin for tissue maintenance and repair [9]. For instance, adipose tissue-derived stromal (ADSCs) cells are known to have abilities to differentiate into bone [10,11], cartilage [12,13], fat [14], myocardium [15], skin [16,17], skeletal muscle [18], and neurons [19]. Significant differences have not been observed between ADSCs and bone marrow-derived mesenchymal stem cells (BMSCs) from the same patient with regard to yield of growth kinetics, adherence, differentiation capacity, cell senescence, or gene transduction efficiency [20]. Furthermore, transplantation of constructs containing human ADSCs significantly stimulates re-epithelialization, granulation formation, and angiogenesis, in athymic mice when compared with those containing human fibroblasts [21]. The multipotent characteristics of ADSCs, as well as their abundance in the human body, make them an attractive potential resource for wound repair and tissue engineering applications. In addition, ADSCs have a homing capability, and migrate to injured tissue when introduced systemically or locally. However, it has been claimed that only a small fraction of the cells can migrate to the target tissue after systemic administration, while the majority of cells accumulate in the kidneys and lung [22,23]. In the case of local injection of ADSCs with an adequate cell-carrier, a substantial proportion of the cells is retained and grows in the target area, while some cells are flushed out into the blood circulation [24,25].

Current techniques for tissue engineering directly implant suitable cells such as ADSCs using adequate solutions including culture medium or plasma as a carrier, or deliver ADSCs in three-dimensional (3D), biocompatible, and biodegradable scaffolds to replace the injured or lesioned tissues [11,16]. However, the repair of large soft- or hard-tissue trauma that predominates in combat-related extremity injuries remains a major challenge. Combat injuries are generally large, and usually involve multiple tissue types, including skin, muscle, tendon, cartilage, and bone, all of which need to be repaired. The 3D scaffold biomaterials should provide a cellular microenvironment suitable for cell survival, adhesion, proliferation, and differentiation to enhance the repopulation and regeneration of the lesioned or damaged tissues. Furthermore, matrix biomaterials present in interstitial tissues should be the favorable substrate for cell morphogenesis, migration, immune defense, and wound repair. ADSCs embedded in 3D matrix materials were also observed to grow and to differentiate into specific cell types in an optimized induction medium [1]. Other scaffolds currently being used to regenerate viable tissues contain materials that vary in their chemical, mechanical, and structural properties. On the other hand, 3D scaffold composed of natural polymers such as collagen, chitin/chitosan, alginate, and gelatin etc. have been used extensively due to highly efficient integration with host tissue and their biocompatibility. Several studies have also reported ADSC transplantation in association with biomaterials in an attempt to enhance the local retention and growth rate of the cells. For example, cell sheets or 3D scaffolds based on chitin/chitosan, fibrin, atelocollagen, and decellularized porcine dermis have been used as inducers of ADSC growth and differentiation [4,12,17]. In this review article, we describe current progress and future directions in the development of 3D scaffold materials for tissue engineering purposes.

2. Methods

2.1. Preparation of ADSCs, LH/P NPs and PRP, and application of ADSCs with scaffolds to generate adipose tissue

A technique of autologous fat grafts has widely applied due to advantages in the field of reconstructive surgery, enabling correction of contour defects and augmentation of soft-tissue volume. Autologous fat is efficiently integrated into the host tissue, and can be harvested with minimal morbidity using direct excision and needle or cannula aspiration techniques to obtain sufficient quantities in most patients. However, the survival rate of fat grafts is still unpredictable, as re-absorption occurs in most grafts to a variable degree, presumably due to poor re-vascularization leading to subsequent death of the fully differentiated adipocytes originally injected [26–28]. Previous studies demonstrated the better efficacy of fat grafts with low molecular weight heparin/protamine nanoparticles (LH/P NPs) prepared with FGF-2 [27] or platelet-rich plasma (PRP) [28] to maintain graft volume and survival in rat model.

In the study, LH/P NPs were prepared by modifying the method as described earlier [29–31]. Briefly, 2.5 mL of LMWH solution (Fragmin: 6.4 mg/mL; Kissei Pharmaceutical Co., Tokyo, Japan), followed by 1.2 mL of protamine solution (10 mg/mL; Mochida Pharmaceutical Co., Tokyo, Japan) was added into 500 mL of saline (Otsuka Normal Saline; Otsuka Pharmaceutical Co., Ltd. Tokyo, Japan). The white solution was mixed vigorously. PRP was prepared as previously described [32,33]. Briefly, before the animal experiment, 40 mL of blood from volunteers was drawn into tube including 4 mL of 2% sodium citrate. The tube was centrifuged for 15 min at 1700 rpm (Table-Top Refrigerated Centrifuge 2800, Roter: RS-240, Kubota, Tokyo, Japan), resulting in three layers: erythrocytes at the bottom of the tube, PRP layer in the middle, and platelet-poor plasma (PPP) layer at the top of the tube. The upper 1 cm of the red blood layer (PRP layer) was collected and centrifuged for 5 min at 3000 rpm to concentrate the platelets. In this procedure, 4 mL of PRP and 16 mL of PPP were finally obtained from 40 mL of blood.

Figure 1 shows preparation of LH/P NPs by mixing LH with protamine (Fig. 1A) and mechanism of formation of ADSCs and LH/P NP-aggregates. LH/P NPs bind to the ADSC surface through heparin-binding cell surface proteins, such as integrin. The interaction of ADSCs with LH/P NPs results in an LH/P NP aggregate formation (Fig. 1B).

Plasma-medium gel provides an extracellular matrix for the culture of different cell types by forming a massive capsule with semi-permeable properties that allows the diffusion of medium components into cells and elimination of waste. Both exogenous and endogenous GFs efficiently bind to LH/P NPs, with their activities remaining stable. Human ADSCs can be grown in two-dimensional (2D) culture using low human serum (HS) (1%–2%) and DMEM with sufficient cytokines on LH/P NPs-coated tissue culture plates [34] (Fig. 2B). Furthermore, ADSCs can also be grown efficiently in three-dimensional (3D) culture using low human plasma (HP) (2%)-DMEM gel containing 0.1 mg/mL LH/P NPs without animal serum [30,34] (Fig. 2A). Furthermore, the phenotypes of both cell types were positive for CD44, CD90 and CD105 (>80%) and negative for CD34 and CD45 (<1%) [30]. Thus, those cells were well maintained in 2D and 3D cultures after seven days. The 3D-proliferated ADSCs and BMSCs maintained their multipotent differentiation capacity, that is, they were able to differentiate into adipocytes and osteoblasts [30]. Those results demonstrated the superior proliferation of both cell types using the 3D culture system in low-concentration HP-DMEM gel with LH/P NPs and FGF-2. The 3D culture system for cultures of ADSCs or BMSCs is composed of low-concentration HP-medium gel with LH/P NPs and FGF-2. ADSCs grew better in 3D culture (Fig. 2A) than in 2D culture (Fig. 2B). Furthermore, the presented 3D culture methods required no animal serum, because the low concentration (2%) of autologous plasma was sufficient [30].

