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.     

A familiar stranger:CD34 expression and putative functions in SVF cells of adipose tissue

Human adipose tissue obtained by liposuction is easily accessible and an abundant potential source of autologous cells for regenerative medicine applications. After digestion of the tissue and removal of differentiated adipocytes, the so-called stromal vascular fraction (SVF) of adipose, a mix of various cell types, is obtained. SVF contains mesenchymal fibroblastic cells, able to adhere to culture plastic and to generate large colonies in vitro , that closely resemble bone marrow-derived colony forming units-fibroblastic, and whose expanded progeny, adipose mesenchymal stem/stromal cells (ASC), show strong similarities with bone marrow mesenchymal stem cells. The sialomucin CD34, which is well known as a hematopoietic stem cell marker, is also expressed by ASC in native adipose tissue but its expression is gradually lost upon standard ASC expansion in vitro . Surprisingly little is known about the functional role of CD34 in the biology and tissue forming capacity of SVF cells and ASC. The present editorial provides a short introduction to the CD34 family of sialomucins and reviews the data from the literature concerning ex- pression and function of these proteins in SVF cells and their in vitro expanded progeny.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

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。     

Translational products of adipose tissue-derived mesenchymal stem cells: Bench to bedside applications

With developments in the field of tissue engineering and regenerative medicine, the use of biological products for the treatment of various disorders has come into the limelight among researchers and clinicians. Among all the available biological tissues, research and exploration of adipose tissue have become more robust. Adipose tissue engineering aims to develop by-products and their substitutes for their regenerative and immunomodulatory potential. The use of biodegradable scaffolds along with adipose tissue products has a major role in cellular growth, proliferation, and differentiation. Adipose tissue, apart from being the powerhouse of energy storage, also functions as the largest endocrine organ, with the release of various adipokines. The progenitor cells among the heterogeneous population in the adipose tissue are of paramount importance as they determine the capacity of regeneration of these tissues. The results of adipose-derived stemcell assisted fat grafting to provide numerous growth factors and adipokines that improve vasculogenesis, fat graft integration, and survival within the recipient tissue and promote the regeneration of tissue are promising. Adipose tissue gives rise to various by-products upon processing. This article highlights the significance and the usage of various adipose tissue by-products, their individual characteristics, and their clinical applications.     

Adipose stromal/stem cells in regenerative medicine:Potentials and limitations

This article presents the stem and progenitor cells from subcutaneous adipose tissue,briefly comparing them with their bone marrow counterparts,and discussing their potential for use in regenerative medicine.Subcutaneous adipose tissue differs from other mesenchymal stromal/stem cells(MSCs) sources in that it contains a pre-adipocyte population that dwells in the adventitia of robust blood vessels.Pre-adipocytes are present both in the stromal-vascular fraction(SVF;freshly isolated cells) and in the adherent fraction of adipose stromal/stem cells(ASCs;in vitro expanded cells),and have an active role on the chronic inflammation environment established in obesity,likely due their monocyticmacrophage lineage identity.The SVF and ASCs have been explored in cell therapy protocols with relative success,given their paracrine and immunomodulatory effects.Importantly,the widely explored multipotentiality of ASCs has direct application in bone,cartilage and adipose tissue engineering.The aim of this editorial is to reinforce the peculiarities of the stem and progenitor cells from subcutaneous adipose tissue,revealing the spheroids as a recently described biotechnological tool for cell therapy and tissue engineering.Innovative cell culture techniques,in particular 3 D scaffold-free cultures such as spheroids,are now available to increase the potential for regeneration and differentiation of mesenchymal lineages.Spheroids are being explored not only as a model for cell differentiation,but also as powerful 3 D cell culture tools to maintain the stemness and expand the regenerative and differentiation capacities of mesenchymal cell lineages.     

Obesity and weight loss could alter the properties of adipose stem cells?

