|Year : 2017 | Volume
| Issue : 1 | Page : 4-18
Recent advances in bone regeneration: The role of adipose tissue-derived stromal vascular fraction and mesenchymal stem cells
Yasir Alabdulkarim1, Bayan Ghalimah2, Mohammad Al-Otaibi1, Hadil F Al-Jallad3, Mina Mekhael4, Bettina Willie5, Reggie Hamdy5
1 Department of Experimental Surgery, McGill University; Department of Orthopedic Surgery, Faculty of Medicine, King Fahad Medical City, Riyadh; Division of Experimental Surgery, Department of Surgery, Shriners Hospital for Children, Canadan Unit, Quebec, Canada
2 Department of Experimental Surgery, McGill University; Division of Experimental Surgery, Department of Surgery, Shriners Hospital for Children, Canadan Unit, Quebec, Canada; Department of Orthopaedic Surgery, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
3 Division of Experimental Surgery, Department of Surgery, Shriners Hospital for Children, n Unit, Quebec, Canada
4 Division of Experimental Surgery, Department of Surgery, Shriners Hospital for Children, n Unit; Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
5 Department of Experimental Surgery, McGill University; Division of Experimental Surgery, Department of Surgery, Shriners Hospital for Children, n Unit; Department of Pediatric Surgery, Division of Paediatric Orthopaedic Surgery, Montreal Children Hospital, McGill University, Montreal, Quebec, Canada
|Date of Web Publication||15-Mar-2017|
Shriners Hospital for Children, 1003, Boulevard Decarie, Montreal, Quebec H4A 0A9
Source of Support: None, Conflict of Interest: None
The management of large bone defects, atrophic nonunions, and other conditions with poor bone formation presents a formidable challenge to the treating physician, as all available techniques of bone reconstruction have drawbacks. Recent advances in stem cell biology, specifically adipose tissue-derived mesenchymal stem cells (ASCs) and adipose tissue stromal vascular fraction (SVF), have opened up new horizons by providing a reliable and abundant source of stem cells with osteogenic potential that can be used in various bone tissue engineering techniques. In this review, several aspects related to the use of ASCs are addressed, such as harvesting and processing of adipose tissue, advantages of ASCs over bone marrow-derived mesenchymal stem cells, mechanism of action and safety of ASCs, and factors affecting the differentiation of ASCs. Published reports on the use of ASCs in critical size defects, nonunions, and distraction osteogenesis are also reviewed. Innovative trends in stem cell research on musculoskeletal pathologies are highlighted, with special emphasis on the increasing evidence that the direct application of freshly prepared SVF processed from adipose tissue into the bone defect to be treated without a prior differentiation or an ex vivo expansion and culture is possible. This highly promising approach may lead to the development of a one-step intraoperative cell therapy.
Keywords: Adipose-derived stem cells, bone regeneration, critical size defect, distraction osteogenesis, stem cells, stromal vascular fraction
|How to cite this article:|
Alabdulkarim Y, Ghalimah B, Al-Otaibi M, Al-Jallad HF, Mekhael M, Willie B, Hamdy R. Recent advances in bone regeneration: The role of adipose tissue-derived stromal vascular fraction and mesenchymal stem cells. J Limb Lengthen Reconstr 2017;3:4-18
|How to cite this URL:|
Alabdulkarim Y, Ghalimah B, Al-Otaibi M, Al-Jallad HF, Mekhael M, Willie B, Hamdy R. Recent advances in bone regeneration: The role of adipose tissue-derived stromal vascular fraction and mesenchymal stem cells. J Limb Lengthen Reconstr [serial online] 2017 [cited 2018 Apr 24];3:4-18. Available from: http://www.jlimblengthrecon.org/text.asp?2017/3/1/4/202207
| Introduction|| |
Bone has the intrinsic capacity of self-repair and the ability to generate new vascularized tissue that is indistinguishable from the surrounding native bone in both its micro and macro architecture. In certain circumstances, however, this innate capacity to heal spontaneously is impaired and external intervention using various bone reconstruction techniques becomes necessary. These conditions include delayed and atrophic nonunion of fractures, poor bone regeneration during distraction osteogenesis and massive bone loss secondary to trauma, postdebridement of osteomyelitis, and postresection of bone tumors [Figure 1]. The clinical importance of these conditions of poor bone formation is reflected by the ever-increasing incidence of cases requiring bone reconstruction. There are reportedly over 2 million bone grafts used in orthopedic procedures worldwide annually in both pediatric and adult populations. Furthermore, besides an unparalleled increase in civilian trauma, war injuries are more common and more severe, due to the nature of weapons used in these conflicts and improvements in prehospital trauma care. This enormous and mounting demand for long-bone and craniofacial skeletal reconstruction represents a major source of financial burden globally.
|Figure 1: Segmental bone defects caused by congenital pseudarthrosis of the tibia after excision of the defect (a-c), tumors (d), posttraumatic (e and f), postinfectious (g and h)|
Click here to view
The current gold-standard procedure for repair and reconstruction of most of these conditions remains autologous bone grafts (ABGs). Harvested most commonly from the iliac crest, ABGs are the only graft material that contains all three elements required for successful bone regeneration: osteoconduction, osteoinduction, and osteogenesis. However, ABGs have several disadvantages, including a limited supply, especially in children, and donor site morbidity (pain, paresthesia, infection, and blood loss)., The Reamer-Irrigator-Aspirator technique is an alternative technique of autogenous bone graft harvesting , that allows for the possibility of harvesting large volumes of cancellous bone (about 60 cc). This method is, however, invasive and includes the potential complications of femoral fractures and cortical penetration. Vascularized, free fibular bone graft is another type of autogenous bone graft, currently being used in the reconstruction of long bone defects of more than 5–7 cm.,,, The advantage of using this method is the availability of a large vascularized corticocancellous autogenous bone graft. On the other hand, it is technically demanding, requires a microvascular technique, results in longer surgical procedure, and may lead to an increase in morbidity and adverse effects on the donor limb site, most notably, gait changes. Bone transport is a technique applying the Ilizarov principles of distraction osteogenesis where large amounts of segmental bone loss are replaced with vascularized native bone. However, the disadvantages include a long period where the external fixator has to be kept in place until the new bone consolidates, and problems related to external fixation, such as pin site infection, pain, and the need for another surgery for bone grafting the docking site.,, Although Ilizarov himself rejected the concept of intramedullary interference, nevertheless, some surgeons recommend bone transport over nails, which diminish the external fixator time by removing the external fixator after the distraction period without increasing the healing time. The Masquelet or induced membrane technique, recently introduced by Masquelet, is used to treat large segmental bone defects as much as 25 cm long.,, It involves the complete removal of all devitalized bone, internal or external fixation, and the application of a cement spacer. This allows for the formation of a periosteal-like membrane around the cement spacer, which is the most important advantage of this technique. After 4–6 weeks, the cement spacer is removed and the defect is filled with ABG or other bone graft materials if necessary. Other advantages include protection against autograft resorption, maintenance of graft position, and prevention of soft tissue interposition. However, the outcome of this technique is not always satisfactory. Allografts offer an alternative to ABGs by providing large amounts of morselized or structural grafts without the need for harvesting. Allografts have major limitations: rejection, transmission of diseases, and cost. They also have lower incorporating properties with the host healing tissues compared to autografts. Demineralized bone matrix (DBM) is a chemically processed allograft product where inorganic materials are removed while the organic collagen matrix is preserved. It has osteoconductive and osteoinductive properties with no osteogenic effect., DBM also does not evoke any immunological reaction due to the fact that most surface antigens are destroyed during the preparation process. Numerous studies have shown that DBM is a biocompatible scaffold and is widely used in several forms alone or to augment other techniques of bone reconstruction. It has also been safely used to deliver adipose stem cells (ASCs).,, Bone graft substitutes (BGSs) include several categories: natural or organic, such as collagen and chitosan, and synthetic, such as polymers, ceramics and glass, and composite grafts. BGSs are not only used as “filler” but also act as “scaffolds” which are porous in nature. Recent advances, particularly in three-dimensional (3D) printing, have enabled the control of pore size and interpore connectivity. It is very porosity that allows for cells to adhere, proliferate, and differentiate, and more importantly, for blood vessels to invade the scaffold. Altogether, they promote bone regeneration. Various osteogenic-inducing factors, such as bone morphogenetic proteins, may be used either alone or in combination with other BGSs. However, all types of allografts and BGSs lack an essential element of bone reconstruction – progenitor osteogenic cells, which is the cornerstone of all the latest techniques of bone tissue engineering. This has sparked the search for other sources of osteogenic cells.