The discovery of ADSCs has attracted attentions in using these cells as cell sources for fat regenerations. ADSCs from inbred mice were prepared as previously described [17,31] with several modifications. Briefly, BALB/c mice adipose tissue from inguinal region was removed, miced, and digested with 0.1% Collagenase type I (Wako Pure Chemical Industries Ltd., Osaka, Japan) and 0.2% Dipase type II (life Technology Oriental, Tokyo, Japan) for 1 h at $37^\circ$ . The digested tissue was mechanically and gently dispersed. The suspension was passed through a 100-μm filter, centrifuged at 420 g for 5 min, and resuspended. The ADSCs were maintained and expanded in DMEM with 1% heat-inactivated FBS and antibiotics [34,35], and adipocyte differentiations were induced by plating the expanded ADSCs in an adipogenic differentiation medium (Cambrex Bio Science Walkersville, MD). Furthermore, human adipose tissues from the abdoment of three patients who gave informed consent were obtained in Otsuka Academy of Cosmetic and Plastic Surgery, Tokyo, Japan. We examined the ADSCs from the three patients, but there were few differences in the osteogenic potential [10,11].

2.2. Application of ADSCs with scaffolds to generate bone and cartilage

A gold standard for bone reconstruction is autologous bone grafting for bone reconstruction because of its essential compatibility and integration capacity. However, a production of exactly fitted 3D replication of missing bone segments is very difficult when transplanting bone from a site dissimilar to the recipient area. Furthermore, serious difficulties were encountered in obtaining bone grafts [36]. Tissue-engineered bone constructs with some rigid and porous scaffold biomaterials represent an alternative for reconstruction of defective bone, in which the scaffold biomaterial infiltrated with osteogenic cells stimulates bone formation [37,38]. BMSCs have been used as osteogenic progenitor cells for this purpose, but are more difficult to harvest enough amount of cells. ADSCs have attracted attentions because of their abundance and ease of isolation [39].

In our previous study, osteogenic differentiations were induced by plating the expanded inbred ADSCs from mice or expanded human ADSCs in an osteogenic differentiation medium (Cambrex Bio Science Walkersville, MD). The stored human ADSCs were seeded at a density (2 × 10⁵ cells/scaffold) into round 𝛽-TCP disks (diameter 11 mm; thickness 2 mm) as a scaffold in 48-well plates (Sumitomo Bakelite Corp., Tokyo, Japan) and then cultured in control medium. Osteogenic differentiation was induced by culturing ADSCs within 𝛽-TCP scaffold in osteogenic differentiation medium. [10,11] (Fig. 3A).

Application of the cartilage regeneration could potentially have major social and economic benefits compared to joint replacement, Autogenous chondrocyte-based therapies have been developed as a promising treatment for degenerative joints. Autogenous chondrocyte transplantation has proved to be an effective method of cartilage repair, but it had a few problems, including defect size constraints, donor site morbidity, and a restricted monolayer culturing period [40]. On the other hand, ADSCs for ease of their abundant preparations might be used to regenerate cartilage in a clinical treatment of cartilage defects [12].

For the differentiation of the chondrocytes in vitro, the micromass pellets (2.5 × 10⁵ cells) were cultured in chondrogenic differentiation medium (Cambrex Bio Science Walkersville, MD). Under low-speed centrifugation, a dense mass of cells formed at the bottom of the conical centrifuge tube. Within 1 day, the cells consolidated to a sigle mass, and the mass gradually increased with achieving the differentiation of the chondrocytes. For in vivo rabbit study, prepared ADSCs (2 × 10⁶ cells/scaffold) were directly seeded into a atelocollagen honeycomb-shaped scaffold with membrane seal (ACHMS) scaffold (diameter: 11 mm; thickness/2 mm) in 48 well plates by being centrifuged at 40 g for 5 min and cultured in F12/DMEM supplemented with 10% FBS and 50 μg/ml ascorbic acid at $37^\circ$ in atmosphere of 5% CO₂ for 3 days [12,40,41]. The ACHMS containing ADSCs were implanted into deficit of rabbit knee cartilage (Fig. 3B).

Cell sheet engineering has recently been developed as a new method for cell transplantation. In the field of cartilage regeneration, transplantation of a chondrocyte sheet using a temperature-sensitive cell culture surface has been demonstrated to be effective and safe for cartilage defects. Poly-N-isoproplyacryamide (PIPAAm) and its copolymer have been grafted onto the surfaces of numerous materials to promote temperature-sensitivity [42,43]. At $37^\circ$ , cells adhere and spread on the hydrophobic PIPAAm-grafted surface. By lowering the temperature to $20^\circ$ , the cells detach from the surface spontaneously as intact cell sheets due to a change in surface characteristics from hydrophobic to hydrophilic. So far, various cell types, including chondrocytes, epithelial cells, skeletal myoblasts, epidermal keratinocytes, periodontal ligament cells, mucosal cells, pancreatic islet cells, hepatocytes, and thyroid cells have been successfully utilized to produce cell sheets in this way [44].

2.3. Application of ADSCs with scaffolds for wound healing, skin restoration, and therapeutic angiogenesis

The potential of ADSCs to self-renew and regenerate tissue has great implications for wound healing, skin restoration and therapeutic angiogenesis. The mechanisms by which ADSCs aid in wound healing and angiogenesis are unclear, but possibilities include direct differentiation of ADSCs within the epithelium, support for angiogenesis in the local tissue, and other paracrine effects from the release of cytokines and growth factors (GFs) to the region. In addition to direct transplantation, differentiation of ADSCs to epithelial cells may be another useful application of ADSCs in wound healing. Indeed, there exist varying protocols to differentiate ADSCs to cells with epithelial characteristics using a combination of conditioned media, GFs, contact with an extracellular matrix, and sometimes specific chemical factors, such as retinoic acid. However, the use of ADSCs in skin restoration is still in its experimental stages, and has been predominantly limited to in vitro investigations [45,46]. Nevertheless, the relative ease of access, high cell yield, and putative anti-inflammatory and angiogenesis effects of ADSCs make them attractive targets for skin engineering, in which a large amount of tissue must be supplied for reconstruction [24].

LH/P NPs as cell carriers can enhance cell viability as well as control the release of GFs. LH/P NPs could substantially enhance the cellular viability of various suspension cultures, including ADSCs [34]. It was observed that LH/P NPs could bind to the surface of cells and the interaction induced ADSCs/LH/P NPs-aggregate formation, and substantially promoted cell viability for at least 3 days in cell suspensions. The ADSC/LH/P NPs-aggregates adhered and grew on suspension culture plates. Similarly, injectable ADSC-delivery technology using gelatin-based microspheres [47,48] significantly improved the healing of healing-impaired wounds. Three-dimensionally (3D) cultured ADSCs derived from inbred male Fisher 344 rats using injectable low Plasma (3%)-DMEM gel with LH/P NPs were applied for cell transplantation [49] (Fig. 4A). In addition, when we applied ADSCs using plasma (6%)-DMEM gel with FGF-2 containing LH/P NPs were administered to full thickness skin excisions of healing-impaired wounds on the backs of streptozotocin-induced diabetic rats, the wound closures and healing were significantly enhanced on post-wounding [50] (Fig. 4B).

