Nanofat: A therapeutic paradigm in regenerative medicine

Adipose tissue is a compact and well-organized tissue containing a heterogeneous cellular population of progenitor cells, including mesenchymal stromal cells. Due to its availability and accessibility, adipose tissue is considered a “stem cell depot.” Adipose tissue products possess anti-inflammatory, anti-fibrotic, anti-apoptotic, and immunomodulatory effects. Nanofat, being a compact bundle of stem cells with regenerative and tissue remodeling potential, has potential in translational and regenerative medicine. Considering the wide range of applicability of its reconstructive and regenerative potential, the applications of nanofat can be used in various disciplines. Nanofat behaves on the line of adipose tissue-derived mesenchymal stromal cells. At the site of injury, these stromal cells initiate a site-specific reparative response comprised of remodeling of the extracellular matrix, enhanced and sustained angiogenesis, and immune system modulation. These properties of stromal cells provide a platform for the usage of regenerative medicine principles in curbing various diseases. Details about nanofat, including various preparation methods, characterization, delivery methods, evidence on practical applications, and ethical concerns are included in this review. However, appropriate guidelines and preparation protocols for its optimal use in a wide range of clinical applications have yet to be standardized.     

Adipose-derived stem cells: isolation, expansion and differentiation

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.     

脂肪干细胞在再生医学中的应用

再生医学是一门研究如何促进创伤与组织再生及功能重建的新兴学科,主要通过研究干细胞分化、机体等正常组织创伤修复与再生等机制来维持、修复、再生或改善损伤组织和器官功能。脂肪干细胞（adipose-derived stem cells,ASCs）是近年来从脂肪组织中分离得到的一种具有多向分化潜能的干细胞,是一种足量的、可用于实际的、有一定吸引力的自体细胞代替的供体资源,并能够广泛的用于组织修复、再生、发育的可塑性及细胞治疗等研究中。阐述了脂肪干细胞在旁分泌、软组织重建及损伤修复、骨骼肌重建、心血管重建、神经系统重建及癌症转移与入侵方面的作用模式,概括总结了目前利用脂肪干细胞参与的临床治疗方法,以期对脂肪干细胞在再生医学中应用研究提供参考。     

Adipose tissue in bone regeneration - stem cell source and beyond

Adipose tissue (AT) is recognized as a complex organ involved in major home-ostatic body functions, such as food intake, energy balance, immunomodulation, development and growth, and functioning of the reproductive organs. The role of AT in tissue and organ homeostasis, repair and regeneration is increasingly recognized. Different AT compartments (white AT, brown AT and bone marrow AT) and their interrelation with bone metabolism will be presented. AT-derived stem cell populations - adipose-derived mesenchymal stem cells and pluripotent-like stem cells. Multilineage differentiating stress-enduring and dedifferentiated fat cells can be obtained in relatively high quantities compared to other sources. Their role in different strategies of bone and fracture healing tissue engineering and cell therapy will be described. The current use of AT- or AT-derived stem cell populations for fracture healing and bone regenerative strategies will be presented, as well as major challenges in furthering bone regenerative strategies to clinical settings.     

Proteomic characterization of adipose tissue constituents, a necessary step for understanding adipose tissue complexity

The original concept of adipose tissue as an inert storage depot for the excess of energy has evolved over the last years and it is now considered as one of the most important organs regulating body homeostasis. This conceptual change has been supported by the demonstration that adipose tissue serves as a major endocrine organ, producing a wide variety of bioactive molecules, collectively termed adipokines, with endocrine, paracrine and autocrine activities. Adipose tissue is indeed a complex organ wherein mature adipocytes coexist with the various cell types comprising the stromal-vascular fraction (SVF), including preadipocytes, adipose-derived stem cells, perivascular cells, and blood cells. It is known that not only mature adipocytes but also the components of SVF produce adipokines. Furthermore, adipokine production, proliferative and metabolic activities and response to regulatory signals (i.e. insulin, catecholamines) differ between the different fat depots, which have been proposed to underlie their distinct association to specific diseases. Herein, we discuss the recent proteomic studies on adipose tissue focused on the analysis of the separate cellular components and their secretory products, with the aim of identifying the basic features and the contribution of each component to different adipose tissue-associated pathologies.     