Stem cells, with their capacity for unlimited self-renewal and differentiation into various cell types, have emerged as an attractive cell source for bone reconstruction., They are uncommitted progenitor cells, present in almost every tissue of the body, and are designed to preserve the structural and functional integrity of tissues, replacing mature, injured or diseased cells. They are the “maintenance and repair shop” of the body. Stem cells can be isolated from either embryonic or postnatal tissues. Adult stem cells obtained from postnatal tissues include hematopoietic stem cells (HSC), mesenchymal stem cells (MSCs), and induced pluripotent stem cells.
| Discovery of Stem Cells|| |
In 1909, Dr. Alexander Maximow described a unique type of cells present in bone marrow (BM) that had the potential to self-renewal and to differentiate into mature blood cells. These cells were transplantable, and their discovery set the foundation for BM transplantation and the treatment of lymphoproliferative disorders. They are known as hematopoietic stem cells. The other common type of adult stem cells, the MSCs, is the focus of this review.
Mesenchymal stem cells
MSCs are non-HSCs discovered in the early 1960s when it was demonstrated that ectopic transplantation of BM fragments in animals leads to the formation of de novo bone., However, it was Caplan, in 1991, who coined for the first time the term “MSCs.” The discovery of MSCs attracted much attention due to their potential to differentiate into cells of mesodermal origin, such as osteoblasts, chondrocytes, and adipocytes. MSCs were initially discovered in BM (BM-derived mesenchymal stem cells [BM-MSCs]). They were then isolated from several other tissues, including adipose tissue, dermis, periosteum, umbilical cord blood, placenta and amniotic fluid, and synovial fluid.,,,,, Besides the capacity for self-renewal and the power to differentiate into multiple cell types, the International Society for Cellular Therapy also recommends the following minimal criteria for defining a cell as a MSC: (1) cells must be plastic adherent when they are maintained in standard culture conditions; (2) cells must express surface markers CD105, CD73, CD34, CD29, and CD90; and (3) cells must lack surface markers CD45, CD31, CD235a, CD11b, CD79, CD19, and human leukocyte antigen-antigen D related surface molecules.,
Osteogenic potential of bone marrow-derived mesenchymal stem cell
Numerous studies have reported the successful use of MSCs obtained from BM aspirate in cases of distraction osteogenesis,, delayed union and nonunion of fractures, critical size defects,, spinal fusions, and a vascular necrosis of the femoral head., The use of BM-MSCs was also reported to improve clinical outcome in patients with mild to moderate osteoarthrosis. BM-MSCs are still the most frequently used stem cells in cell-based bone tissue engineering.
Problems associated with bone marrow-derived mesenchymal stem cells
Despite numerous reports of successful bone healing with BM-MSCs, there are, however, several disadvantages: (1) it is a painful and invasive procedure; (2) it often results in inconsistent and low yield of MSCs; and (3) in vitro culture and expansion of BM-MSCs is required to obtain an adequate number of stem cells for clinical application. Cell expansion and culture for clinical application must be carried out in a laborious, expensive, and time-consuming good manufacturing practice (GMP) laboratory. Moreover, the ability of MSCs to proliferate and differentiate declines after extensive passages.,, There is also an increased risk for pathogen contamination and genetic transformation with repeated cultures. Furthermore, culturing for a few weeks necessitates an additional surgery for reimplantation. Eliminating the need for an extra surgery strongly motivated the development of intraoperative techniques to avoid time – consuming, expensive and laborious GMP handling. These techniques include the use of specific machines such as Bone Marrow Aspirate Concentrate (BMAC™) System (Harvest Technologies GmbH). This system is designed to be used in the operating room, where BM aspirate is centrifuged and concentrated in about 20 min. However, besides the issues associated with BM-MSCs mentioned previously, the large volume of BM aspirate – about 300–400 cc – required to obtain the optimal amount of BM concentrate as recommended by Hernigou (20 cc),, may represent too large a volume of aspirate for many patients, especially in children.
Most importantly, however, it is the paucity of published reports on the results of using BMAC technique with immediate reimplantation into the patient in a one-step surgery. Apart from the studies reported by Hernigou et al. on the effectiveness of using bone mineral content (BMC) in various bone pathologies,,, we were able to find only few clinical studies reporting the results of these techniques. Most clinical trials are Phase 1 or 2 (clinicaltrials.gove-NCT01581892, NCT0178805, and NCT01206179). A Phase 3 clinical trial was started in 2007 and was completed by 2013 (clinicaltrials.gove-NCT00512434). This trial involved multiple centers in France and recruited 186 patients with open tibial fractures. The patients were randomized into two groups of 93 patients each. One group received BMAC and standard treatment, while the other group received the standard treatment only. The results, however, are still unpublished.
Most published reports on the use of BMAC are either retrospective in nature, conducted in a single center, or included a small number of patients., Other studies combined the use of BMC with scaffolds, growth factors, low-intensity pulse ultrasound or allograft, and other inducing agents. This makes it more difficult to reach any significant conclusion on the efficacy of BMC. Furthermore, most of these studies are nonrandomized and noncontrolled., In a retrospective study by Le Nail et al., 43 cases of delayed and nonunion of open tibial fractures were treated with BMAC. Only 23 cases who received BMAC had successful healing (53.5%). The success of the procedure depended on the number of colony-forming unit-fibroblast (CFU-F) received (469 vs. 153.103, P = 0.013, success vs. failure). In this report, it was also mentioned that BMAC if prepared within the first 110 days of the fracture, the success was 47%, while if prepared 110 days after fracture, the success increases to 73%. It was also reported in that paper that there was no healing if the gap was >4 mm. In another retrospective study, 19 patients with long bone nonunion were evaluated following the use of BMC and complete healing occurred in 15 cases (78%). Recently, Stanovici et al. reported that aspirating BM followed by centrifugation to concentrate mononuclear cells and then immediate implantation into the bone defect with or without the use of a synthetic bone substitute has not led to reproducible results. Therefore, there remain major limitations with the use of BMAC regarding the number of BM-MSCs available for reinjection. These disadvantages prompted scientists to search for other reliable sources of MSCs.
In 2001, Zuk et al. made a major breakthrough in stem cell biology when they discovered that adipose tissue contained a rich source of MSC, adipose-derived stem cells (ADSc). This discovery opened new horizons in regenerative medicine and bone tissue engineering techniques.
| The Adipose Tissue as an Attractive Source for Mesenchymal Stem Cell|| |
The adipose tissue is a specialized form of connective tissue that serves an important role in energy storage and metabolism. It also provides other vital mechanical and metabolic functions, such as hormone synthesis and secretion, endogenous heat production and thermal insulation, and internal organs support and lubrication. On average, adipose tissue comprises around 15%–20% of the total body weight. There are two types of adipose tissue in the human body, the white adipose tissue and brown adipose tissue. Both types are characterized by the presence of adipocytes, but each has its unique physiological functions. The brown adipose tissue, mainly present during the first 10 years of human life, functions as a source for endogenous heat production. The white adipose tissue is present in the human body throughout life and specializes in long-term energy storage. In histological sections, the adipose tissue is formed by two compartments, the parenchyma (functional part) and stroma (supportive component). Adipocytes, the fat-storing cells, make up the parenchyma and are considered the predominant cells in the adipose tissue. They are embedded in an extracellular matrix (ECM) formed by a connective tissue. Stromal cells refer to the connective tissue cells  and constitute the stromal vascular fraction (SVF) of adipose tissue with large amounts of MSCs. [Figure 2] represents a schematic for the constituents of adipose tissue.
|Figure 2: The cellular and extracellular constituents of adipose tissue. The cellular population comprises mature adipocytes, preadipocytes, postadipocytes, mesenchymal stem cells, endothelial cells, pericytes, mast cells, macrophages, fibroblasts, circulating blood cells, reticulocytes, and nervous system elements. Half of the cell population is made up of mature adipocytes ranging from 25 to 200 μm in size, where each adipocyte is in direct contact with a blood vessel. Extracellular matrix is made of Collagen type IV and VI, laminin, proteoglycan, perlecan, and heparin sulfate, all are required for the protection and compartmentalization of the cellular population|
Click here to view
| Adipose Tissue-Derived Mesenchymal Stromal Cells (ASCS)|| |
ASCs have opened up promising new possibilities in adult stem cell therapies. Similar to BM-MSCs, ASCs can differentiate into various cells of mesodermal origin, including adipocytes, osteoblasts, and chondrocytes., More recently, it has been shown that their capacity to differentiate is not only limited to cells of mesodermal originbut also able to differentiate into cells of ectodermal and endodermal origin under appropriate conditions. ASCs show many similarities with BM-MSC in surface marker profiles, multilineage differentiation potential, and growth properties.,,
| Advantages of ASCS over Bone Marrow-Derived Mesenchymal Stem Cells|| |
[Table 1] provides a comparison between BM-MSC and ASCs.