Heparinoids, such as heparin, LH, or heparan sulfate, are well-known cofactors that enhance the activities of heparin-binding GFs which have angiogenic activity [51,52]. Heparinoids are able to adsorb and enhance the activities of various heparin-binding GFs and cytokines that are released from ADSCs, including IL-6, GM-CSF, FGF-2, PDGF-bb, VEGF, and HGF [53]. Similarly, LH shows high affinities FGF-2 [29,54], HGF [55], and various GFs derived from PRP [33]. They were also efficiently adsorbed by LH/P NP enhancing their activities and protecting the GFs from inactivation by acidic or high heat environments, and protease degradation in vitro [33]. Furthermore, LH/P NPs bind to the ADSC surface through heparin-binding cell surface proteins, such as integrins. The interaction of ADSCs with LH/P NPs results in ADSCs seeded in an LH/P NP aggregate formation, which appears to promote cellular viability in vitro (Fig. 1B). Thus, subcutaneous injection of mouse ADSC/LH/P NP aggregates could stimulate cell proliferation and migration, resulting in neovascularization in vivo [31].

3. Results and discussion

3.1. ADSCs with scaffolds to generate adipose tissue

Recently, cell-based approaches utilizing ADSCs in combination with cell carriers such as fibrin [56,57] and decellularized adipose tissue bioscaffolds [58] for adipose tissue engineering have been developed, and were reported to promote both short-term adipogenesis in vivo and to repair defect sites [59]. Furthermore, studies on fat tissue engineering were performed using ADSCs in combination with collagen-gelatin sponge [60], and 3D porous silk-based fibroin [61].

Our studies demonstrated the better efficacy of fat grafts with low molecular weight heparin/protamine nanoparticles (LH/P NPs) prepared with platelet-rich plasma (PRP) or FGF-2 to maintain graft volume and survival in rat model [29–31]. On the other hand, the discovery of ADSCs has attracted attentions in using these cells as sources of adipocytes for fat regeneration. Unsurprisingly, it has been demonstrated that ADSCs have a great ability to differentiate into mature adipocytes when cultured with medium containing steroids, a cyclic AMP inducer, and fatty acids to promote terminal differentiation [9]. In the study, staining with oil red O or Nile red has performed to confirm a differentiation into adipocytes. These dyes stain cytoplasmic lipid droplets red, and specifically label cells performing a defining function of adipocytes. On the other hand, expression of adipocyte-specific genes has also been used to demonstrate ADSC differentiation into adipocytes. Expression of the lipoprotein lipase and fatty acid binding protein 4 genes is required for fatty acid metabolism, and both gene products are adipocyte markers [2,3] signaling a change in cell function to lipid accumulation. The expression has proved a liable and sensitive indicator of adipocyte differentiation [2].

3.2. Application of ADSCs with scaffolds to generate bone and cartilage

The demonstration of ADSC differentiation towards the osteogenic lineage is more complex than that of adipogenic differentiation as described above. The simplest method is staining for calcified extracellular matrix components. Alizarin red or osteocalcin immunostaining as a simple method was used to evaluate the presence of calcium-rich deposits produced by cells in culture. However, this type of positive staining does not represent differentiation of ADSCs into cells capable of forming bone tissue, it simply shows that the cells are increasing calcification. However, analyses for gene expressions of osteocalcin and/or osteopontin are required to confirm the differentiation [2,3]. Figure 3A shows tissue-engineered bone driven by ADSCs in a beta-tricalcium phosphate (𝛽-TCP) scaffold in a nude mouse model [10,11].

Scaffolds for osteogenesis need to mimic bone morphology and structure to optimize integration into the surrounding tissue, and to provide a suitable microenvironment for ADSC survival, proliferation, adherence, and differentiation to osteoblasts [62,63]. Hydroxyapatite (HA: Ca₁₀(PO₄)₆(OH)₂) is clinically used in different forms as a well-characterized biomaterial. HA is suitable for integrating into diseased or damaged bone tissues since it mimics the mineralized bone phase and supplies calcium ions for the newly forming bone during resorption [64]. Also, 𝛽-TCP (Ca₃(PO₃)₂) was considered to be suitable for clinical use as a carrier for ADSCs because of its chemical and crystallographic similarities to the inorganic phase of native bone [11,65]. Biphasic calcium phosphate (BCP) refers to homogenous composites of HA and 𝛽-TCP [66]. However, BCP tends to have poor mechanical properties [67]. Porous 𝛽-TCP was gradually degraded and finally replaced by new bone after approximately 24 weeks when it was buried in the bone defect where it provided a 3D structure and microenvironment for bone regeneration [68]. As reported for other scaffolds, akermanite (Ca₂MgSi₂O₂) showed better mechanical properties and degradation rates than other bioceramics [65,69]. Furthermore, Liao et al. [70] compared the osteogenic potential of porcine ADSCs using three scaffolds, PCI (polycaprolactone), PCI-𝛽-TCP, and collagen I-coated PCI-𝛽-TCP, and found that the osteogenic ability of ADSCs in vitro and in vivo was enhanced by coating collagen onto the PCI-𝛽-TCP [70].

Chondrogenesis using ADSCs has been demonstrated in 3D culture with chondrogenic factors such as transforming growth factor 𝛽 and/or bone morphogenic protein. ADSCs have been encapsulated in various biomaterials, including collagen, chitin/chitosan, gelatin, alginate, and agarose, and cultured in media including chondrogenic factors that facilitate cartilage formation [42]. Scaffolds for cartilage tissue engineering are expected to fulfill the prerequisites concerning structure, cytocompatibility, and shape prior to transplantation [40]. As an example, Fig. 3B shows tissue-engineered cartilage with autologous ADSCs using an atelocollagen honeycomb-shaped scaffold with membrane sealing (ACHMS) in the rabbit [12].

The demonstration of chondrogenic differentiation of ADSCs faces the same problems as osteogenic differentiation as described above. Method used to demonstrate ADSC differentiation into a chondrogenic lineage is staining for increased expression of proteoglycans using Toluidine blue, Alcian blue, or Safranin-O [71]. This type of positive staining does not represent differentiation of ADSCs into cells capable of forming cartilage tissue, it simply represents that the cells are increasing expression of proteoglycans. Therefore, confirmation of chondrogenic differentiation would be required to evaluate mRNA profiling of differentiated cells for expression of cartilage-specific transcripts such as type II collagen and/or type X collagen that are cartilage-specific proteins and analysis of the extracellular matrix produced by differentiated cells for cartilage-specific proteins.

3.3. ADSCs with scaffolds for wound healing and skin restoration

The potential of ADSCs to self-renew and regenerate tissue has great implications for wound healing and skin restoration. The mechanisms by which ADSCs aid in wound healing are unclear, but possibilities include direct differentiation of ADSCs within the epithelium, support for angiogenesis in the local tissue, and other paracrine effects from the release of angiogenic cytokines and growth factors (GFs) to the region. In addition to direct transplantation, differentiation of ADSCs to epithelial cells may be another useful application of ADSCs in wound healing. Indeed, there exist varying protocols to differentiate ADSCs to cells with epithelial characteristics using a combination of conditioned media, GFs, contact with an extracellular matrix, and sometimes specific chemical factors, such as retinoic acid. However, the use of ADSCs in skin restoration is still in its experimental stages, and so far has been predominantly limited to in vitro investigations. Nevertheless, the relative ease of access, high cell yield, and putative anti-inflammatory effects of ADSCs make them attractive targets for skin engineering, in which a large amount of tissue must be supplied for reconstruction [24].