The discovery that adipose tissue represents an interesting source of multipotent stem cells has led to many studies exploring the clinical potential of these cells in cell-based therapies. Recent advances in understanding the secretory capacity of adipose tissue and the role of adipokines in the development of obesity and associated disorders have added a new dimension to the study of adipose tissue biology in normal and diseased states. Subcutaneous adipose tissue forms the interface between the clinical application of regenerative medicine and the establishment of the pathological condition of obesity. These two facets of adipose tissue should be understood as potentially related phenomena. Because of the functional characteristics of adipose stem cells, these cells represent a fundamental tool for understanding how these two facets are interconnected and could be important for therapeutic applications. In fact, adipose tissue stem cells have multiple functions in obesity related to adipogenic, angiogenic and secretory capacities. In addition, we have also previously described a predominance of larger blood vessels and an adipogenic memory in the subcutaneous adipose tissue after massive weight loss subsequent to bariatric surgery(ex-obese patients). Understanding the reversibility of the behavior of adipose stem cells in obeses and in weight loss is relevant to both physiological studies and the potential use of these cells in regenerative medicine.     

猪脂肪基质细胞成骨与成脂分化潜能的研究

目的:探索猪脂肪基质细胞体外培养和向成骨与脂肪细胞分化的条件。方法:常规方法培养猪脂肪基质细胞,分别向成骨细胞与脂肪细胞进行诱导,应用免疫组化(碱性磷酸酶法、茜素红)及油红O染色对诱导分化的细胞进行鉴定。结果:在体外培养条件下,猪的脂肪基质细胞呈成纤维样,生长旺盛,在一定的条件下,可分别被诱导分化为成骨细胞与脂肪细胞,向成骨分化的细胞表达碱性磷酸酶,在培养皿中可形成钙化斑。而在向脂肪细胞诱导分化过程中,细胞中可见有小脂滴生成,用油红O染色呈橘红色。结论:脂肪基质细胞是一种混合细胞,除了能向脂肪细胞分化外,在一定的诱导条件下,也能向成骨细胞分化。     

Stem cells from adipose tissue

This is a review of the growing scientific interest in the developmental plasticity and therapeutic potential of stromal cells isolated from adipose tissue. Adipose-derived stem/stromal cells (ASCs) are multipotent somatic stem cells that are abundant in fat tissue. It has been shown that ASCs can differentiate into several lineages, including adipose cells, chondrocytes, osteoblasts, neuronal cells, endothelial cells, and cardiomyocytes. At the same time, adipose tissue can be harvested by a minimally invasive procedure, which makes it a promising source of adult stem cells. Therefore, it is believed that ASCs may become an alternative to the currently available adult stem cells (e.g. bone marrow stromal cells) for potential use in regenerative medicine. In this review, we present the basic information about the field of adipose-derived stem cells and their potential use in various applications.     

Autologous stromal vascular fraction cells: A tool for facilitating tolerance in rheumatic disease

Since the days of Medawar, the goal of therapeutic tolerogenesis has been a “Holy Grail” for immunologists. While knowledge of cellular and molecular mechanisms of this process has been increasing at an exponential rate, clinical progress has been minimal. To provide a mechanistic background of tolerogenesis, we overview common processes in the naturally occurring examples of: pregnancy, cancer, oral tolerance and anterior chamber associated immune deviation. The case is made that an easily accessible byproduct of plastic surgery, the adipose stromal vascular fraction, contains elements directly capable of promoting tolerogenesis such as T regulatory cells and inhibitory macrophages. The high content of mesenchymal and hematopoietic stem cells from this source provides the possibility of trophic/regenerative potential, which would augment tolerogenic processes by decreasing ongoing inflammation. We discuss the application of this autologous cell source in the context of rheumatoid arthritis, concluding with some practical examples of its applications.     

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.     