|Table 1: Stem cells isolated from adipose tissue are identical to the bone marrow counterparts with no postharvest complications|
Click here to view
Compared to BM, adipose tissue can be harvested with minimal patient discomfort. It is readily available in large, almost unlimited quantities., It has a high stem cell to volume ratio., 70, ,,,, In fact, adipose tissue contains the highest concentration of MSC of all tissues. One gram of adipose tissue holds about 500-fold greater the number of MSCs than 1 g of BM.,, Harvesting can easily be scaled up according to the need. ASCs possess more immunomodulation capacity than BM-MSCs. Most importantly, adipose tissue can be processed within a short time frame (<2 h) to obtain highly enriched ASC preparations (residing in the SVF), thus allowing to obtain clinically relevant stem cell quantities that can be applied immediately after adipose tissue processing in a one-step surgical procedure.,, The proven capacity of ASCs for robust osteogenesis has placed it at the forefront of bone regenerative medicine and bone tissue engineering as the ideal cell source to regenerate bone. This is reflected in the important funding allocated to ASC research as well as the number of clinical trials underway. Over the past years, the California Institute of Regenerative Medicine have invested US$3 billion and the National Institutes of Health US$1.4 over the past years (total of US$8.4 billion) in the field of stem cell therapy and regenerative medicine. There are over sixty ongoing clinical trials investigating the use of SVF and ASCs in various conditions.
Processing of adipose tissue and isolation of stromal vascular fraction
Several techniques of harvesting adipose tissue have been described. [Table 2] summarizes the different techniques used to isolate the SVF, comprising resection, tumescent liposuction, and ultrasound-assisted liposuction. It has been shown in a human study that resection and tumescent liposuction are preferable to ultrasound-assisted liposuction in the yield and growth characteristics of ASCs. After harvesting, the adipose tissue is washed, rinsed, followed by an enzymatic digestion with collagenase, and then centrifugation, leading to the formation of SVF [Figure 3].,, Nonenzymatic mechanical techniques have also been developed. However, the disadvantage of mechanical techniques is the very low yield of ASCs. In a recent international meeting on ASCs held in San Diego, USA, in November 2016, the International Federation for Adipose Therapeutics and Science agreed that the gold standard for isolation of SVF and ASCs remained the collagenase enzymatic digestion [Table 3].
|Table 2: Harvesting and Isolation of adipose-derived stem cells, enzymatic versus mechanical methods of isolation|
Click here to view
|Figure 3: The stromal vascular fraction is obtained after adipose tissue resection, washing, mincing, tissue collagenase digestion, and centrifugation. For functional studies, the stromal vascular fraction can be cultured where the ASCs adhere to the plastic and assume a spindle-like shape. Used with permission Gir P, et al.|
Click here to view
|Table 3: Key differences between the various methods of adipose-derived stem cells isolation [Summary for Table 1]|
Click here to view
SVF [Figure 3] contains a mixture of heterogeneous progenitor and stem cells – MSCs (10%) and endothelial progenitor cells (2%) – that stimulate angiogenesis and release angiogenic growth factors, such as vascular endothelial growth factor and insulin-like growth factor. It also contains hepatocyte growth factor-1, macrophages and monocytes (10%),, lymphocytes (10%–15% of cells, including regulatory T-cells), interleukin (IL)-1 and IL-10 receptor antagonists, and matrix-secreting fibroblasts.
Mechanism of action of stromal vascular fraction
The regenerative capacity of SVF is likely derived from the heterogeneity of its constituents that provide numerous mechanisms for regeneration to occur [Figure 4]. It is this “cocktail” of cells and growth factors that gives the SVF a huge regenerative potential. MSCs residing in the SVF possess strong osteogenic potential not only by their ability to differentiate into osteogenic cells following induction, but also by their paracrine effects through the secretion of numerous growth factors and cytokines with angiogenic,, anti-inflammatory, and immunosuppressive properties. However, MSCs represent only 10% of cells in the SVF. Other cells residing in the SVF (as mentioned above) also have powerful regenerative potential, including angiogenic, anti-inflammatory, and immunomodulation effects. One of the most abundant cell types, the preadipocyte (59% of the total SVF population),, shares many of the same phenotypic markers and characteristics of MSCs, indicating an involvement in the regenerative process. Pericytes possess high proliferative potential and express typical MSC markers. Macrophages have immunological  and anti-inflammatory  effects.
MSCs present in the SVF can be further isolated from the SVF by plastic adherence in culture, expanded in vitro,,, and induced to differentiate into osteogenic lineages., Isolated ASCs can also be cryopreserved in a media of serum and dimethyl sulfoxide without losing their ability to differentiate and proliferate. Their capacity for cryopreservation could pave the way to an “Adipose Stem Cell Bank” in the future.
| Is There a Need to Isolate and Culture Ascs from the Stromal Vascular Fraction? Problems With Ex Vivo Expansion Cultures|| |
Several studies have reported that there is an adequate number of MSCs residing in the SVF to elicit a positive osteogenic response and that further ex vivo culture is not necessary. Ex vivo cultures of ASCs share similar disadvantages with cultures of BMCs, namely, the need for an expensive and time-consuming GMP laboratory, the increased risk for pathogen contamination and genetic transformation after repeated cultures (after passage 4), and the need for an additional surgery for reimplantation. It has also been shown that the characteristics of ASCs, in particular the osteogenic and angiogenic potential, might be altered  with repeated cultures.
Safety profile of stromal vascular fraction, ASCs, and carcinogenic potential
The safety profile of SVF and ASCs isolated from SVF has been well documented in several preclinical and clinical studies , in various pathologies: plastic surgery, burn wounds, diabetic ulcers,, and osteoarthritis. No adverse events have been reported in two Phase 1 trials involving SVF and osteoconductive scaffolds., However, concerns have been raised regarding the carcinogenic potential of ASCs upon transplantation as in vitro and preclinical studies report that ASCs can interact with tumor cells and induce tumor progression. This should be taken into consideration with patients previously diagnosed with cancer.
Differentiation of ASCs
Unlike BM and HSCs that will differentiate into bone cells or blood cells, respectively, without the need for induction, ASCs need to be induced to differentiate into bone. Numerous factors can direct the differentiation of ASCs into bone tissue, including specific culture media, growth factors (such as BMPs), platelet-rich plasma, alendronate, mechanical factors, and the surface topography and chemical structure of various scaffolds [Figure 5]., Until recently, it was thought that ASCs needed to be differentiated before implantation. Most reported preclinical studies on ASCs have therefore involved ex vivo culture and differentiation of ASCs before reimplantation [Figure 6].
|Figure 5: Stimulating factors affecting ASCs differentiation into osteoblasts|
Click here to view
|Figure 6: Adipose-derived stem cells. In the old paradigm, were used to be reinjected into the patients for regenerative purposes after ex vivo cellular expansion and cell culture|
Click here to view
Scaffolds, as proposed in the “diamond concept,” are one of the four critical elements to be considered for achieving successful bone regeneration along with growth factors, MSCs, and the mechanical environment. The role of scaffolds has evolved over the past few decades from just providing 3D structural support to cells, to guiding specific cellular behavior through the release of growth factors, incorporation of surface ligands, and engineered physicochemical properties.
Scaffolds used for bone tissue engineering must be biocompatible, biodegradable, and osteoconductive. Therefore, a wide range of natural and synthetic polymers have emerged as promising biomaterials. Examples of natural polymers include chitosan, collagen, alginate, and hyaluronic acid. Synthetic polymers, which have been explored more than their natural counterpart, include polycaprolactone, poly lactic-co-glycolic acid, polymethylmethacrylate, and poly L-lactic acid., Investigators have also examined the benefits of blending natural and synthetic polymers to obtain specific mechanical or biodegradation properties. Moreover, fabricating biocomposite scaffolds by incorporating an inorganic component (tricalcium phosphate, hydroxyapatite, etc.) has been investigated to improve cell adhesion, induce osteogenic differentiation, and increase mineralization. More recently, there has been revitalized interest in using magnesium to fabricate 3D biodegradable scaffolds for improving bone healing, which has demonstrated promising results.