Many factors, including GFs, cytokines, and chemokines influence a wound healing that is a complex and dynamic process [72], and an adequate 3D-matrix for the stimulation of ADSC proliferation and the formation of new skin tissue and blood vessels. To achieve this goal, scaffolds must meet specific requirements such as mimicking the native extracellular matrix of the target tissue; allowing cell attachment, migration, proliferation, and differentiation to, and maintenance of the target phenotype; promoting vascularization and nutrient delivery; and having a biodegradation rate and mechanical properties that are adequate to support the formation of the new skin tissue [73]. Tissue regeneration is based on a fundamental triangle composed of three fundamental elements – cells, GFs, and scaffolds – which act synergistically. Adequate scaffolds provide a conducive matrix for supporting the genic capability of progenitor cells mediated by the inductive capability of GFs [3]. ADSC therapy using polymeric micro- and nano-carriers based on synthetic polymers (e.g., poly lactide co-glycolic acid (PLGA) and poly-L-lysine) or natural polymers (e.g., collagen, chitin/chitosan, gelatin, alginate, and fibrin) have been successfully used as cell delivery devices. These biomaterials have been shown to be conducive for cell, growth, adherences, migration, and differentiation. Numerous researches have been performed for treating skin defects as temporary skin substitutes such as porcine xenografts, synthetic membranes, atelocollagen sponge, and allogenic substitutes. Furthermore, permanent skin substitutes containing cultured epidermis and dermal substitutes have been studied [73,74]. Atelocollagen matrix (ACM; PELNAC; Johnson & Johnson, Tokyo, Japan) as artificial dermal substitutes, are structurally optimized to incorporate into surrounding wound, and to allow cell invasion by fibroblasts and capillaries for subsequent dermal remodeling [16,17]. Figure 5 shows healing of a healing-impaired wound with autologous ADSCs using an atelocollagen scaffold (PELNAC) in diabetic mice [16,17].

We previously reported the ability of injectable LH/P NPs to adsorb, protect, and activate fibroblast growth factor (FGF)-2 [29,54], hepatocyte growth factor (HGF) [55], and GFs derived from PRP [32] that were also involved in cell proliferation, migration, and angiogenesis. The studies suggested that LH/P NPs serve as an effective nano-carrier for various GFs, particularly for local application. Thus, GFs containing LH/P NPs have a substantial ability to induce vascularization and fibrous tissue formation because of the gradual controlled release, protection and activation of GF molecules from GF-containing LH/P NPs [32].

LH/P NPs as cell carriers can enhance cell viability as well as control the release of GFs. LH/P NPs could substantially enhance the cellular viability of various suspension cultures, including ADSCs [34]. It was observed that LH/P NPs could bind to the surface of cells and the interaction induced ADSCs/LH/P NPs-aggregate formation, and substantially promoted cell viability for at least 3 days in cell suspensions. The ADSC/LH/P NPs-aggregates adhered and grew on suspension culture plates. Similarly, injectable ADSC-delivery technology using gelatin-based microspheres [47,48] significantly improved the healing of healing-impaired wounds. Three-dimensionally (3D) cultured ADSCs derived from inbred male Fisher 344 rats using injectable low Plasma (3%)-DMEM gel with LH/P NPs were applied for cell transplantation [49] (Fig. 4A). In addition, when we applied ADSCs using plasma (6%)-DMEM gel with FGF-2 containing LH/P NPs were administered to full thickness skin excisions of healing-impaired wounds on the backs of streptozotocin-induced diabetic rats (Fig. 4B), the wound closures and healing were significantly enhanced on post-wounding [50].

3.4. ADSCs with cell carriers for therapeutic angiogenesis

It is well known that new blood vessels grow into the ischemic tissues to prevent further damage and eventual necrosis, when a tissue is exposed to severe ischemia. A clinical strategy termed “therapeutic angiogenesis” is an important means to salvage tissues in severe ischemic diseases [15]. Researchers in the cardiovascular field are testing the hypothesis that is facilitating angiogenesis with GFs such as FGFs, HGFs, and vascular endothelial growth factors (VEGFs) and/or transgenes encoding these factors will improve tissue perfusion and function in severe ischemic diseases [75,76]. Although a number of preclinical studies supported the safety and feasibility of clinical application of therapeutic angiogenesis using GFs and/or transgenes, recent larger clinical randomized placebo-controlled trials often failed to show sufficient improvement of angiogenesis, function, or symptoms [77].

Putative endothelial progenitor cells (EPCs) in adult peripheral blood has been identified by Asahara et al. [78]. Transplantation of cultured EPCs could successfully augment angiogenesis and tissue blood supply in experimental animal models with ischemia [79]. However, the utility of EPCs was limited because of difficulties with culture expansion techniques and problems with cell numbers. Another interesting cell source for therapeutic angiogenesis is ADSCs. The ADSCs are considered an attractive candidate for therapeutic application in regenerative medicines because of their abundance and ease of isolation from subcutaneous fat, expansion, and established regenerative and angiogenic potential [80]. However, the application of ADSCs for therapeutic angiogenesis and vasculogenesis requires microcarriers as adequate injectable vehicles necessary for ADSC transplantation.

The promise of therapeutic angiogenesis using ADSCs relies on the administration of appropriate numbers of cells. However, the approach continues to be complicated by cell availability and uncontrollable cell loss and death at lesion sites. Furthermore, the production of large numbers of ADSCs is costly and side effects from large numbers of cells introduced to the lesion site. Therefore, optimal cell delivery strategies are necessary to enhance the specificity, efficacy, and reproducibility of cell therapy leading to optimized cell dosing and reduced side effects [30,46]. Recently, two strategies to efficiently deliver ADSCs to target tissues, using LH/P NPs [53] and primed 3D injectable micro-niches [81], were developed for the treatment of critical limb ischemia.

Heparinoids, such as heparin, LH, or heparan sulfate, are well-known cofactors that enhance the activities of heparin-binding GFs. Heparinoids are able to adsorb and enhance the activities of various heparin-binding GFs and cytokines that are released from ADSCs, including IL-6, GM-CSF, b-FGF, PDGF-bb, VEGF, and HGF [53]. Similarly, LH shows high affinities FGF-2, HGF, and various GFs derived from PRP [32]. They were also efficiently adsorbed by LH/P NP enhancing their activities and protecting the GFs from inactivation by acidic or high heat environments, and protease degradation in vitro [30]. Furthermore, LH/P NPs bind to the ADSC surface through heparin-binding cell surface proteins, such as integrins. The interaction of ADSCs with LH/P NPs results in ADSCs seeded in an LH/P NP aggregate formation, which appears to promote cellular viability in vitro (Fig. 3). Thus, subcutaneous injection of mouse ADSC/LH/P NP aggregates could stimulate cell proliferation and migration, resulting in neovascularization in vivo [31].