大鼠脂肪间充质干细胞的鉴定及肝内移植的研究

目的:从脂肪组织中获取间充质干细胞(ADMSCs)并验证其多向分化潜能,探讨ADMSCs在肝再生中的作用。方法:获取大鼠脂肪组织,用胶原酶消化法获取干细胞,并进行体外扩增、传代,取第3代细胞分别用不同诱导培养液进行成骨、成脂诱导,诱导后通过细胞形态学和特殊染色观察诱导效果。用PKH26标记细胞,制作部分肝切除模型,将标记的自体ADMSCs经门静脉植入体内,2周后切下取肝脏制成冰冻切片,荧光显微镜观察植入细胞在肝脏的定位,免疫荧光染色观察其白蛋白的表达。结果:从脂肪组织中分离出的细胞能在体外大量扩增,能被诱导分化为成骨细胞、脂肪细胞,ADMSCs移植2周后,可见PKH26标记细胞散在分布于肝内,免疫荧光染色显示标记细胞白蛋白染色阳性。结论:大鼠脂肪组织中可以获取具有多向分化潜能的间充质干细胞,该细胞在肝再生环境中能向肝细胞分化,参与肝再生。     

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: Towards the definition of a mesenchymal stem cell proteomic signature

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2‐DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo‐natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.     

Human adipose stromal vascular cell delivery in a fibrin spray

Background aimsAdipose tissue represents a practical source of autologous mesenchymal stromal cells (MSCs) and vascular-endothelial progenitor cells, available for regenerative therapy without in vitro expansion. One of the problems confronting the therapeutic application of such cells is how to immobilize them at the wound site. We evaluated in vitro the growth and differentiation of human adipose stromal vascular fraction (SVF) cells after delivery through the use of a fibrin spray system.MethodsSVF cells were harvested from four human adult patients undergoing elective abdominoplasty, through the use of the LipiVage system. After collagenase digestion, mesenchymal and endothelial progenitor cells (pericytes, supra-adventitial stromal cells, endothelial progenitors) were quantified by flow cytometry before culture. SVF cells were applied to culture vessels by means of the Tisseel fibrin spray system. SVF cell growth and differentiation were documented by immunofluorescence staining and photomicrography.ResultsSVF cells remained viable after application and were expanded up to 3 weeks, when they reached confluence and adipogenic differentiation. Under angiogenic conditions, SVF cells formed endothelial (vWF+, CD31+ and CD34+) tubules surrounded by CD146+ and α-smooth muscle actin+ perivascular/stromal cells.ConclusionsHuman adipose tissue is a rich source of autologous stem cells, which are readily available for regenerative applications such as wound healing, without in vitro expansion. Our results indicate that mesenchymal and endothelial progenitor cells, prepared in a closed system from unpassaged lipoaspirate samples, retain their growth and differentiation capacity when applied and immobilized on a substrate using a clinically approved fibrin sealant spray system.     

Mature adipocytes may be a source of stem cells for tissue engineering

Adipose tissue contains a large portion of stem cells. These cells appear morphologically like fibroblasts and are primarily derived from the stromal cell fraction. Mature (lipid-filled) adipocytes possess the ability to become proliferative cells and have been shown to produce progeny cells that possess the same morphological (fibroblast-like) appearance as the stem cells from the stromal fraction. A closer examination of mature adipocyte-derived progeny cells may prove to be an emerging area of growth/metabolic physiology that may modify present thinking about adipose tissue renewal capabilities. Knowledge of these cells may also prove beneficial in cell-based therapies for tissue repair, regeneration, or engineering.     

骨髓间充质干细胞在组织工程研究中的应用新进展

骨髓间充质干细胞又称为骨髓源性间充质干细胞,是指存在于骨髓基质细胞系统中的一类干细胞,具有高度稳定的体外扩增能力和多向分化潜能等特点。骨髓间充质干细胞因其取材方便,易于分离和培养,以及在适当条件下可诱导分化为皮肤、骨骼、内脏、血液、神经等多种组织细胞的独特优势,目前被广泛应用于药物开发、免疫调节、组织修复、器官重建等多个研究领域。近年来,骨髓间充质干细胞作为种子细胞在组织工程领域有着非常诱人的潜在应用前景。本文就骨髓间充质干细胞在组织工程学研究中应用的最新进展作一综述。     

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.     