Dedifferentiated fat cells: A cell source for regenerative medicine

The identification of an ideal cell source for tissue regeneration remains a challenge in the stem cell field. The ability of progeny cells to differentiate into other cell types is important for the processes of tissue reconstruction and tissue engineering and has clinical, biochemical or molecular implications. The adaptation of stem cells from adipose tissue for use in regenerative medicine has created a new role for adipocytes. Mature adipocytes can easily be isolated from adipose cell suspensions and allowed to dedifferentiate into lipid-free multipotent cells, referred to as dedifferentiated fat (DFAT) cells. Compared to other adult stem cells, the DFAT cells have unique advantages in their abundance, ease of isolation and homogeneity. Under proper condition in vitro and in vivo, the DFAT cells have exhibited adipogenic, osteogenic, chondrogenic, cardiomyogenc, angiogenic, myogenic, and neurogenic potentials. In this review, we first discuss the phenomena of dedifferentiation and transdifferentiation of cells, and then dedifferentiation of adipocytes in particular. Understanding the dedifferentiation process itself may contribute to our knowledge of normal growth processes, as well as mechanisms of disease. Second, we highlight new developments in DFAT cell culture and summarize the current understanding of DFAT cell properties. The unique features of DFAT cells are promising for clinical applications such as tissue regeneration.     

Adipokines in inflammation and metabolic disease

The worldwide epidemic of obesity has brought considerable attention to research aimed at understanding the biology of adipocytes (fat cells) and the events occurring in adipose tissue (fat) and in the bodies of obese individuals. Accumulating evidence indicates that obesity causes chronic low-grade inflammation and that this contributes to systemic metabolic dysfunction that is associated with obesity-linked disorders. Adipose tissue functions as a key endocrine organ by releasing multiple bioactive substances, known as adipose-derived secreted factors or adipokines, that have pro-inflammatory or anti-inflammatory activities. Dysregulated production or secretion of these adipokines owing to adipose tissue dysfunction can contribute to the pathogenesis of obesity-linked complications. In this Review, we focus on the role of adipokines in inflammatory responses and discuss their potential as regulators of metabolic function.     

Adipose tissue and the vascularization of biomaterials: Stem cells,microvascular fragments and nanofat—a review

Tissue defects in the human body after trauma and injury require precise reconstruction to regain function. Hence, there is a great demand for clinically translatable approaches with materials that are both biocompatible and biodegradable. They should also be able to adequately integrate within the tissue through sufficient vascularization. Adipose tissue is abundant and easily accessible. It is a valuable tissue source in regenerative medicine and tissue engineering, especially with regard to its angiogenic potential. Derivatives of adipose tissue, such as microfat, nanofat, microvascular fragments, stromal vascular fraction and stem cells, are commonly used in research, but also clinically to enhance the vascularization of implants and grafts at defect sites. In plastic surgery, adipose tissue is harvested via liposuction and can be manipulated in three ways (macro-, micro- and nanofat) in the operating room, depending on its ultimate use. Whereas macro- and microfat are used as a filling material for soft tissue injuries, nanofat is an injectable viscous extract that primarily induces tissue remodeling because it is rich in growth factors and stem cells. In contrast to microfat that adds volume to a defect site, nanofat has the potential to be easily combined with scaffold materials due to its liquid and homogenous consistency and is particularly attractive for blood vessel formation. The same is true for microvascular fragments that are easily isolated from adipose tissue through collagenase digestion. In preclinical animal models, it has been convincingly shown that these vascular fragments inosculate with host vessels and subsequently accelerate scaffold perfusion and host tissue integration. Adipose tissue is also an ideal source of stem cells. It yields larger quantities of cells than any other source and is easier to access for both the patient and doctor compared with other sources such as bone marrow. They are often used for tissue regeneration in combination with biomaterials. Adipose-derived stem cells can be applied unmodified or as single cell suspensions. However, certain pretreatments, such as cultivation under hypoxic conditions or three-dimensional spheroids production, may provide substantial benefit with regard to subsequent vascularization in vivo due to induced growth factor production. In this narrative review, derivatives of adipose tissue and the vascularization of biomaterials are addressed in a comprehensive approach, including several sizes of derivatives, such as whole fat flaps for soft tissue engineering, nanofat or stem cells, their secretome and exosomes. Taken together, it can be concluded that adipose tissue and its fractions down to the molecular level promote, enhance and support vascularization of biomaterials. Therefore, there is a high potential of the individual fat component to be used in regenerative medicine.     