ADSCs can potentially release various angiogenesis GFs factors that can be efficiently immobilized on LH/P NPs [53] or LH/P NP-coated culture plates [34]. Injection of PRP containing LH/P NPs alleviated limb loss in an adult BALB/c-nu/nu male mouse-induced ischemic hind limb model [82]. Thus, LH/P NPs could enhance the angiogenesis effects of GFs released ADSCs through their ability to immobilize and activate various angiogenesis factors. It has been suggested that a mechanism of the angiogenesis involve not only ADSCs, but also endogenous inflammatory cells to induce the release of various angiogenesis factors [72,83]. Clearly, there is considerable demand for the development of a therapeutic angiogenesis strategy using ADSCs with LH/P NPs. Enhanced specificity, efficacy, safety and reproducibility will provide concomitant improvements in cell survival and therapeutic function at lesion sites.

4. Conclusion and prospects

The potential of adipose tissue to be a source of multipotent adult stem cells has garnered a great deal of attention in the field of regenerative medicine. With the increasing number of overweight and obese individuals, isolation of ADSCs from lipo-aspirate samples may prove to be a clinically feasible option. Given the relative abundance of ADSCs, their ease of harvest and culture, and their high yield relative to other stem cell pools such as bone marrow, it is likely that the research and clinical usage of ADSCs will continue to grow. While some initial reports show positive clinical outcomes, well-designed and controlled studies, as well as long-term post-treatment follow-ups will be paramount to ensure the safety and efficacy of procedures for patients. For ADSCs, the development of detailed and efficient differentiation protocols for various cell types, optimization of in vivo delivery methods with adequate biomaterials, and mitigation of the immune response in allogeneic transplantations are challenges that need to be overcome. Thus, many additional works are necessary in order to bridge the gap between findings in basic science and the clinical treatment of diseases with stem cell-based regenerative medicine.

Despite some clinical trials in humans and existing data from animal experiments, the various risks and safety concerns associated with ADSC transplant have not been fully elucidated. Since adipose tissue serves important auxiliary endocrine functions, transplantation of ADSCs may exert unintended paracrine and endocrine effects on peripheral tissues. For instance, the use of ADSCs with adequate scaffold for cell-based therapy may induce a proinflammatory response [48,83], and prolonged inflammation may ultimately cause or exacerbate chronic inflammatory disease, such as rheumatoid arthritis and osteoarthritis. Thus, the use of ADSCs with cell carriers will require stringent monitoring in patients with a history of chronic inflammatory disease. Another important consideration for ADSC transplants is the potential for tumorigenicity [84,85]. ADSCs were shown to aid in chronic inflammation and facilitated the transformation of gastritis to gastric cancer [84]. Thus, further basic studies on the interaction between ADSCs and various cancers will need to be performed to elucidate any potential hazards such as de novo tumorigenesis, or stimulated growth of existing tumors.

The first generation of cell-based therapy, involving the direct injection of cell suspensions, has been investigated in various clinical trials, but problems were encountered with cellular localization and survival in host tissue [72]. Longer and Vacanti in 1993 proposed the tissue engineering approach as a second generation of cell-based therapy, using 3D biodegradable and porous polymer scaffolds seeded with cultured cells [86]. In addition, decellularized scaffolds, which provide an acellular, naturally occurring 3D biologic scaffold for cell culture are a new approach for tissue engineering that has been used to construct whole-organs [87]. However, transplantation of scaffold-based tissue often results in fibrosis, necrosis due to compromised angiogenesis, and inflammatory responses driven by the biodegradation of scaffolds. Although additional work is necessary to improve the efficacy and safety of ADSC transplantation, including the use of bio-scaffolds, sheets, hydrogel, and cell aggregation inducers, the available data provide tangible grounds for optimism about the future of ADSC based tissue regeneration therapies.

Footnotes

Acknowledgements

We thank the personnel of the Institute of Laboratory Animals, Graduate School of Medicine, National Defense Medical College for their expert care of animals. This study was partially supported by the Ministry of Education, Culture, Sports, Science and Technology of the Government of Japan (grant no. 1058500).

Authors’ contributions

Masayuki Ishihara, Satoko Kishimoto, Shingo Nakamura, Koichi Fukuda, Yoko Sato, and Hidemi Hattori contributed to the concept, the writing, the illustrations and the revision of this manuscript.

Conflict of interest

None to report.

References

Zuk

P.A.

, The adipose-derived stem sell: Looking back and looking ahead, Mol. Biol. Cell 21(11) (2010), 1783. doi:10.1091/mbc.E09-07-0589.

Locke

Feisst

and P.R.

Dunbar

, Concise review: Human adipose-derived stem cell: Separating promise from clinical need, Stem Cells 29(3) (2011), 404. doi:10.1002/stem.593.

Wankhade

U.D.

Shen

Kolhe

and Fulzele

, Advances in adipose-derived stem cells isolation, characterization, and application in regenerative tissue engineering, Stem Cell Int. (2016), ID 3206807, 9 pages. doi:10.1155/2016/3206807.

Bel

Planat-Bernard

Saitoh

Bonnevie

Ballamy

Sabbah

, Composites cell sheets, A further step toward safe and effective myocardial regeneration by cardiac progenitors derived from embryonic stem cells, Circulation 122(suppl 1) (2010), S118–S123. doi:10.1161/CIRCULATIONAHA.109.927293.

Pengfei

J.I.

Manupipatpong

Xie

and Li

, Induced pluripotent stem cells: Generation strategy and epigenetic mystery behind reprogramming, Stem Cell Int. (2016), ID 8415010: 11 pages. doi:10.1155/2016/8416010.

Mirshekar-Syahkal

Fitch

and Ottersbach

, Concise review: From greenhouse to Garden: The changing soil of hematopoietic stem cell microenvironment during development, Stem Cells 32(7) (2014), 1691. doi:10.1002/stem.1680.

Crisan

Yap

Casteilla

Chem

C.W.

Corselli

Park

T.S.

, A perivascular origin for mesenchymal stem cells in multiple human organs, Cell Stem Cell 3(3) (2008), 301. doi:10.1016/j.stem.2008.07.003.

Kishi

Imanishi

Ohara

Ninomiya

Okabe

Hattori

, Distribution of adipose-derived stem cells in adipose tissues from human cadavers, J. Plast. Reconstr. Aethet. Surg. 63(10) (2010), 1717. doi:10.1016/j.bjps.2009.10.020.

Zuk

P.A.

Zhu

Mizuno

Huang

Futrell

J.W.

Katz

A.J.

, Multilineage cells from human adipose tissue: Implications for cell-based therapies, Tissue Eng. 7(2) (2001), 211. doi:10.1089/107632701300062859.

10.

Hattori

Sato

Masuoka

Ishihara

Kikuchi

Matui

, Osteogenic potential of human adipose tissue-derived stromal cells as alternative stem cell source, Cells Tissues Organs 178(1) (2004), 2. doi:10.1159/000081088.

11.

Hattori

Masuoka

Sato

Ishihara

Asazuma

Takase

, Bone formation using human adipose tissue-derived stromal cells and a biodegradable scaffold, J. Biomed. Mater. Res. B 76(1) (2006), 230. doi:10.1002/jbm.b.30357.

12.

Masuoka

Asazuma

Hattori

Yoshihara

Sato

Matsumura

, Tissue engineering of articular cartilage with autologous cultured adipose tissue-derived stromal cells using atelocollagen honeycomb-shaped scaffold with membrane sealing in rabbits, J. Biomed. Mater. Res. B 79(1) (2006), 25. doi:10.1002/jbm.b.30507.