Osteogenic differentiation of amniotic fluid mesenchymal stromal cells and their bone regeneration potential

In orthopedics, tissue engineering approach using stem cells is a valid line of treatment for patients with bone defects. In this context, mesenchymal stromal cells of various origins have been extensively studied and continue to be a matter of debate. Although mesenchymal stromal cells from bone marrow are already clinically applied, recent evidence suggests that one may use mesenchymal stromal cells from extra-embryonic tissues, such as amniotic fluid, as an innovative and advantageous resource for bone regeneration. The use of cells from amniotic fluid does not raise ethical problems and provides a sufficient number of cells without invasive procedures. Furthermore, they do not develop into teratomas when transplanted, a consequence observed with pluripotent stem cells. In addition, their multipotent differentiation ability, low immunogenicity, and anti-inflammatory properties make them ideal candidates for bone regenerative medicine. We here present an overview of the features of amniotic fluid mesenchymal stromal cells and their potential in the osteogenic differentiation process. We have examined the papers actually available on this regard, with particular interest in the strategies applied to improve in vitro osteogenesis. Importantly, a detailed understanding of the behavior of amniotic fluid mesenchymal stromal cells and their osteogenic ability is desirable considering a feasible application in bone regenerative medicine.     

Adipose tissue,angiogenesis and angio-MIR under physiological and pathological conditions

Angiogenesis is a crucial process for the maintenance of normal tissue physiology and it is involved in tissue remodeling and regeneration. This process is essential for adipose tissue maintenance. The adipose tissue is composed by different cell types including stromal vascular cells as well as adipose stem cells (ASCs). In particular, ASCs are multipotent somatic stem cells that are able to differentiate and secrete several growth factors; they are recently emerging as a new cell reservoir for novel therapies and strategies in many diseases. Several studies suggest that ASCs have peculiar properties and participate in different disease-related processes such as angiogenesis. Furthermore, pathological expansion of adipose tissue brings to hypoxia, a major condition of unhealthy angiogenesis.Recent evidences have shown that microRNAs (miRNAs) play a crucial role also on ASCs as they take part in stemness maintenance, proliferation, and differentiation. It has been suggested that some miRNAs (MIR126, MIR31, MIR221 MIR222, MIR17-92 cluster, MIR30, MIR100 and MIR486) are directly involved in the angiogenic process by controlling multiple genes involved in this pathway. With the present review, we aim at providing an updated summary of the importance of adipose tissue under physiological and pathological conditions and of its relationship with neovascularization process. In particular, we report an overview of the most important miRNAs involved in angiogenesis focusing on ASCs. Hopefully the data presented will bring benefit in developing new therapeutic strategies.     

Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro

Adipose tissue is a rich, ubiquitous and easily acces-sible source for multipotent stromal/stem cells and has, therefore, several advantages compared to other sourc-es of mesenchymal stromal/stem cells. Several studies have tried to identify the origin of the stromal/stem cell population within adipose tissue in situ. This is a complicated attempt because no marker has currently been described which unambiguously identifies native adipose-derived stromal/stem cells(ASCs). Isolated and cultured ASCs are a non-uniform preparation consisting of several subsets of stem and precursor cells. Cultured ASCs are characterized by their expression of a panel of markers(and the absence of others), whereas their in vitro phenotype is dynamic. Some markers were ex-pressed de novo during culture, the expression of some markers is lost. For a long time, CD34 expression was solely used to characterize haematopoietic stem and progenitor cells, but now it has become evident that it is also a potential marker to identify an ASC subpopula-tion in situ and after a short culture time. Nevertheless, long-term cultured ASCs do not express CD34, perhaps due to the artificial environment. This review gives an update of the recently published data on the origin and phenotype of ASCs both in vivo and in vitro. In addition, the composition of ASCs(or their subpopula-tions) seems to vary between different laboratories andpreparations. This heterogeneity of ASC preparationsmay result from different reasons. One of the main problems in comparing results from different laborato-ries is the lack of a standardized isolation and culture protocol for ASCs. Since many aspects of ASCs, suchas the differential potential or the current use in clinical trials, are fully described in other recent reviews, this review further updates the more basic research issues concerning ASCs' subpopulations, heterogeneity andculture standardization.