Choosing the biomaterial(s) is the first step. The second step involves the fabrication of a 3D porous scaffold with specific pore sizes, excellent pore interconnectivity, and desirable mechanical properties. Porosity and pore-interconnectivity are essential to promote cellular infiltration into the scaffold, and to allow for nutrient/waste exchange. Mechanical properties, on the other hand, can be tailored to allow for load bearing or to promote osteogenic differentiation of MSCs. Various techniques have been developed to fabricate scaffolds for bone tissue engineering such as freeze-drying, self-assembly, phase separation, electrospinning, and 3D printing. Moreover, injectable hydrogels have been used to address a clinical need where minimally invasive surgery could be used to fill a critical size defect or bone cavity.,
Finally, incorporating osteogenic growth factors within the scaffolds and modifying the surface chemistry and topography to guide cellular behavior have been employed to improve the overall osteoinductive and osteoconductive properties of scaffolds. For example, controlling the spatiotemporal release of growth factors has been gaining attention recently since it mimics physiological conditions. On the other hand, there has been growing interest in understanding how surface nanotopography can induce MSC differentiation.,
| ASCS in Critical Size Segmental Defects|| |
The effect of ASCs has been extensively studied in animal models with critical size segmental defects (CSSDs), and results show strong evidence that ASCs significantly increased the rate of new bone formation and the overall mechanical strength at the site of defect.,,,,, In a study published by de Girolamo et al., the authors found that culture-expanded ASCs seeded on hydroxyapatite scaffold enhanced good bone formation in a rabbit critical size defect. The effects of ASCs were also assessed on rat ulnar CSSD models. At 24 weeks postimplantation, osteogenic-differentiated ADSs combined with DBM promoted bone formation both at the center and the periphery of the defect. In a case report published in 2004, autologous adipose-derived-mesenchymal stem cells (AD-MSCs) combined with fibrin glue was used to treat widespread calvarial defects in a 7-year-old girl. Three months after the reconstruction, computed tomography scan showed evidence of new bone formation with a near complete calvarial continuity. [Table 4] and [Table 5] review CSSDs in animal models of long bones and craniofacial defects, respectively, while [Table 6] shows the clinical application of ASCs application in human CSSDs.
|Table 4: The use of adipose stem cells in animal models of critical size defect-long bones|
Click here to view
|Table 5: The use of adipose stem cells in animal models of critical size defect-craniofacial bones|
Click here to view
|Table 6: Human: clinical application of adipose stem cell in critical size defect|
Click here to view
| ASCS in Distraction Osteogenesis|| |
ASCs therapy has been shown to accelerate bone formation in Distraction Osteogenesis and hence lower the duration of treatment. Nomura et al. used human uncultured AD-MSCs combined with Collagen Type 1 gel in a rat model of femoral distraction osteogenesis. Cells were injected immediately after termination of the distraction and healing was assessed at 8 weeks postinjection. Radiologic analysis of the samples showed that ASCs significantly promoted new bone formation and shortened the consolidation time compared to the control groups. Furthermore, the ASCs groups showed considerably higher scores on biomechanical tests than the control groups.
| Adipose-Derived-Mesenchymal Stem Cells in Fracture Nonunion|| |
Tawonsawatruk et al. evaluated the ability of human ASCs (hASC) to prevent fracture nonunion in rat models. The animals were assigned to three groups: control, BM-MSCs, and ASCs. Stem cells were injected percutaneously at the fracture site. At 8 weeks posttreatment, both BM-MSC and ASCs groups showed substantial improvement in bone mineralization and maturity of bone tissues at the fracture gap. The authors explained that both cell types prevented fracture nonunion through the paracrine effect rather than the direct proliferation or differentiation of the transplanted cells.
Using osteoconductive scaffolds to initiate osteogenic differentiation of ASCs within the stromal vascular fraction
A major breakthrough in stem cell biology and bone tissue engineering occurred over the last few years. Recent evidence suggests that ASCs do not need to be differentiated toward the osteogenic path before implantation provided that they are implanted on osteoconductive scaffolds ,, or in orthotopic sites. ASCs, cultured on titanium granules, readily differentiated into osteoblasts and showed greater levels of ALP activity with a significant increase in ECM mineralization. Chitosan hydrogels have also been shown to successfully support hASC viability by providing cell-to-cell contact, where oxygen and nutrients can be transported freely through the cells, thereby creating a favorable bioenvironmental condition that would influence hASC differentiation into the desired cell lineage. Differentiation of ASCs was also shown to take place if implanted on other osteogenic scaffolds, including fluoride-coated magnesium and polycaprolactone scaffolds. The multipotent cells within the SVF adhere very quickly to the scaffold material, proliferate rapidly, and are differentiated toward the osteogenic lineage.,
One-step intraoperative cell therapy: Dream or reality?
Many advancements in new techniques involving ASCs constitute a huge step forward in the development of a one-step intraoperative technique, where the harvesting of adipose tissue, processing of the SVF, implantation on a suitable osteogenic scaffold, and local application to a bony defect site, are carried out in one-step within one surgical procedure. The latest evidence from two preliminary reports in literature suggests that this approach is feasible and safe. Two Phase 1 trials , suggest that freshly isolated SVF added onto calcium phosphate ceramics or ceramic granules have the ability to elicit an osteogenic response after implantation without any ex vivo differentiation or application of any inducing agent [Figure 6]. This was observed in eight elderly female patients supplemented with internal fixation for osteoporotic fractures of the humerus  and ten elderly patients who had their maxillary sinus floor elevated in dental surgery. Such an advance would be of tremendous clinical importance as it would obviate the need for pretreatment of ASCs and allow for reconstruction in one-step without tissue leaving the operating room. The use of scaffolds not only allows us to “fill the defect” with an osteoconductive matrix and support, it also permits the adherence of stem cells, their proliferation and differentiation into the desired cells (osteoblasts) in vivo after implantation.
| Conclusion|| |
There is a new paradigm developing in the field of stem cell biology and bone regeneration, as seen in the recent shift to using SVF instead of ASCs. This shift is due to several reasons as follows: first, the tremendous regenerative capacity of SVF eliminates the need for any other step; second, the large amount of stem cells present in freshly isolated SVF removes the need for further expansion and culture; and third, prior differentiation into osteogenic cells before implantation may not be required if osteoconductive scaffolds are used.
The use of freshly prepared SVF-containing ASCs with osteoconductive scaffolds to address challenging conditions of bone loss or deficiencies in children and adults is a novel and promising cell-based bone tissue engineering approach [Figure 7]. We believe that this one-step surgical approach, where the harvesting of adipose tissue, the processing and seeding of SVF onto scaffolds, and the subsequent implantation in the patient is indeed feasible and could take place within 2 h in a single surgery. In order for this cutting edge technology to become available and affordable worldwide, several steps must be taken as follows:First, the safety and feasibility of this approach have to be demonstrated in Phase 1 clinical trials. Second, the efficacy of this one-step surgery has to be evaluated in multicenter randomized clinical trials (RCTs). The development of intraoperative equipment and machines for the processing of the SVF [Figure 8] must also be completed according to GMP guidelines and, more importantly, must address the concerns of the various regulatory agencies. Only then, will this dream come true!
|Figure 7: Stromal vascular fraction cell therapy. In the new paradigm, the autologous freshly isolated stromal vascular fraction is reinjected into the patient without ex vivo culture, expansion, or differentiation|
Click here to view
|Figure 8: Future-d direction. Adipose tissue resection, followed by the isolation of the stromal vascular fraction, will be mixed with the appropriate scaffold under investigation to be implanted into the defect within one surgical step; the duration of the whole procedure will be <2 h|
Click here to view
We are indebted to the Guylaine Bédard for her preparation of the charts and figures. We also wish to thank Shiong-En Chan for her editing.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Polo-Corrales L, Latorre-Esteves M, Ramirez-Vick JE. Scaffold design for bone regeneration. J Nanosci Nanotechnol 2014;14:15-56.
Oryan A, Alidadi S, Moshiri A, Maffulli N. Bone regenerative medicine: Classic options, novel strategies, and future directions. J Orthop Surg Res 2014;9:18.
Silber JS, Anderson DG, Daffner SD, Brislin BT, Leland JM, Hilibrand AS, et al.
Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion. Spine (Phila Pa 1976) 2003;28:134-9.