13.

Huang

J.I.

Beanes

S.R.

Zhu

Lorenz

H.P.

Hedrick

M.H.

Benhaim

, Rat extramedullary adipose tissue as a source of osteo chondrogenic progenitor cells, Plast. Reconstr. Surg. 109(3) (2002), 1033, PMID: 11884830.

14.

Toriyama

Kawaguchi

Kitoh

Tajima

Inou

Kitagawa

, Endogenous adipocyte precursor cells for regenerative soft-tissue engineering, Tissue Eng. 8(1) (2002), 157. doi:10.1089/107632702753503144.

15.

Murohara

, Autologous adipose tissue as a new source of progenitor cells for therapeutic angiogenesis, J. Cardiol. 53(2) (2009), 155. doi:10.1016/j.jjcc.2009.01.003.

16.

Nambu

Ishihara

Nakamura

Mizuno

Yanagibayashi

Kanatani

, Enhanced healing of mitomycin C-treated wounds in rats using inbred adipose tissue-derived stromal cells within an atelocollagen matrix, Wound Rep. Regen. 15(4) (2007), 505. doi:10.1111/j.1524-475X.2007.00258.x.

17.

Nambu

Ishihara

Kishimoto

Yanagibayashi

Yamamoto

Azuma

, Stimulatory effect of autologous adipose tissue-derived stromal cells in an atelocollagen matrix on wound healing in diabetic db/db mice, J. Tissue Eng. 2 (2011), 158105. doi:10.4061/2011/158105.

18.

Mizuno

Zuk

P.A.

Zhu

Lorenz

H.P.

Benhaim

and Hedrick

M.H.

, Myogenic differentiation by human processed lipoaspirate cells, Plast. Reconstr. Surg. 109(1) (2002), 199, PMID: 11786812.

19.

Safford

K.M.

Hicok

K.C.

Safford

S.D.

Halvorsen

Y.D.

Wilkison

W.O.

Gimble

J.M.

, Neurogenic differentiation of murine and human adipose-derived stromal cells, Biochem. Biophys. Res. Commun. 294(2) (2002), 371. doi:10.1016/S0006-291X(02)00469-2.

20.

De Ugarte

D.A.

Morizono

Elbarbary

Alfonso

Zuk

P.A.

Zhu

, Comparison of multi-lineage cells from human adipose tissue and bone marrow, Cells Tissues Organs 174(3) (2003), 101. doi:10.1159/000071150.

21.

Wang

H.J.

Pieper

Schotel

Van Blitterswijk

C.A.

and Lamme

E.N.

, Stimulation of skin repair is dependent on fibroblast source and presence of extracellular matrix, Tissue Engineering 10(7–8) (2004), 1054. doi:10.1089/ten.2004.10.1054.

22.

Kang

S.K.

Shin

I.S.

and Ra

J.C.

, Journey of mesenchymal stem cells for homing: Strategies to enhance efficacy and safety of stem cell therapy, Stem Cells Int. (2012), 342968: 11 pages. doi:10.1155/2012/342968.

23.

Barbash

I.M.

Chouraqui

and Baron

, Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: Feasibility, cell migration, and body distribution, Circulation 108(7) (2003), 863. doi:10.1161/01.CIR.0000084828.503106A.

24.

Wahl

E.A.

Fierro

F.A.

Peavy

T.R.

Hopfner

Dye

J.F.

Machens

H.-G.

, In vitro evaluation of scaffolds for the delivery of mesenchymal stem cells to wounds, BioMed. Res. Int. 2015 (2015), 14 pages. doi:10.1155/2015/108571.

25.

Pittenger

M.F.

, Multilineage potential of adult human mesenchymal stem cells, Science 284(5411) (1999), 143, PMID: 10102814.

26.

Onesti

M.G.

Fioramonti

Carella

Fino

Marchese

and Scuderi

, Improvement of mouth functional disability in systemic sclerosis patients over one year in a trial of fat transplantation versus adipose-derived stromal cells, Stem Cell Intern. 2016 (2016), 9 pages. doi:10.1155/2016/2416192.

27.

Nakamura

S.I.

Ishihara

Takikawa

Murakami

Kishimoto

Nakamura

, Increased survival of free fat grafts and vascularization in rats with local delivery of fragmin/protamine microparticles containing FGF-2 (F/P MP-F), J. Biomed. Mater. Res. B 96(2) (2011), 234. doi:10.1002/jbm.b.31757.

28.

Nakamura

S.I.

Ishihara

Takikawa

Murakami

Kishimoto

and Nakamura

, Platelet-rich plasma (PRP) promotoes survival of fat-grafts in rats, Annal. Plast. Surg. 65(1) (2010), 101. doi:10.1097/SAP.0b013e3181b0273c.

29.

Mori

Nakamura

Kishimoto

Kawakami

Suzuki

Matsui

, Preparation and characterization of low-molecular-weight heparin/protamine nanoparticles (LMW-H/P NPs) as FGF-2 carrier, Int. J. Nanomed. 5(1) (2010), 147, PMID: 20463930.

30.

Ishihara

Kishimoto

Takikawa

Hattori

Nakamura

and Shimizu

, Biomedical application of low molecular weight heparin/protamine nano/micro particles as cell- and growth factor-carriers and coating matrix, Int. J. Mol. Sci. 16(5) (2015), 11785. doi:10.3390/ijms160511785.

31.

Nakamura

Kishimoto

Nakamura

S.-I.

Nambu

Fujita

Tanaka

, Fragmin/protamine microparticles as cell carriers enhance viability of adipose-derived stromal cells and their subsequent effect on in vivo neovascularization, J. Biomed. Mater. Res. A 92(4) (2010), 1614. doi:10.1002/jbm.a.32506.

32.

Takikawa

Sumi

Ishihara

Kishimoto

Nakamura

Yanagibayashi

, PRP&F/P MPs improved survival of dorsal paired pedicle skim flaps in rats, J. Surg. Res. 170(1) (2011), e189. doi:10.1016/j.jss.2011.05.051.

33.

Takikawa

S.-I.

Nakamura

Nambu

Ishihara

Fujita

, Enhancement of vascularization and granulation tissue formation by growth factors in human platelet-rich plasma-containing fragmin/protamine microparticles, J. Biomed. Mater. Res. B 97(2) (2011), 373. doi:10.1002/jbm.b.31824.

34.

Kishimoto

Ishihara

Mori

Takikawa

Hattori

Nakamura

, Effective expansion of human adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles-coated substratum with human platelet-rich plasma, J. Tissue Eng. Regen. Med. 7(12) (2012), 955. doi:10.1002/term.1488.

35.

Hattori

Nogami

Tanaka

Amano

Fukuda

and Kishimoto

, Expansion and characterization of adipose tissue-derived stromal cells cultured with low serum medium, J. Biomed. Mater. Res. B 87B (2008), 229.

36.

A.V.

Mendez

J.J.

Chang

Niklason

L.E.

and Steinbacher

D.M.