Invited review: Pre- and postnatal adipose tissue development in farm animals: from stem cells to adipocyte physiology

Both white and brown adipose tissues are recognized to be differently involved in energy metabolism and are also able to secrete a variety of factors called adipokines that are involved in a wide range of physiological and metabolic functions. Brown adipose tissue is predominant around birth, except in pigs. Irrespective of species, white adipose tissue has a large capacity to expand postnatally and is able to adapt to a variety of factors. The aim of this review is to update the cellular and molecular mechanisms associated with pre- and postnatal adipose tissue development with a special focus on pigs and ruminants. In contrast to other tissues, the embryonic origin of adipose cells remains the subject of debate. Adipose cells arise from the recruitment of specific multipotent stem cells/progenitors named adipose tissue-derived stromal cells. Recent studies have highlighted the existence of a variety of those cells being able to differentiate into white, brown or brown-like/beige adipocytes. After commitment to the adipocyte lineage, progenitors undergo large changes in the expression of many genes involved in cell cycle arrest, lipid accumulation and secretory functions. Early nutrition can affect these processes during fetal and perinatal periods and can also influence or pre-determinate later growth of adipose tissue. How these changes may be related to adipose tissue functional maturity around birth and can influence newborn survival is discussed. Altogether, a better knowledge of fetal and postnatal adipose tissue development is important for various aspects of animal production, including neonatal survival, postnatal growth efficiency and health.     

Therapeutic applications of conditioned medium from adipose tissue

For the past number of decades, adipose tissue has attracted significant interest due to its complicated composition and versatile functions. Adipose tissue is no longer considered to be just an energy‐storing fat pad, but is also a key ring player in interaction networks between various organs and tissues. A wide range of factors released by adipose tissue are responsible for regulation of adipose tissue and other distant target tissues and cells, such as kidneys, skeletal muscle, the cardiovascular system and the immune system, in an auto‐/paracrine manner. A mixture of bioactive molecules makes up the conditioned medium of adipose tissue. The beneficial role played by these bioactive molecules in angiogenesis, wound healing, tissue regeneration and immunomodulation has been demonstrated by various studies. Study of this conditioned medium helps deepen our understanding of underlying mechanisms and broadens the potential for therapeutic applications. In this review, we have aimed to improve fundamental understanding of conditioned medium from adipose tissue and to summarize recent efforts to study its therapeutic applications.     

Perinatal stem cells: A promising cell resource for tissue engineering of craniofacial bone

In facing the mounting clinical challenge and suboptimal techniques of craniofacial bone defects resulting from various conditions, such as congenital malformations, osteomyelitis, trauma and tumor resection, the ongoing research of regenerative medicine using stem cells and concurrent advancement in biotechnology have shifted the focus from surgical reconstruction to a novel stem cell-based tissue engineering strategy for customized and functional craniofacial bone regeneration. Given the unique ontogenetical and cell biological properties of perinatal stem cells, emerging evidence has suggested these extraembryonic tissue-derived stem cells to be a promising cell source for extensive use in regenerative medicine and tissue engineering. In this review, we summarize the current achievements and obstacles in stem cell-based craniofacial bone regeneration and subsequently we address the characteristics of various types of perinatal stem cells and their novel application in tissue engineering of craniofacial bone. We propose the promising feasibility and scope of perinatal stem cell-based craniofacial bone tissue engineering for future clinical application.     

Stem cells: their source,potency and use in regenerative therapies with focus on adipose-derived stem cells – a review

Stem cells can be defined as units of biological organization that are responsible for the development and the regeneration of organ and tissue systems. They are able to renew their populations and to differentiate into multiple cell lineages. Therefore, these cells have great potential in advanced tissue engineering and cell therapies. When seeded on synthetic or nature-derived scaffolds in vitro, stem cells can be differentiated towards the desired phenotype by an appropriate composition, by an appropriate architecture, and by appropriate physicochemical and mechanical properties of the scaffolds, particularly if the scaffold properties are combined with a suitable composition of cell culture media, and with suitable mechanical, electrical or magnetic stimulation. For cell therapy, stem cells can be injected directly into damaged tissues and organs in vivo. Since the regenerative effect of stem cells is based mainly on the autocrine production of growth factors, immunomodulators and other bioactive molecules stored in extracellular vesicles, these structures can be isolated and used instead of cells for a novel therapeutic approach called “stem cell-based cell-free therapy”. There are four main sources of stem cells, i.e. embryonic tissues, fetal tissues, adult tissues and differentiated somatic cells after they have been genetically reprogrammed, which are referred to as induced pluripotent stem cells (iPSCs). Although adult stem cells have lower potency than the other three stem cell types, i.e. they are capable of differentiating into only a limited quantity of specific cell types, these cells are able to overcome the ethical and legal issues accompanying the application of embryonic and fetal stem cells and the mutational effects associated with iPSCs. Moreover, adult stem cells can be used in autogenous form. These cells are present in practically all tissues in the organism. However, adipose tissue seems to be the most advantageous tissue from which to isolate them, because of its abundancy, its subcutaneous location, and the need for less invasive techniques. Adipose tissue-derived stem cells (ASCs) are therefore considered highly promising in present-day regenerative medicine.     