DeOrio JK, Farber DC. Morbidity associated with anterior iliac crest bone grafting in foot and ankle surgery. Foot Ankle Int 2005;26:147-51.
Dawson J, Kiner D, Gardner W nd, Swafford R, Nowotarski PJ. The reamer-irrigator-aspirator as a device for harvesting bone graft compared with iliac crest bone graft: Union rates and complications. J Orthop Trauma 2014;28:584-90.
Dimitriou R, Mataliotakis GI, Angoules AG, Kanakaris NK, Giannoudis PV. Complications following autologous bone graft harvesting from the iliac crest and using the RIA: A systematic review. Injury 2011;42 Suppl 2:S3-15.
Feuvrier D, Sagawa Y Jr., Béliard S, Pauchot J, Decavel P. Long-term donor-site morbidity after vascularized free fibula flap harvesting: Clinical and gait analysis. J Plast Reconstr Aesthet Surg 2016;69:262-9.
Hamdy RC, Rendon JS, Tabrizian M. Distraction Osteogenesis and its Challenges in Bone Regeneration. Rijeka, Croatia: INTECH Open Access Publisher; 2012.
Houdek MT, Bayne CO, Bishop AT, Shin AY. The outcome and complications of vascularised fibular grafts. Bone Joint J 2017;99-B: 134-8.
Allsopp BJ, Hunter-Smith DJ, Rozen WM. Vascularized versus nonvascularized bone grafts: What is the evidence? Clin Orthop Relat Res 2016;474:1319-27.
Borzunov DY. Long bone reconstruction using multilevel lengthening of bone defect fragments. Int Orthop 2012;36:1695-700.
Gubin AV, Borzunov DY, Malkova TA. The Ilizarov paradigm: Thirty years with the Ilizarov method, current concerns and future research. Int Orthop 2013;37:1533-9.
Gubin AV, Borzunov DY, Marchenkova LO, Malkova TA, Smirnova IL. Contribution of G.A. Ilizarov to bone reconstruction: Historical achievements and state of the art. Strategies Trauma Limb Reconstr 2016;11:145-52.
Masquelet AC. Muscle reconstruction in reconstructive surgery: Soft tissue repair and long bone reconstruction. Langenbecks Arch Surg 2003;388:344-6.
Morelli I, Drago L, George DA, Gallazzi E, Scarponi S, Romanò CL. Masquelet technique: Myth or reality? A systematic review and meta-analysis. Injury 2016;47 Suppl 6:S68-76.
Pelissier P, Martin D, Baudet J, Lepreux S, Masquelet AC. Behaviour of cancellous bone graft placed in induced membranes. Br J Plast Surg 2002;55:596-8.
Taylor BC, French BG, Fowler TT, Russell J, Poka A. Induced membrane technique for reconstruction to manage bone loss. J Am Acad Orthop Surg 2012;20:142-50.
Drosos GI, Touzopoulos P, Ververidis A, Tilkeridis K, Kazakos K. Use of demineralized bone matrix in the extremities. World J Orthop 2015;6:269-77.
Tuli SM, Singh AD. The osteoninductive property of decalcified bone matrix. An experimental study. J Bone Joint Surg Br 1978;60:116-23.
Gu H, Xiong Z, Yin X, Li B, Mei N, Li G, et al.
Bone regeneration in a rabbit ulna defect model: Use of allogeneic adipose-derivedstem cells with low immunogenicity. Cell Tissue Res 2014;358:453-64.
Wen C, Yan H, Fu S, Qian Y, Wang D, Wang C. Allogeneic adipose-derived stem cells regenerate bone in a critical-sized ulna segmental defect. Exp Biol Med (Maywood) 2016;241:1401-9.
Dufrane D, Docquier PL, Delloye C, Poirel HA, André W, Aouassar N. Scaffold-free three-dimensional graft from autologous adipose-derived stem cells for large bone defect reconstruction: Clinical proof of concept. Medicine (Baltimore) 2015;94:e2220.
Fillingham Y, Jacobs J. Bone grafts and their substitutes. Bone Joint J 2016;98-B 1 Suppl A:6-9.
Makhdom AM, Hamdy RC. The role of growth factors on acceleration of bone regeneration during distraction osteogenesis. Tissue Eng Part B Rev 2013;19:442-53.
Giannoudis PV, Dinopoulos H, Tsiridis E. Bone substitutes: An update. Injury 2005;36 Suppl 3:S20-7.
Dai R, Wang Z, Samanipour R, Koo KI, Kim K. Adipose-derived stem cells for tissue engineering and regenerative medicine applications. Stem Cells Int 2016;2016:6737345.
Frese L, Dijkman PE, Hoerstrup SP. Adipose tissue-derived stem cells in regenerative medicine. Transfus Med Hemother 2016;43:268-74.
Nicoletti GF, De Francesco F, D'Andrea F, Ferraro GA. Methods and procedures in adipose stem cells: State of the art and perspective for translation medicine. J Cell Physiol 2015;230:489-95.
Spinelli V, Guillot PV, De Coppi P. Induced pluripotent stem (iPS) cells from human fetal stem cells (hFSCs). Organogenesis 2013;9:101-10.
Maximow AA, The lymphocyte as a stem cell common to different blood elements in embryonic development and during the post-fetal life of mammals. Folia Haematol 1909;8:125-34.
Tavassoli M, Crosby WH. Transplantation of marrow to extramedullary sites. Science 1968;161:54-6.
Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV. Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol 1966;16:381-90.
Caplan AI. The mesengenic process. Clin Plast Surg 1994;21:429-35.
Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al.
Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143-7.
Feisst V, Brooks AE, Chen CJ, Dunbar PR. Characterization of mesenchymal progenitor cell populations directly derived from human dermis. Stem Cells Dev 2014;23:631-42.
Toma JG, Akhavan M, Fernandes KJ, Barnabé-Heider F, Sadikot A, Kaplan DR, et al.
Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 2001;3:778-84.
Rogers I, Casper RF. Umbilical cord blood stem cells. Best Pract Res Clin Obstet Gynaecol 2004;18:893-908.
Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24:1294-301.
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al.
Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315-7.
Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, et al.
Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy 2013;15:641-8.
Kitoh H, Kitakoji T, Tsuchiya H, Katoh M, Ishiguro N. Distraction osteogenesis of the lower extremity in patients with achondroplasia/hypochondroplasia treated with transplantation of culture-expanded bone marrow cells and platelet-rich plasma. J Pediatr Orthop 2007;27:629-34.
Hatzokos I, Stavridis SI, Iosifidou E, Karataglis D, Christodoulou A. Autologous bone marrow grafting combined with demineralized bone matrix improves consolidation of docking site after distraction osteogenesis. J Bone Joint Surg Am 2011;93:671-8.
Healey JH, Zimmerman PA, McDonnell JM, Lane JM. Percutaneous bone marrow grafting of delayed union and nonunion in cancer patients. Clin Orthop Relat Res 1990; 256:280-5.
Hernigou P, Poignard A, Beaujean F, Rouard H. Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am 2005;87:1430-7.
Eltorai AE, Susai CJ, Daniels AH. Mesenchymal stromal cells in spinal fusion: Current and future applications. J Orthop 2016;14:1-3.
Ma XW, Cui DP, Zhao DW. Vascular endothelial growth factor/bone morphogenetic protein-2 bone marrow combined modification of the mesenchymal stem cells to repair the avascular necrosis of the femoral head. Int J Clin Exp Med 2015;8:15528-34.
Hernigou P, Trousselier M, Roubineau F, Bouthors C, Chevallier N, Rouard H, et al.
Stem cell therapy for the treatment of hip osteonecrosis: A 30-year review of progress. Clin Orthop Surg 2016;8:1-8.
Mardones R, Via AG, Jofré C, Minguell J, Rodriguez C, Tomic A, et al.
Cell therapy for cartilage defects of the hip. Muscles Ligaments Tendons J 2016;6:361-6.
Seong JM, Kim BC, Park JH, Kwon IK, Mantalaris A, Hwang YS. Stem cells in bone tissue engineering. Biomed Mater 2010;5:062001.
Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997;64:278-94.
Siddappa R, Licht R, van Blitterswijk C, de Boer J. Donor variation and loss of multipotency during in vitro
expansion of human mesenchymal stem cells for bone tissue engineering. J Orthop Res 2007;25:1029-41.
Agata H, Asahina I, Watanabe N, Ishii Y, Kubo N, Ohshima S, et al.
Characteristic change and loss of in vivo
osteogenic abilities of human bone marrow stromal cells during passage. Tissue Eng Part A 2010;16:663-73.