, Osteogenic performance of donor-matched human adipose and bone marrow mesenchymal cells under dynamic culture, Tissue Engineer. 21(9 and 10) (2015), 1621. doi:10.1089/ten.tea.2014.0115.

37.

Grayson

W.L.

Frohlich

Yeager

Bhumiratana

Chan

M.E.

Cannizzaro

, Engineering anatomically shaped human bone grafts, Proc. Natl Acad. Sci. USA 107(8) (2010), 3299. doi:10.1073/pnas.0905439106.

38.

L.L.

Wang

D.S.

Wang

J.Z.

, The interactions between rat-adipose-derived stromal cells, recombinant human bone morphogenetic protein-2, and beta-tricalcium phosphate play an important role in bone tissue engineering, Tissue Eng. Part A 16(9) (2010), 2927. doi:10.1089/ten.TEA.2010.0018.

39.

Folgiero

Migliano

Tedesco

Iacovelli

Bon

Torre

M.L.

, Purification and characterization of adipose-derived stem cells from patients with lipoaspirate transplant, Cell Transplant. 19(10) (2010), 1225. doi:10.3727/09638910X519265.

40.

Masuoka

Asazuma

Ishihara

Sato

Hattori

Ishihara

, Tissue engineering of articular cartilage using allograft of cultured chondrocytes in a membrane-sealed atelocollagen honeycomb-shaped scaffold, J. Biomed. Mater. Res. B 75(1) (2005), 177. doi:10.1002/jbm.b.30284.

41.

Sato

Kikuchi

Ishihara

Asazuma

Kikuchi

, Tissue engineering of the intervertebral disc with cultured annulus fibrosus cells using atelocollagen honeycomb-shaped scaffold with membrane seal (ACHMS scaffold), Med. Biol. Eng. Comput. 41 (2003), 365.

42.

Dang

P.N.

Solorio

L.D.

and Alsberg

, Driving cartilage formation in high-density human adipose-derived stem cell aggregate and sheet construct without exogenous growth factor delivery, Tissue Engineering Part A 20(23,24) (2014), 3163. doi:10.1089/ten.tea.2012.0551.

43.

Shimizu

Kamei

Adachi

Hamanishi

Kamei

Mahmoud

E.E.M.

, Repair mechanism of osteochondrocyte defect promoted by bioengineered chondrocyte sheet, Tissue Engineering 21(5,6) (2015), 1131. doi:10.1089/ten.tea.2014.0310.

44.

Tang

and Okano

, Recent development of temperature-responsive surfaces and their application for cell sheet engineering, Regenerative Biomaterials 1(1) (2014), 91. doi:10.1093/rb/rbu011.

45.

Kishimoto

Ishihara

Takikawa

Mori

Hattori

Fujita

, Novel experimental and clinical therapeutic uses of low-molecular-weight heparin/protamine microparticles, Pharmaceutics 4 (2012), 42. doi:10.3390/pharmaceutics4010042.

46.

Nemeno

J.G.E.

Lee

Yang

Lee

K.M.

and Lee

J.I.K.

, Applications and implications of heparin and protamine in tissue engineering and regenerative medicine, BioMed. Res. Int. 2014 (2014), 10 pages. doi:10.1155/2014/936196.

47.

Nguyen

A.H.

McKinney

Miller

Bongiorno

and McDevitt

T.C.

, Gelatin methacrylate microspheres for growth factor controlled release, Acta Biomater. 13(101-10) (2015), 101. doi:10.1016/j.actbio.2014.11.028.

48.

Zhou

Yan

Zhang

Liu

Huang

, Expansion and delivery of adipose-derived mesenchymal stem cells on three microcarriers for soft tissue regeneration, Tissue Eng. Part A 17(23–24) (2011), 2981. doi:10.1089/ten.tea.2010.0707.

49.

Sumi

Ishihara

Kishimoto

Takikawa

Doumoto

Azuma

, Transplantation of inbred adipose-derived stromal cells in rats with plasma gel containing fragmin/protamine microparticles and FGF-2, J. Biomed. Mater. Res. B 101(5) (2013), 784. doi:10.1002/jbm.b.32882.

50.

Sumi

Ishihara

Kishimoto

Takikawa

Hattori

Takikawa

, Effective wound healing in streptozotocin-induced diabetic rats by adipose-derived stromal cell-transplantation in plasma-gel containing fragmin/protamine microparticles, Ann. Plast. Surg. 72(1) (2014), 113. doi:10.1097/SAP.0000000000000014.

51.

Lindahl

Lidholt

Spillmann

and Kjellen

, More to heparin than anti-coagulation, Thromb. Res. 75 (1994), 1–32.

52.

Ishihara

and Ono

, Structure and function of heparin and heparan sulfate: Heparinoid library anmodification of FGF-activities, Trends Glycosci. Glycotechnol. 10(52) (1998), 223.

53.

Kishimoto

Inoue

Nakamura

Hattori

Ishihara

Sakuma

, Low-molecular weight heparin protamine complex augmented the potential of adipose-derived stromal cells to ameliorate limb ischemia, Atherosclerosis 249 (2016), 132. doi:10.1016/j.atherosclosis.2016.04.003.

54.

Nakamura

Kanatani

Kishimoto

Nambu

Ohno

Hattori

, Controlled release of FGF-2 using fragmin/protamine microparticles and effect on neovascularization, J. Biomed. Mater. Res. A 91(3) (2009), 814. doi:10.1002/jbm.a.32265.

55.

Kishimoto

Ishihara

Nakamura

Takikawa

Fujita

Sumi

, Fragmin/protamine microparticles to absorb and protect HGF and to function as local HGF carrier in vivo, Acta Biomater. 9(1) (2013), 4763. doi:10.1016/j.actbio.2012.08.003.

56.

Shimojo

A.A.M.

Perez

A.G.M.

Galdames

S.E.M.

Brissac

I.C.S.

and Santana

M.H.A.

, Performance of PRP associated with porous chitosan as a composite scaffold for regenerative medicine, The Scientific World J. 2015 (2015), 12 pages. doi:10.1155/2015/396131.

57.

Verseijden

Posthumus-van

S.S.J.

vanNeck

J.W.

Hofer

S.O.

Hovius

S.E.

and vanOsch

G.J.

, Comparing scaffold-free and fibrin-based adipose-derived stromal cell constructs for adipose tissue engineering: An invitro and in vivo study, Cell Transplant 21(10) (2012), 2283. doi:10.3727/096368912X653129.

58.

Han

T.T.

Toutounji

Amesden

B.G.

and Flynn

L.E.

, Adipose-derived stromal cells mediate in vivo adipogenesis, angiogenesis and inflammation in decellularized adipose tissue bioscaffolds, Biomaterials 72(125-37) (2015), 125–137. doi:10.1016/j.biomaterials.2015.08.053.

59.

Hong

Peptan

I. A.

Coptan

I. A.

and Daw

J. L.

, Adipose tissue engineering by human adipose-derived stromal cells, Cells Tissues Organs 183(3) (2006), 133. doi:10.1159/000095987.

60.

Lin

S.D.

Huang

S.H.

Lin

Y.N.

S.H.

Chang

H.W.

Lin

T.M.

, Engineering adipose tissue from uncultured human adipose stromal vascular fraction on collagen matrix and gelatin sponge scaffolds, Tissue Eng. Part A 17(11–12) (2011), 1489. doi:10.1089/ten.TEA.2010.0688.