Lipid peroxidation of poly-unsaturated fatty acids in normal and obese adipose tissues

Adipose tissues function as the primary storage compartment of fatty acids and as an endocrine organ that affects peripheral tissues. Many of adipose tissue-derived factors, often termed adipokines, have been discovered in recent years. The synthesis and secretion of these factors vary in different depots of adipose tissues. Excessive lipid accumulation in adipocytes induces inflammatory processes by up-regulating the expression and release of pro-inflammatory cytokines. In addition, activated macrophages in the obese adipose tissue release inflammatory cytokines. Adipose tissue inflammation has also been linked to an enhanced metabolism of polyunsaturated fatty acids (PUFAs). The non-enzymatic peroxidation of PUFAs and of their 12/15-lipoxygenase-derived hydroperoxy metabolites leads to the generation of the reactive aldehyde species 4-hydroxyalkenals. This review shows that 4-hydroxyalkenals, in particular 4-hydroxynonenal, play a key role in lipid storage homeostasis in normal adipocytes. Nonetheless, in the obese adipose tissue an increased production of 4-hydroxyalkenals contributes to the inflamed phenotype.     

Decellularized adipose matrix provides an inductive microenvironment for stem cells in tissue regeneration

Stem cells play a key role in tissue regeneration due to their self-renewal and multidirectional differentiation, which are continuously regulated by signals from the extracellular matrix (ECM) microenvironment. Therefore, the unique biological and physical characteristics of the ECM are important determinants of stem cell behavior. Although the acellular ECM of specific tissues and organs (such as the skin, heart, cartilage, and lung) can mimic the natural microenvironment required for stem cell differentiation, the lack of donor sources restricts their development. With the rapid development of adipose tissue engineering, decellularized adipose matrix (DAM) has attracted much attention due to its wide range of sources and good regeneration capacity. Protocols for DAM preparation involve various physical, chemical, and biological methods. Different combinations of these methods may have different impacts on the structure and composition of DAM, which in turn interfere with the growth and differentiation of stem cells. This is a narrative review about DAM. We summarize the methods for decellularizing and sterilizing adipose tissue, and the impact of these methods on the biological and physical properties of DAM. In addition, we also analyze the application of different forms of DAM with or without stem cells in tissue regeneration (such as adipose tissue), repair (such as wounds, cartilage, bone, and nerves), in vitro bionic systems, clinical trials, and other disease research.     

Adipokines and the cardiovascular system: mechanisms mediating health and disease

This review focuses on the role of adipokines in the maintenance of a healthy cardiovascular system, and the mechanisms by which these factors mediate the development of cardiovascular disease in obesity. Adipocytes are the major cell type comprising the adipose tissue. These cells secrete numerous factors, termed adipokines, into the blood, including adiponectin, leptin, resistin, chemerin, omentin, vaspin, and visfatin. Adipose tissue is a highly vascularised endocrine organ, and different adipose depots have distinct adipokine secretion profiles, which are altered with obesity. The ability of many adipokines to stimulate angiogenesis is crucial for adipose tissue expansion; however, excessive blood vessel growth is deleterious. As well, some adipokines induce inflammation, which promotes cardiovascular disease progression. We discuss how these 7 aforementioned adipokines act upon the various cardiovascular cell types (endothelial progenitor cells, endothelial cells, vascular smooth muscle cells, pericytes, cardiomyocytes, and cardiac fibroblasts), the direct effects of these actions, and their overall impact on the cardiovascular system. These were chosen, as these adipokines are secreted predominantly from adipocytes and have known effects on cardiovascular cells.     

Differentiation of human adipose-derived adult stem cells into neuronal tissue: Does it work?

