Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice

Recent studies suggest that human adipose tissue contains pluripotent stem cells similar to bone marrow-derived stem cells. Taking advantage of homogeneously marked cells from green fluorescent protein (GFP) transgenic mice, we have previously demonstrated that bone marrow-derived stromal cells (BSCs) differentiate into a variety of cell lineages both in vitro and in vivo. In the present study, we extend this approach to characterize adipose tissue-derived stromal cells, sometimes called processed lipoaspirate (PLA) cells. Adipose-derived stromal cells (ASCs) were isolated from inguinal fat pads of GFP transgenic mice after extensive washing with phosphate-buffered saline and treatment with collagenase. After primary culture in a control medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum) and expansion to two passages, the cells were incubated in either an osteogenic medium (Dulbecco's modified Eagle's medium+10% fetal bovine serum+dexamethasone+ascorbate-2-phosphate+beta-glycerophosphate) or a chondrogenic medium (Dulbecco's modified Eagle's medium+1% fetal bovine serum+insulin+ascorbate-2-phosphate+transforming growth factor-beta1) for 2-4 weeks to induce osteogenesis and chondrogenesis, respectively. Osteogenic differentiation was assessed by von Kossa and alkaline phosphatase staining, while chondrogenic differentiation was assessed by Alcian blue staining. Expression of osteocyte specific osteopontin, osteocalcin, and alkaline phosphatase, and chondrocyte specific aggrecan and type II/X collagen was confirmed by RT-PCR. ASCs incubated in the osteogenic medium were stained positively for von Kossa and alkaline phosphatase staining. Expression of osteocyte specific genes, except osteocalcin, was also detected. Incubation with chondrogenic medium induced Alcian blue positive cells and expression of aggrecan and type II/X collagen genes. No osteochondrogenic differentiation was observed in cells incubated in the control medium. ASCs from GFP transgenic mice have both osteogenic and chondrogenic potential in vitro. Since this cell population can be easily identified through fluorescence microscopy, it may be an ideal source of ASCs for further experiments on stem cell biology and tissue engineering.     

From heterogeneity to plasticity in adipose tissues: site-specific differences

In mammals, two types of adipose tissues are present, brown (BAT) and white (WAT). WAT itself can be divided into subcutaneous and internal fat deposits. All these tissues have been shown to present a great tissue plasticity, and recent data emphasized on the multiple differentiation potentials obtained from subcutaneous WAT. However, no study has compared the heterogeneity of stroma-vascular fraction (SVF) cells and their differentiation potentials according to the localization of the fat pad. This study clearly demonstrates that WAT and BAT present different antigenic features and differentiation potentials. WAT by contrast to BAT contains a large population of hematopoietic cells composed essentially of macrophages and hematopoietic progenitor cells. In WAT, the non-hematopoietic population is mainly composed of mesenchymal stem cell (MSC)-like but contains also a significant proportion of immature cells, whereas in BAT, the stromal cells do not present the same phenotype. Internal and subcutaneous WAT present some discrete differences in the phenotype of their cell populations. WAT derived SVF cells give rise to osteoblasts, endothelial cells, adipocytes, hematopoietic cells, and cardiomyoblasts only from inguinal cells. By contrast, BAT derived SVF cells display a reduced plasticity. Adipose tissues thus appear as complex tissues composed of different cell subsets according to the location of fat pads. Inguinal WAT appears as the most plastic adipose tissue and represents a potential and suitable source of stem cell, considering its easy sampling as a major advantage for cell therapy.     

The potential of adipose-derived adult stem cells as a source of neuronal progenitor cells

Adipose-derived adult stem cells are a population of mesenchymal stem cells extracted from discarded adipose tissue. Many have reported the differentiation of adipose-derived stem cells into chondrocytes, myocytes, osteoblasts, and, most recently, neural progenitor cells. This article covers the current state of the potential of the differentiation of adipose-derived stem cells into neuronal cells and an overview of their potential as adult stem therapies for neurological disorders. It has been reported that adipose-derived stem cells are capable of undergoing neuronal differentiation using protocols similar to that of Woodbury et al., which reported the differentiation of bone marrow stromal cells specifically into neurons. However, the transdifferentiation of bone marrow stromal cells into neuronal cells has recently fallen under intense criticism, which will likely place the plasticity of adipose-derived stem cells under scrutiny as well. To date, no group has produced evidence that adipose-derived stem cells are capable of differentiating to mature, functional neuronal cells in vitro. However, recent in vivo studies with adipose-derived stem cells are promising.     