61.

Mauney

J.R.

Nguyen

Kirker-Head

Gimble

J.M.

and Kaplan

D.R.

, Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose-derived mesenchymal stem cell with silk fibroin 3D scaffolds, Biomaterials 28(25) (2007), 5280. doi:10.1016/j.biomaterials.2007.08.017.

62.

Barba

Cicione

Bernardini

Michetti

and Lattanzi

, Adipose-derived mesenchymal cells for bone regeneration: State of the art, BioMed. Research Intern. 2013 (2013), 11 pages, ID 416391. doi:10.1155/2013/416391.

63.

Hattori

Ishihara

Fukuda

Suda

and Katagiri

, Establishment of a novel method for enriching osteoblast projenitors from adipose tissue using a difference in cell adhesive properties, Biochem. Biophys. Res. Commun. 343(4) (2006), 1118. doi:10.1016/j.bbrc.2006.03.061.

64.

Schmits

J.P.

Holliger

J.O.

and Milam

S.B.

, Reconstruction of bone using using calcium phosphate bone cements: A critical review, J. Oral Maxillofacial Surg. 57(9) (1999), 1122, PMID: 10484115.

65.

Liu

Cen

Yin

, A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and 𝛽-TCP ceramics, Biomaterials 29(36) (2008), 4792. doi:10.1016/j.biomaterials.2008.08.039.

66.

Kruyt

M.C.

Wilson

C.E.

deBruijn

J.D.

, The effect of cell-based bone tissue engineering in a goat transverse process model, Biomaterials 27(29) (2006), 5099. doi:10.1016/j.biomaterials.2006.05.048.

67.

Lopez

M.J.

and Daigle

P.R.

, Adult multipotent stromal cell technology for bone regeneration: A review, Veterinary Surg. 42(1) (2013), 1. doi:10.1111/j.1532-950X.2012.01077.x.

68.

Dong

Z.J.

Uemura

Shirasaki

and Tateishi

, Promotion of bone formation using highly pure porous 𝛽-TCP combined with bone marrow-derived osteoprogenitos cells, Biomaterials 23(23) (2002), 4493, PMID: 12322969.

69.

Hing

K.A.

Wilson

C.E.

and Buckland

, Comparative performance of three ceramic bone graft substitutes, Spine J. 7(4) (2007), 475.

70.

Liao

H.T.

Lee

M.Y.

Tsai

W.W.

Wang

H.C.

and Lu

W.C.

, Osteogenesis of adipose-derived stem cells on poly-caprolactone-beta-tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I, J. Tissue Eng. Regen. Med. (2013), PMID: 23955935. doi:10.1002/term.1811.

71.

Rosenberg

, Chemical basis for the histological use of saframin O in the study of articular cartilage, J. Bone Joint Surg. Am. 53(1) (1971), 69, PMID: 4250366.

72.

Hattori

and Ishihara

, Altered protein secretions during interactions between adipose tissue- or bone marrow-derived stromal cells and inflammatory cells, Stem Cell Res. Ther. 6 (2015), 70. doi:10.1186/s3287-015-0052-y.

73.

Natesan

Baer

D.G.

Walters

T.J.

Walters

T.J.

Babu

and Christy

R.J.

, Adipose derived stem cell delivery into collagen gels using chitosan microspheres, Tissue Eng. A 16(4) (2010), 1369. doi:10.1089/ten.tea.2009.0404.

74.

Shokrgozar

M.A.

Fattahi

Bonakdar

Kashani

I.R.

Majidi

Haghighipour

, Healing potential of mesenchymal stem cells cultured on a collagen-based scaffold for skin regeneration, Iran Biomed. J. 16(2) (2012), 68. doi:10.6091/ibj.1053.2012.

75.

Takeshita

Zhengm

L.P.

Brogi

Kearney

L.Q.

Bunting

, Therapeutic angiogenesis. A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model, J. Clin. Invest. 93(2) (1994), 662. doi:10.1172/JCI117018.

76.

Morishita

Nakamura

Hayashi

Taniyama

Moriguchi

Nagano

, Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in rabbit hind limb ischemia model as cytokine supplement therapy, Hypertension 33(6) (1999), 1379, PMID: 10373220.

77.

Losordo

D.W.

and Dimmeler

, Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: Angiogenic cytokines, Circulation 109(21) (2004), 2487. doi:10.1161/01.CIR.0000128595.79378.FA.

78.

Asahara

Murohara

Sullivan

Silver

van der Zee

, Isolation of putative progenitor endothelial cells for angiogenesis, Science 275(5302) (1997), 964, PMID: 9020076.

79.

Kalka

Masuda

Takahashi

Kalka-Moll

W.M.

Silver

Kearney

, Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization, Proc. Natl Acad. Sci. USA 97(7) (2000), 3422. doi:10.1073/pnas.070046397.

80.

Makarevich

P.I.

Boldyreva

M.A.

Gluhanyuk

E.V.

Efimenko

A.U.

Dergilev

K.V.

Shevchenko

E.K.

, Enhanced angiogenesis in ischemic skeletal muscle after transplantation of cell sheets from baculovirus-transduced adipose-derived stromal cells expressing VEGF165, Stem Cell Res. Ther. 6 (2015), 204. doi:10.1186/s13287-015-0199-6.

81.

Liu

Zeng

Zuo

Feng

, Primed 3D injectable microniches enabling low-dosage cell therapy for critical limb ischemia, Proc. Natl Acad. Sci. USA 111(37) (2014), 13511. doi:10.1073/pnas.1411295111.

82.

Nakamura

Takikawa

Ishihara

Nakayama

Kishimoto

Isoda

, Delivery system for autologous growth factors fabricated with low-molecular- weight heparin and protamine to attenuate ischemic hin-limb loss in a mouse model, J. Artif. Organ 15(4) (2012), 375. doi:10.1007/s10047-012-0658-0.

83.

Hattori

Amano

Ogawa

H.Y.

Ando

Takase

and Ishihara

, Angiogenesis following cell injection by an excess inflammatory response coordinated by bone marrow cells, Cell Transpl. 22(12) (2013), 2381. doi:10.3727/096368912X658863.

84.

Hall

Andreeff

and Marini

, The participation of mesenchymal stem cells in tumor stroma formation and their aplication as targeted-gene delivery vehicles, Handbook of Exp. Pharmacol. 180 (2007), 263. doi:10.1007/978-3-540-68976-8_12.

85.

Schweizer

Tsuji

Gorantla

V.S.

Marra

K.G.

Rubin

J.P.

and Plock

J.A.

, The role of adipose-derived stem cells in breast cancer progression and metastasis, Stem Cells Intern. 2015 (2015), 120949, 17 pages. doi:10.1155/2015/120949.

86.

Langer

and Vacanti

J.P.

, Tissue engineering, Science 260(5110) (1993), 920, PMID: 8493529.

87.

Badylak

S.F.

Taylor

and Uygun

, Whole-organ tissue engineering: Decellularization and recellularization of three-dimensional matrix scaffolds, Annu. Rev. Biomed. Eng. 13 (2011), 27. doi:10.1146/annurev-bioeng-071910-124743.