Jäger M, Jelinek EM, Wess KM, Scharfstädt A, Jacobson M, Kevy SV, et al.
Bone marrow concentrate: A novel strategy for bone defect treatment. Curr Stem Cell Res Ther 2009;4:34-43.
Hernigou P, Poignard A, Manicom O, Mathieu G, Rouard H. The use of percutaneous autologous bone marrow transplantation in nonunion and avascular necrosis of bone. J Bone Joint Surg Br 2005;87:896-902.
Flouzat-Lachaniette CH, Heyberger C, Bouthors C, Roubineau F, Chevallier N, Rouard H, et al.
Osteogenic progenitors in bone marrow aspirates have clinical potential for tibial non-unions healing in diabetic patients. Int Orthop 2016;40:1375-9.
Hernigou P, Trousselier M, Roubineau F, Bouthors C, Chevallier N, Rouard H, et al.
Local transplantation of bone marrow concentrated granulocytes precursors can cure without antibiotics infected nonunion of polytraumatic patients in absence of bone defect. Int Orthop 2016;40:2331-8.
Le Nail LR, Stanovici J, Fournier J, Splingard M, Domenech J, Rosset P. Percutaneous grafting with bone marrow autologous concentrate for open tibia fractures: Analysis of forty three cases and literature review. Int Orthop 2014;38:1845-53.
Scaglione M, Fabbri L, Dell'Omo D, Gambini F, Guido G. Long bone nonunions treated with autologous concentrated bone marrow-derived cells combined with dried bone allograft. Musculoskelet Surg 2014;98:101-6.
Sugaya H, Mishima H, Aoto K, Li M, Shimizu Y, Yoshioka T, et al.
Percutaneous autologous concentrated bone marrow grafting in the treatment for nonunion. Eur J Orthop Surg Traumatol 2014;24:671-8.
ThuaTrungHau Le, Bui DP, Duy TN, Nhat PD, QuyBao Le, Huy NP, et al
. Autologous bone marrow stem cells combined with allograft cancellous bone in treatment of nonunion. Biomed Res Ther 2015;2:29.
Stanovici J, Le Nail LR, Brennan MA, Vidal L, Trichet V, Rosset P, et al.
Bone regeneration strategies with bone marrow stromal cells in orthopaedic surgery. Curr Res Transl Med 2016;64:83-90.
Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al.
Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 2001;7:211-28.
Heaton JM. The distribution of brown adipose tissue in the human. J Anat 1972;112(Pt 1):35-9.
Eroschenko VP, di Fiore MS. DiFiore's Atlas of Histology with Functional Correlations. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins; 2008.
Rebelatto CK, Aguiar AM, Moretão MP, Senegaglia AC, Hansen P, Barchiki F, et al.
Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue. Exp Biol Med (Maywood) 2008;233:901-13.
Gimble JM, Guilak F. Differentiation potential of adipose derived adult stem (ADAS) cells. Curr Top Dev Biol 2003;58:137-60.
Zhu Y, Liu T, Song K, Fan X, Ma X, Cui Z. Adipose-derived stem cell: A better stem cell than BMSC. Cell Biochem Funct 2008;26:664-75.
Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, Zandiehdoulabi B, Schouten TE, Kuik DJ, et al.
Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: Implications for cell-based therapies. Cell Tissue Res 2008;332:415-26.
Prins HJ, Schulten EA, Ten Bruggenkate CM, Klein-Nulend J, Helder MN. Bone regeneration using the freshly isolated autologous stromal vascular fraction of adipose tissue in combination with calcium phosphate ceramics. Stem Cells Transl Med 2016;5:1362-74.
Kuehlfluck P, Moghaddam A, Helbig L, Child C, Wildemann B, Schmidmaier G; HTRG-Heidelberg Trauma Research Group. RIA fractions contain mesenchymal stroma cells with high osteogenic potency. Injury 2015;46 Suppl 8:S23-32.
Aust L, Devlin B, Foster SJ, Halvorsen YD, Hicok K, du Laney T, et al.
Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy 2004;6:7-14.
Gimble J, Guilak F. Adipose-derived adult stem cells: Isolation, characterization, and differentiation potential. Cytotherapy 2003;5:362-9.
Katz AJ, Tholpady A, Tholpady SS, Shang H, Ogle RC. Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells. Stem Cells 2005;23:412-23.
Nakagami H, Morishita R, Maeda K, Kikuchi Y, Ogihara T, Kaneda Y. Adipose tissue-derived stromal cells as a novel option for regenerative cell therapy. J Atheroscler Thromb 2006;13:77-81.
Tholpady SS, Llull R, Ogle RC, Rubin JP, Futrell JW, Katz AJ. Adipose tissue: Stem cells and beyond. Clin Plast Surg 2006;33:55-62, vi.
Varma MJ, Breuls RG, Schouten TE, Jurgens WJ, Bontkes HJ, Schuurhuis GJ, et al.
Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells. Stem Cells Dev 2007;16:91-104.
Faustini M, Bucco M, Chlapanidas T, Lucconi G, Marazzi M, Tosca MC, et al.
Nonexpanded mesenchymal stem cells for regenerative medicine: Yield in stromal vascular fraction from adipose tissues. Tissue Eng Part C Methods 2010;16:1515-21.
Mizuno H. Adipose-derived stem cells for tissue repair and regeneration: Ten years of research and a literature review. J Nippon Med Sch 2009;76:56-66.
Gomillion CT, Burg KJ. Stem cells and adipose tissue engineering. Biomaterials 2006;27:6052-63.
Kolaparthy LK, Sanivarapu S, Moogla S, Kutcham RS. Adipose tissue – Adequate, accessible regenerative material. Int J Stem Cells 2015;8:121-7.
Melief SM, Geutskens SB, Fibbe WE, Roelofs H. Multipotent stromal cells skew monocytes towards an anti-inflammatory function: The link with key immunoregulatory molecules. Haematologica 2013;98:e121-2.
Helder MN, Knippenberg M, Klein-Nulend J, Wuisman PI. Stem cells from adipose tissue allow challenging new concepts for regenerative medicine. Tissue Eng 2007;13:1799-808.
Farré-Guasch E, Prins HJ, Overman JR, Ten Bruggenkate CM, Schulten EA, Helder MN, et al.
Human maxillary sinus floor elevation as a model for bone regeneration enabling the application of one-step surgical procedures. Tissue Eng Part B Rev 2013;19:69-82.
Fraser JK, Hicok KC, Shanahan R, Zhu M, Miller S, Arm DM. The Celution ®
system: Automated processing of adipose-derived regenerative cells in a functionally closed system. Adv Wound Care (New Rochelle) 2014;3:38-45.
Lin K, Matsubara Y, Masuda Y, Togashi K, Ohno T, Tamura T, et al.
Characterization of adipose tissue-derived cells isolated with the Celution system. Cytotherapy 2008;10:417-26.
Aronowitz JA, Ellenhorn JD. Adipose stromal vascular fraction isolation: A head-to-head comparison of four commercial cell separation systems. Plast Reconstr Surg 2013;132:932e-9e.
Doi K, Tanaka S, Iida H, Eto H, Kato H, Aoi N, et al.
Stromal vascular fraction isolated from lipo-aspirates using an automated processing system: Bench and bed analysis. J Tissue Eng Regen Med 2013;7:864-70.
Güven S, Karagianni M, Schwalbe M, Schreiner S, Farhadi J, Bula S, et al.
Validation of an automated procedure to isolate human adipose tissue-derived cells by using the Sepax ®
technology. Tissue Eng Part C Methods 2012;18:575-82.
Kakudo N, Tanaka Y, Morimoto N, Ogawa T, Kushida S, Hara T, et al.
Adipose-derived regenerative cell (ADRC)-enriched fat grafting: Optimal cell concentration and effects on grafted fat characteristics. J Transl Med 2013;11:254.
Granel B, Daumas A, Jouve E, Harlé JR, Nguyen PS, Chabannon C, et al.
Safety, tolerability and potential efficacy of injection of autologous adipose-derived stromal vascular fraction in the fingers of patients with systemic sclerosis: An open-label phase I trial. Ann Rheum Dis 2015;74:2175-82.
Domenis R, Lazzaro L, Calabrese S, Mangoni D, Gallelli A, Bourkoula E, et al.
Adipose tissue derived stem cells: In vitro
and in vivo
analysis of a standard and three commercially available cell-assisted lipotransfer techniques. Stem Cell Res Ther 2015;6:2.