Bidirectional induction toward paraxial mesodermal derivatives from mouse ES cells in chemically defined medium

Embryonic stem cells (ESCs) are a renewable cell source of tissue for regenerative therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ESC cultures can induce primitive streak-like mesodermal cells. In differentiated mouse ESCs, platelet-derived growth factor receptor-α (PDGFR-α) and E-cadherin (ECD) are useful markers to distinguish between paraxial mesodermal progenitor cells and undifferentiated and endodermal cells, respectively. Here, we demonstrate methods for BMP4-mediated induction of paraxial mesodermal progenitors using PDGFR-α and ECD as markers for purification and characterization. Serum-free monolayers of ESCs cultured with BMP4 could efficiently promote paraxial mesodermal differentiation akin to embryonic mesodermal development. BMP4 treatment alone induced paraxial mesodermal progenitors that could differentiate into osteochondrogenic cells in vitro and in vivo. Furthermore, early removal of BMP4 followed by lithium chloride (LiCl) promoted the differentiation to myogenic progenitor cells. These myogenic progenitors were able to differentiate further in vitro into mature skeletal muscle cells. Thus, we successfully induced the efficient bidirectional differentiation of mouse ESCs toward osteochondrogenic and myogenic cell types using chemically defined conditions.     

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.     

Cell kinetics,DNA integrity,differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells

Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.     

Current applications of adipose-derived stem cells and their future perspectives

Adult stem cells have a great potential to treat various diseases. For these cell-based therapies, adipose-derived stem cells(ADSCs) are one of the most promising stem cell types, including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs). ESCs and iPSCs have taken center stage due to their pluripotency. However, ESCs and iPSCs have limitations in ethical issues and in identification of characteristics, respectively. Unlike ESCs and iPSCs, ADSCs do not have such limitations and are not only easily obtained but also uniquely expandable. ADSCs can differentiate into adipocytes, osteoblasts, chondrocytes, myocytes and neurons under specific differentiation conditions, and these kinds of differentiation potential of ADSCs could be applied in regenerative medicine e.g., skin reconstruction, bone and cartilage formation, etc. In this review, the current status of ADSC isolation, differentiation and their therapeutic applications are discussed.     

Viscoelastic properties of human mesenchymally-derived stem cells and primary osteoblasts, chondrocytes, and adipocytes

The mechanical properties of single cells play important roles in regulating cell-matrix interactions, potentially influencing the process of mechanotransduction. Recent studies also suggest that cellular mechanical properties may provide novel biological markers, or "biomarkers," of cell phenotype, reflecting specific changes that occur with disease, differentiation, or cellular transformation. Of particular interest in recent years has been the identification of such biomarkers that can be used to determine specific phenotypic characteristics of stem cells that separate them from primary, differentiated cells. The goal of this study was to determine the elastic and viscoelastic properties of three primary cell types of mesenchymal lineage (chondrocytes, osteoblasts, and adipocytes) and to test the hypothesis that primary differentiated cells exhibit distinct mechanical properties compared to adult stem cells (adipose-derived or bone marrow-derived mesenchymal stem cells). In an adherent, spread configuration, chondrocytes, osteoblasts, and adipocytes all exhibited significantly different mechanical properties, with osteoblasts being stiffer than chondrocytes and both being stiffer than adipocytes. Adipose-derived and mesenchymal stem cells exhibited similar properties to each other, but were mechanically distinct from primary cells, particularly when comparing a ratio of elastic to relaxed moduli. These findings will help more accurately model the cellular mechanical environment in mesenchymal tissues, which could assist in describing injury thresholds and disease progression or even determining the influence of mechanical loading for tissue engineering efforts. Furthermore, the identification of mechanical properties distinct to stem cells could result in more successful sorting procedures to enrich multipotent progenitor cell populations.     

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies

Background: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem‐cell exhibits tri‐lineage differentiation potential and is able to maintain its proliferation potential and cell‐surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad‐derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. Materials and methods: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell‐surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. Results: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell‐surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad‐derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator‐activated receptor γ2 and lipoprotein lipase, and oil red O staining. Discussion: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem‐cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.     

Differentiation of mesenchymal stem cells into osteoblasts on honeycomb collagen scaffolds

Tissue engineering using living cells is emerging as an alternative to tissue or organ transplantation. The adult mesenchymal stem cells can be differentiated into multilineage cells, such as adipocytes, chondrocytes, or osteoblasts when cultured with specific growth factors. In the present investigation, we have studied the effect of honeycomb collagen scaffolds for the adhesion, differentiation and proliferation of bone marrow-derived mesenchymal stem cells into osteoblasts. Mesenchymal stem cells were isolated from 6-week old albino rat femur bone marrow, and cultured in alpha-MEM medium without beta-glycerophosphate and dexamethasone. Honeycomb collagen discs were prepared from bovine dermal atelocollagen, cross-linked by UV-irradiation and sterilized by heat. The honeycomb discs were placed on the culture dishes before seeding the stem cells. The cells attached quickly to the honeycomb collagen scaffold, differentiated and proliferated into osteoblasts. The differentiated osteoblasts were characterized by morphological examination and alkaline phosphatase activity. The osteoblasts also synthesized calcium-deficient hydroxyapatite (pseudo-hydroxyapatite) crystals in the culture. The mineralization was confirmed by Von Kossa staining and the crystals were analyzed by X-ray diffraction. Light microscopy and DNA measurements showed that the differentiated osteoblasts multiplied into several layers on the honeycomb collagen scaffold. The results demonstrated that the honeycomb collagen sponge is an excellent scaffold for the differentiation and proliferation of mesenchymal stem cells into osteoblasts. The data further proved that honeycomb collagen is an effective substrate for tissue engineering applications, and is very useful in the advancing field of stem cell technology and cell-based therapy.     