Adipose tissue contains many cells and proteins that are of value not only for their potential therapeutic applications, but also for the low cost of their harvest and delivery. Mesenchymal stem cells (MSC) were originally isolated from the bone marrow, although similar populations have been isolated from adipose and other tissues. At one time, neural tissues were not regarded as regenerative populations of cells. Therefore, the identification of cell populations capable of neuronal differentiation has generated immense interest. Adipose tissue may represent an alternative source of cells that are capable of neuronal differentiation, potentially enhancing its use in the treatment of neurological disease. The aim of this review is to cover the current state of knowledge of the differentiation potential of human adipose-derived stem (ADAS) cells, specifically their ability to give rise to neuronal cells in vitro. This review presents and discusses different protocols used for inducing human ADAS cells to differentiate in vitro, and the neuronal markers utilized in each system.     

Differential Regulation of Adipokines May Influence Migratory Behavior in the White-Throated Sparrow (Zonotrichia albicollis)

White-throated sparrows increase fat deposits during pre-migratory periods and rely on these fat stores to fuel migration. Adipose tissue produces hormones and signaling factors in a rhythmic fashion and may be controlled by a clock in adipose tissue or driven by a master clock in the brain. The master clock may convey photoperiodic information from the environment to adipose tissue to facilitate pre-migratory fattening, and adipose tissue may, in turn, release adipokines to indicate the extent of fat energy stores. Here, we present evidence that a change in signal from the adipokines adiponectin and visfatin may act to indicate body condition, thereby influencing an individual''s decision to commence migratory flight, or to delay until adequate fat stores are acquired. We quantified plasma adiponectin and visfatin levels across the day in captive birds held under constant photoperiod. The circadian profiles of plasma adiponectin in non-migrating birds were approximately inverse the profiles from migrating birds. Adiponectin levels were positively correlated to body fat, and body fat was inversely related to the appearance of nocturnal migratory restlessness. Visfatin levels were constant across the day and did not correlate with fat deposits; however, a reduction in plasma visfatin concentration occurred during the migratory period. The data suggest that a significant change in the biological control of adipokine expression exists between the two migratory conditions and we propose a role for adiponectin, visfatin and adipose clocks in the regulation of migratory behaviors.     

Human adult pluripotency: Facts and questions

Cellular reprogramming and induced pluripotent stem cell(IPSC) technology demonstrated the plasticity of adult cell fate, opening a new era of cellular modelling and introducing a versatile therapeutic tool for regenerative medicine.While IPSCs are already involved in clinical trials for various regenerative purposes, critical questions concerning their medium-and long-term genetic and epigenetic stability still need to be answered. Pluripotent stem cells have been described in the last decades in various mammalian and human tissues(such as bone marrow, blood and adipose tissue). We briefly describe the characteristics of human-derived adult stem cells displaying in vitro and/or in vivo pluripotency while highlighting that the common denominators of their isolation or occurrence within tissue are represented by extreme cellular stress. Spontaneous cellular reprogramming as a survival mechanism favoured by senescence and cellular scarcity could represent an adaptative mechanism. Reprogrammed cells could initiate tissue regeneration or tumour formation dependent on the microenvironment characteristics. Systems biology approaches and lineage tracing within living tissues can be used to clarify the origin of adult pluripotent stem cells and their significance for regeneration and disease.     

Secretome analysis of rat adipose tissues shows location-specific roles for each depot type

Obesity prevalence is reaching pandemic proportions becoming a major public health threat for many industrialized nations. It is especially worrying as it causes a higher risk of premature death due to associated diseases such as type 2 diabetes, cardiovascular disease, and some cancers. Current evidence shows biological and genetic differences between adipose tissues depending on its anatomical location. Particularly, upper body/visceral fat distribution in obesity is closely linked to metabolic complications. In this report, we characterize for the first time the secretome of rat adipose tissue explants from different anatomical localizations and its differential analysis. Visceral, subcutaneous, and gonadal fat specific secretomes and differentially secreted proteins among the three fat depots were analyzed by 2-DE and MS. Reference maps for location-specific adipose tissue secretomes are shown and the 45 most significant differences are listed. Identified proteins include classical adipokines and novel secreted proteins. Interestingly, our results show that the type of proteins and their role in different biological processes diverge significantly when comparing the set of proteins identified from visceral, subcutaneous and gonadal fat explants. This study emphasizes and supports the differential role of adipose tissue in accordance to its anatomical localization.     

Tooth-derived stem cells: Update and perspectives

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context,stem cells(SCs) present the characteristics of selfrenewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation(CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs(TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.