Baptista LS, do Amaral RJ, Carias RB, Aniceto M, Claudio-da-Silva C, Borojevic R. An alternative method for the isolation of mesenchymal stromal cells derived from lipoaspirate samples. Cytotherapy 2009;11:706-15.
Shah FS, Wu X, Dietrich M, Rood J, Gimble JM. A non-enzymatic method for isolating human adipose tissue-derived stromal stem cells. Cytotherapy 2013;15:979-85.
Markarian CF, Frey GZ, Silveira MD, Chem EM, Milani AR, Ely PB, et al.
Isolation of adipose-derived stem cells: A comparison among different methods. Biotechnol Lett 2014;36:693-702.
Mitchell JB, McIntosh K, Zvonic S, Garrett S, Floyd ZE, Kloster A, et al.
Immunophenotype of human adipose-derived cells: Temporal changes in stromal-associated and stem cell-associated markers. Stem Cells 2006;24:376-85.
Yoshimura K, Shigeura T, Matsumoto D, Sato T, Takaki Y, Aiba-Kojima E, et al.
Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates. J Cell Physiol 2006;208:64-76.
Condé-Green A, Rodriguez RL, Slezak S, Singh DP, Goldberg NH, McLenithan J. Comparison between stromal vascular cells' isolation with enzymatic digestion and mechanical processing of aspirated adipose tissue. PlastReconstrSurg 2014;134:54.
Millan A, Landerholm T, Chapman J. Comparison between collagenase adipose digestion and Stromacell mechanical dissociation for mesenchymal stem cell separation. McNair Sch J CSUS 2014;15:86-101.
Dos-Anjos Vilaboa S, Navarro-Palou M, Llull R. Age influence on stromal vascular fraction cell yield obtained from human lipoaspirates. Cytotherapy 2014;16:1092-7.
Van Pham P, Hong-Thien Bui K, Quoc Ngo D, Tan Khuat L, Kim Phan N. Transplantation of nonexpanded adipose stromal vascular fraction and platelet-rich plasma for articular cartilage injury treatment in mice model. J Med Eng 2013;2013:832396.
Michalek J, Moster R, Lukac L, Proefrock K, Petrasovic M, Rybar J, et al.
Autologous adipose tissue-derived stromal vascular fraction cells application in patients with osteoarthritis. Cell Transplant 2015;20:1-36.
Raposio E, Caruana G, Bonomini S, Libondi G. A novel and effective strategy for the isolation of adipose-derived stem cells: Minimally manipulated adipose-derived stem cells for more rapid and safe stem cell therapy. Plast Reconstr Surg 2014;133:1406-9.
Ferguson RE, Cui X, Fink BF, Vasconez HC, Pu LL. The viability of autologous fat grafts harvested with the LipiVage system: A comparative study. Ann Plast Surg 2008;60:594-7.
Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, Helder MN, Klein-Nulend J, Schouten TE, et al.
Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Cytotherapy 2006;8:166-77.
Blackshear CP, Longaker MT, Wan DC. Commentary on: The effects of fat harvesting and preparation, air exposure, obesity, and stem cell enrichment on adipocyte viability prior to graft transplantation. Aesthet Surg J 2016;36:1174-5.
Gir P, Oni G, Brown SA, Mojallal A, Rohrich RJ. Human adipose stem cells: Current clinical applications. Plast Reconstr Surg 2012;129:1277-90.
Condé-Green A, Kotamarti VS, Sherman LS, Keith JD, Lee ES, Granick MS, et al.
Shift toward mechanical isolation of adipose-derived stromal vascular fraction: Review of upcoming techniques. Plast Reconstr Surg Glob Open 2016;4:e1017.
Astori G, Vignati F, Bardelli S, Tubio M, Gola M, Albertini V, et al.
” and multicolor phenotypic characterization of cell subpopulations identified in fresh human adipose tissue stromal vascular fraction and in the derived mesenchymal stem cells. J Transl Med 2007;5:55.
Guo J, Nguyen A, Banyard DA, Fadavi D, Toranto JD, Wirth GA, et al.
Stromal vascular fraction: A regenerative reality? Part 2: Mechanisms of regenerative action. J Plast Reconstr Aesthet Surg 2016;69:180-8.
Han S, Sun HM, Hwang KC, Kim SW. Adipose-derived stromal vascular fraction cells: Update on clinical utility and efficacy. Crit Rev Eukaryot Gene Expr 2015;25:145-52.
Colazzo F, Chester AH, Taylor PM, Yacoub MH. Induction of mesenchymal to endothelial transformation of adipose-derived stem cells. J Heart Valve Dis 2010;19:736-44.
Du WJ, Reppel L, Leger L, Schenowitz C, Huselstein C, Bensoussan D, et al.
Mesenchymal stem cells derived from human bone marrow and adipose tissue maintain their immunosuppressive properties after chondrogenic differentiation: Role of HLA-G. Stem Cells Dev 2016;25:1454-69.
Pierantozzi E, Badin M, Vezzani B, Curina C, Randazzo D, Petraglia F, et al.
Human pericytes isolated from adipose tissue have better differentiation abilities than their mesenchymal stem cell counterparts. Cell Tissue Res 2015;361:769-78.
Zimmerlin L, Donnenberg VS, Rubin JP, Donnenberg AD. Mesenchymal markers on human adipose stem/progenitor cells. Cytometry A 2013;83:134-40.
Navarro A, Marín S, Riol N, Carbonell-Uberos F, Miñana MD. Human adipose tissue-resident monocytes exhibit an endothelial-like phenotype and display angiogenic properties. Stem Cell Res Ther 2014;5:50.
Zeyda M, Farmer D, Todoric J, Aszmann O, Speiser M, Györi G, et al.
Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production. Int J Obes (Lond) 2007;31:1420-8.
Baer PC. Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo
and in vitro
. World J Stem Cells 2014;6:256-65.
Aronowitz JA, Lockhart RA, Hakakian CS, Hicok KC. Clinical safety of stromal vascular fraction separation at the point of care. Ann Plast Surg 2015;75:666-71.
Miyagi-Shiohira C, Kurima K, Kobayashi N, Saitoh I, Watanabe M, Noguchi Y, et al.
Cryopreservation of adipose-derived mesenchymal stem cells. Cell Med 2015;8:3-7.
Bellotti C, Stanco D, Ragazzini S, Romagnoli L, Martella E, Lazzati S, et al.
Analysis of the karyotype of expanded human adipose-derived stem cells for bone reconstruction of the maxillo-facial region. Int J Immunopathol Pharmacol 2013;26 1 Suppl: 3-9.
Tzouvelekis A, Paspaliaris V, Koliakos G, Ntolios P, Bouros E, Oikonomou A, et al.
A prospective, non-randomized, no placebo-controlled, phase Ib clinical trial to study the safety of the adipose derived stromal cells-stromal vascular fraction in idiopathic pulmonary fibrosis. J Transl Med 2013;11:171.
Loder S, Peterson JR, Agarwal S, Eboda O, Brownley C, DeLaRosa S, et al.
Wound healing after thermal injury is improved by fat and adipose-derived stem cell isografts. J Burn Care Res 2015;36:70-6.
Shen T, Pan ZG, Zhou X, Hong CY. Accelerated healing of diabetic wound using artificial dermis constructed with adipose stem cells and poly (L-glutamic acid)/chitosan scaffold. Chin Med J (Engl) 2013;126:1498-503.
Lee HC, An SG, Lee HW, Park JS, Cha KS, Hong TJ, et al.
Safety and effect of adipose tissue-derived stem cell implantation in patients with critical limb ischemia: A pilot study. Circ J 2012;76:1750-60.
Koh YG, Jo SB, Kwon OR, Suh DS, Lee SW, Park SH, et al.
Mesenchymal stem cell injections improve symptoms of knee osteoarthritis. Arthroscopy 2013;29:748-55.
Saxer F, Scherberich A, Todorov A, Studer P, Miot S, Schreiner S, et al.
Implantation of stromal vascular fraction progenitors at bone fracture sites: From a rat model to a first-in-man study. Stem Cells 2016;34:2956-66.
Chandler EM, Seo BR, Califano JP, Andresen Eguiluz RC, Lee JS, Yoon CJ, et al.
Implanted adipose progenitor cells as physicochemical regulators of breast cancer. Proc Natl Acad Sci U S A 2012;109:9786-91.
Senarath-Yapa K, McArdle A, Renda A, Longaker MT, Quarto N. Adipose-derived stem cells: A review of signaling networks governing cell fate and regenerative potential in the context of craniofacial and long bone skeletal repair. Int J Mol Sci 2014;15:9314-30.
