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 Table of Contents  
REVIEW ARTICLE
Year : 2017  |  Volume : 3  |  Issue : 1  |  Page : 4-18

Recent advances in bone regeneration: The role of adipose tissue-derived stromal vascular fraction and mesenchymal stem cells


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 Publication15-Mar-2017

Correspondence Address:
Reggie Hamdy
Shriners Hospital for Children, 1003, Boulevard Decarie, Montreal, Quebec H4A 0A9
Canada
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jllr.jllr_1_17

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  Abstract 

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 2017 Aug 23];3:4-18. Available from: http://www.jlimblengthrecon.org/text.asp?2017/3/1/4/202207


  Introduction Top


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.[1] 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)

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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.[2] However, ABGs have several disadvantages, including a limited supply, especially in children, and donor site morbidity (pain, paresthesia, infection, and blood loss).[3],[4] The Reamer-Irrigator-Aspirator technique is an alternative technique of autogenous bone graft harvesting [5],[6] 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.[6] 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.[7],[8],[9],[10] 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.[7] 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.[8] 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.[8],[11],[12] Although Ilizarov himself rejected the concept of intramedullary interference,[13] 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.[14],[15],[16] 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.[17] 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.[2] They also have lower incorporating properties with the host healing tissues compared to autografts.[2] Demineralized bone matrix (DBM) is a chemically processed allograft product where inorganic materials are removed while the organic collagen matrix is preserved.[18] It has osteoconductive and osteoinductive properties with no osteogenic effect.[18],[19] DBM also does not evoke any immunological reaction due to the fact that most surface antigens are destroyed during the preparation process.[19] 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).[20],[21],[22] 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.[23] 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.[24] However, all types of allografts and BGSs lack an essential element of bone reconstruction – progenitor osteogenic cells,[25] 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.[26],[27] 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.[28] 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.[29]


  Discovery of Stem Cells Top


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.[30] 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.[31],[32] 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.[33] 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.[33],[34],[35],[36],[37],[38] 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.[39],[40]

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,[41],[42] delayed union and nonunion of fractures, critical size defects,[43],[44] spinal fusions,[45] and a vascular necrosis of the femoral head.[46],[47] The use of BM-MSCs was also reported to improve clinical outcome in patients with mild to moderate osteoarthrosis.[48] BM-MSCs are still the most frequently used stem cells in cell-based bone tissue engineering.[49]

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;[34] 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.[50],[51],[52] There is also an increased risk for pathogen contamination and genetic transformation with repeated cultures.[50] 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).[53] 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),[44],[54] 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,[54],[55],[56] 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.[57],[58] 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.[59],[60] 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.[57] In another retrospective study,[58] 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.[61] 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).[62] This discovery opened new horizons in regenerative medicine and bone tissue engineering techniques.


  The Adipose Tissue as an Attractive Source for Mesenchymal Stem Cell Top


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.[63] 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.[64] The white adipose tissue is present in the human body throughout life and specializes in long-term energy storage.[63] In histological sections, the adipose tissue is formed by two compartments, the parenchyma (functional part) and stroma (supportive component).[65] 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.[63] Stromal cells refer to the connective tissue cells [40] 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

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  Adipose Tissue-Derived Mesenchymal Stromal Cells (ASCS) Top


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.[66],[67] 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.[27] ASCs show many similarities with BM-MSC in surface marker profiles, multilineage differentiation potential, and growth properties.[68],[69],[70]


  Advantages of ASCS over Bone Marrow-Derived Mesenchymal Stem Cells Top


[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

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Compared to BM, adipose tissue can be harvested with minimal patient discomfort. It is readily available in large, almost unlimited quantities.[72],[73] It has a high stem cell to volume ratio.[68], 70, [74],[75],[76],[77],[78] 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.[79],[80],[81] Harvesting can easily be scaled up according to the need. ASCs possess more immunomodulation capacity than BM-MSCs.[82] 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.[70],[83],[84] 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.[105] 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].[105],[106],[107] Nonenzymatic mechanical techniques have also been developed.[108] 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

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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[107] Gir P, et al.

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Table 3: Key differences between the various methods of adipose-derived stem cells isolation [Summary for Table 1]

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SVF [Figure 3] contains a mixture of heterogeneous progenitor and stem cells – MSCs (10%)[109] and endothelial progenitor cells (2%)[109] – 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%),[109],[110] lymphocytes (10%–15% of cells, including regulatory T-cells), interleukin (IL)-1 and IL-10 receptor antagonists,[111] 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,[27],[112] anti-inflammatory, and immunosuppressive properties.[27] 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.[113] One of the most abundant cell types, the preadipocyte (59% of the total SVF population),[114],[115] 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.[114] Macrophages have immunological [116] and anti-inflammatory [117] effects.
Figure 4: Stem cell mechanism of action

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MSCs present in the SVF can be further isolated from the SVF by plastic adherence in culture, expanded in vitro,[40],[109],[118] and induced to differentiate into osteogenic lineages.[110],[119] Isolated ASCs can also be cryopreserved in a media of serum and dimethyl sulfoxide without losing their ability to differentiate and proliferate.[120] 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 Top


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),[121] 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 [111] 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 [119],[122] in various pathologies: plastic surgery, burn wounds,[123] diabetic ulcers,[124],[125] and osteoarthritis.[126] No adverse events have been reported in two Phase 1 trials involving SVF and osteoconductive scaffolds.[70],[127] 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.[128] 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].[129],[130] 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

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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

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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.[131] 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.[132] Synthetic polymers, which have been explored more than their natural counterpart, include polycaprolactone, poly lactic-co-glycolic acid, polymethylmethacrylate, and poly L-lactic acid.[133],[134] 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.[135] More recently, there has been revitalized interest in using magnesium to fabricate 3D biodegradable scaffolds for improving bone healing, which has demonstrated promising results.[136]

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.[137] 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.[138],[139]

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.[140] On the other hand, there has been growing interest in understanding how surface nanotopography can induce MSC differentiation.[141],[142]


  ASCS in Critical Size Segmental Defects Top


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.[143],[144],[145],[146],[147],[148] 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.[149] 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.[21] 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.[150] [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

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Table 5: The use of adipose stem cells in animal models of critical size defect-craniofacial bones

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Table 6: Human: clinical application of adipose stem cell in critical size defect

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  ASCS in Distraction Osteogenesis Top


ASCs therapy has been shown to accelerate bone formation in Distraction Osteogenesis and hence lower the duration of treatment.[156] 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.[157]


  Adipose-Derived-Mesenchymal Stem Cells in Fracture Nonunion Top


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.[158]

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 [110],[159],[160] 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.[161] 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.[162] 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.[163],[164]

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 [70],[127] 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 [70] and ten elderly patients who had their maxillary sinus floor elevated in dental surgery.[127] 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 Top


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.[165]

Future directions

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

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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

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Acknowledgments

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

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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Abstract
Introduction
Discovery of Ste...
The Adipose Tiss...
Adipose Tissue-D...
Advantages of AS...
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ASCS in Critical...
ASCS in Distract...
Adipose-Derived-...
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