Morcos MW, Al-Jallad H, Hamdy R. Comprehensive review of adipose stem cells and their implication in distraction osteogenesis and bone regeneration. Biomed Res Int 2015;2015:842975.
Giannoudis PV, Einhorn TA, Marsh D. Fracture healing: The diamond concept. Injury 2007;38 Suppl 4:S3-6.
Jahan K, Tabrizian M. Composite biopolymers for bone regeneration enhancement in bony defects. Biomater Sci 2016;4:25-39.
Rezwan K, Chen QZ, Blaker JJ, Boccaccini AR. Biodegradable and bioactive porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials 2006;27:3413-31.
Yun YP, Kim SJ, Lim YM, Park K, Kim HJ, Jeong SI, et al.
The effect of alendronate-loaded polycarprolactone nanofibrous scaffolds on osteogenic differentiation of adipose-derived stem cells in bone tissue regeneration. J Biomed Nanotechnol 2014;10:1080-90.
Swetha M, Sahithi K, Moorthi A, Srinivasan N, Ramasamy K, Selvamurugan N. Biocomposites containing natural polymers and hydroxyapatite for bone tissue engineering. Int J Biol Macromol 2010;47:1-4.
Zhang Y, Xu J, Ruan YC, Yu MK, O'Laughlin M, Wise H, et al.
Implant-derived magnesium induces local neuronal production of CGRP to improve bone-fracture healing in rats. Nat Med 2016;22:1160-9.
Lu T, Li Y, Chen T. Techniques for fabrication and construction of three-dimensional scaffolds for tissue engineering. Int J Nanomedicine 2013;8:337-50.
Kim J, Kim IS, Cho TH, Lee KB, Hwang SJ, Tae G, et al.
Bone regeneration using hyaluronic acid-based hydrogel with bone morphogenic protein-2 and human mesenchymal stem cells. Biomaterials 2007;28:1830-7.
Nayef L, Mekhail M, Benameur L, Rendon JS, Hamdy R, Tabrizian M A combinatorial approach towards achieving an injectable, self-contained, phosphate-releasing scaffold for promoting biomineralization in critical size bone defects. Acta Biomater 2016;29:389-97.
Kinney MA, McDevitt TC. Emerging strategies for spatiotemporal control of stem cell fate and morphogenesis. Trends Biotechnol 2013;31:78-84.
Stevens MM, George JH. Exploring and engineering the cell surface interface. Science 2005;310:1135-8.
Tsimbouri P, Gadegaard N, Burgess K, White K, Reynolds P, Herzyk P, et al.
Nanotopographical effects on mesenchymal stem cell morphology and phenotype. J Cell Biochem 2014;115:380-90.
Daei-Farshbaf N, Ardeshirylajimi A, Seyedjafari E, Piryaei A, Fadaei Fathabady F, Hedayati M, et al.
Bioceramic-collagen scaffolds loaded with human adipose-tissue derived stem cells for bone tissue engineering. Mol Biol Rep 2014;41:741-9.
Arrigoni E, de Girolamo L, Di Giancamillo A, Stanco D, Dellavia C, Carnelli D, et al.
Adipose-derived stem cells and rabbit bone regeneration: Histomorphometric, immunohistochemical and mechanical characterization. J Orthop Sci 2013;18:331-9.
Walmsley GG, Senarath-Yapa K, Wearda TL, Menon S, Hu MS, Duscher D, et al.
Surveillance of stem cell fate and function: A system for assessing cell survival and collagen expression in situ
. Tissue Eng Part A 2016;22:31-40.
Tajima S, Tobita M, Orbay H, Hyakusoku H, Mizuno H. Direct and indirect effects of a combination of adipose-derived stem cells and platelet-rich plasma on bone regeneration. Tissue Eng Part A 2015;21:895-905.
König MA, Canepa DD, Cadosch D, Casanova E, Heinzelmann M, Rittirsch D, et al.
Direct transplantation of native pericytes from adipose tissue: A new perspective to stimulate healing in critical size bone defects. Cytotherapy 2016;18:41-52.
Niemeyer P, Fechner K, Milz S, Richter W, Suedkamp NP, Mehlhorn AT, et al.
Comparison of mesenchymal stem cells from bone marrow and adipose tissue for bone regeneration in a critical size defect of the sheep tibia and the influence of platelet-rich plasma. Biomaterials 2010;31:3572-9.
de Girolamo L, Arrigoni E, Stanco D, Lopa S, Di Giancamillo A, Addis A, et al.
Role of autologous rabbit adipose-derived stem cells in the early phases of the repairing process of critical bone defects. J Orthop Res 2011;29:100-8.
Lendeckel S, Jödicke A, Christophis P, Heidinger K, Wolff J, Fraser JK, et al.
Autologous stem cells (adipose) and fibrin glue used to treat widespread traumatic calvarial defects: Case report. J Craniomaxillofac Surg 2004;32:370-3.
Lee MK, DeConde AS, Lee M, Walthers CM, Sepahdari AR, Elashoff D, et al.
Biomimetic scaffolds facilitate healing of critical-sized segmental mandibular defects. Am J Otolaryngol 2015;36:1-6.
Liu G, Zhang Y, Liu B, Sun J, Li W, Cui L. Bone regeneration in a canine cranial model using allogeneic adipose derived stem cells and coral scaffold. Biomaterials 2013;34:2655-64.
Linero I, Chaparro O. Paracrine effect of mesenchymal stem cells derived from human adipose tissue in bone regeneration. PLoS One 2014;9:e107001.
Saçak B, Certel F, Akdeniz ZD, Karademir B, Ercan F, Özkan N, et al.
Repair of critical size defects using bioactive glass seeded with adipose-derived mesenchymal stem cells. J Biomed Mater Res B Appl Biomater 2016. [Epub ahead of print].
Vériter S, André W, Aouassar N, Poirel HA, Lafosse A, Docquier PL, et al.
Human adipose-derived mesenchymal stem cells in cell therapy: Safety and feasibility in different “hospital exemption” clinical applications. PLoS One 2015;10:e0139566.
Tee BC, Sun Z. Mandibular distraction osteogenesis assisted by cell-based tissue engineering: A systematic review. Orthod Craniofac Res 2015;18 Suppl 1:39-49.
Nomura I, Watanabe K, Matsubara H, Hayashi K, Sugimoto N, Tsuchiya H. Uncultured autogenous adipose-derived regenerative cells promote bone formation during distraction osteogenesis in rats. Clin Orthop Relat Res 2014;472:3798-806.
Tawonsawatruk T, West CC, Murray IR, Soo C, Péault B, Simpson AH. Adipose derived pericytes rescue fractures from a failure of healing – Non-union. Sci Rep 2016;6:22779.
Levi B, Longaker MT. Concise review: Adipose-derived stromal cells for skeletal regenerative medicine. Stem Cells 2011;29:576-82.
Nguyen A, Guo J, Banyard DA, Fadavi D, Toranto JD, Wirth GA, et al.
Stromal vascular fraction: A regenerative reality? Part 1: Current concepts and review of the literature. J Plast Reconstr Aesthet Surg 2016;69:170-9.
Dahl M, Syberg S, Jørgensen NR, Pinholt EM. Adipose derived mesenchymal stem cells-their osteogenicity and osteoblast in vitro
mineralization on titanium granule carriers. J Craniomaxillofac Surg 2013;41:e213-20.
Debnath T, Ghosh S, Potlapuvu US, Kona L, Kamaraju SR, Sarkar S, et al.
Proliferation and differentiation potential of human adipose-derived stem cells grown on chitosan hydrogel. PLoS One 2015;10:e0120803.
Jurgens WJ, Kroeze RJ, Bank RA, Ritt MJ, Helder MN. Rapid attachment of adipose stromal cells on resorbable polymeric scaffolds facilitates the one-step surgical procedure for cartilage and bone tissue engineering purposes. J Orthop Res 2011;29:853-60.
Overman JR, Farré-Guasch E, Helder MN, ten Bruggenkate CM, Schulten EA, Klein-Nulend J. Short (15 minutes) bone morphogenetic protein-2 treatment stimulates osteogenic differentiation of human adipose stem cells seeded on calcium phosphate scaffolds in vitro
. Tissue Eng Part A 2013;19:571-81.
Gimble JM, Bunnell BA, Guilak F. Human adipose-derived cells: An update on the transition to clinical translation. Regen Med 2012;7:225-35.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]