Basic science review on adipose tissue for clinicians

The recognition that fat contains stem cells has driven further examination into the potential uses of fat and adipose-derived stem cells in a wide number of clinical situations. New information about the harvesting, isolation, and subsequent differentiation properties of isolated adipose-derived stem cells has led to new research into novel tissue-engineered constructs and the transformation of adipose-derived stem cells to induced pluripotent stem cells. Clinically, use of fat grafts and adipose-derived stem cells worldwide and in the United States has dramatically increased in parallel to questions concerning the safety and efficacy of adipose-derived stem cell-based treatments. Currently, the U.S. Food and Drug Administration has not approved the use of isolated adipose-derived stem cells for medical indications.     

Staphylococcal enterotoxin C injection in combination with ascorbic acid promotes the differentiation of bone marrow-derived mesenchymal stem cells into osteoblasts in vitro

Staphylococcal enterotoxin C injection is established as a clinical therapy for delayed healing or disunion of bone fractures. In the present study, the effects of staphylococcal enterotoxin C injection in combination with ascorbic acid (SEC-AA) on the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) and their influences on the mineralization of osteoblasts were investigated. SEC-AA treatment induced increased levels of alkaline phosphatase activity in MSCs and increased numbers of alizarin red-stained calcified nodules, indicating enhanced differentiation of MSCs into osteoblasts. The findings demonstrated that SEC-AA promoted the differentiation of MSCs into osteoblasts and accelerated the cytopoiesis of osteoblasts. Our data provide a cytological model for bone fracture therapy aimed at shortening the time required for healing and improving the clinical outcome, and also provide a theoretical basis for inducible differentiation of MSCs, mineralization of osteoblasts and reconstruction of bone tissues.     

Ucma, a novel secreted cartilage-specific protein with implications in osteogenesis

Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.     

Current applications of adipose-derived stem cells and their future perspectives简

Adult stem cells have a great potential to treat various diseases. For these cell-based therapies, adipose-derived stem cells (ADSCs) are one of the most promising stem cell types, including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs have taken center stage due to their pluripotency. However, ESCs and iPSCs have limitations in ethical issues and in identification of characteristics, respectively. Unlike ESCs and iPSCs, ADSCs do not have such limitations and are not only easily obtained but also uniquely expandable. ADSCs can differentiate into adipocytes, osteoblasts, chondrocytes, myocytes and neurons under specific differentiation conditions, and these kinds of differentiation potential of ADSCs could be applied in regenerative medicine e.g., skin reconstruction, bone and cartilage formation, etc. In this review, the current status of ADSC isolation, differentiation and their therapeutic applications are discussed.     

Adipose tissue: a subtle and complex cell system

White adipose tissue (WAT) represents a large amount of all adult tissues. For a long time, it was considered as a poorly active, overgrown and undesirable tissue. It was mainly studied for its involvement in energy metabolism and disorders, as well as for its endocrine functions. WAT is composed of two main populations, matures adipocytes and stroma vascular fraction (SVF) that can be separated easily. The SVF contains two compartments, stromal and hematopoietic that have been recently characterized. The stromal population (or ADAS for Adipose Derived Stromal Cells) presents functional features of, as well as lineage relationship with, macrophages. These stromal cells, that are able to differentiate into adipocytes, also display endothelial potential, and could be considered as vascular progenitors. Differentiation of various adipose-derived cell subsets towards functional cardiomyocytes, osteoblasts, chondrocytes, muscle, hematopoietic and neural cells was also obtained in vitro or in vivo. Adipose tissue thus appears as a complex tissue composed of different cell subsets that could vary according to the nature and the location of fat pads, or to the physiological or pathological status. WAT appears as a very plastic and heterogeneous tissue that is very easy to sample. This represents a great advantage when considering adipose tissue as a potential and suitable source of stem cell for cell therapy. Further investigations in this way have to lead to the emergence of new insights fundamental to progress in our knowledge of adipose tissue biology.     

Differential expression of CCN-family members in primary human bone marrow-derived mesenchymal stem cells during osteogenic, chondrogenic and adipogenic differentiation

BACKGROUND: The human cysteine rich protein 61 (CYR61, CCN1) as well as the other members of the CCN family of genes play important roles in cellular processes such as proliferation, adhesion, migration and survival. These cellular events are of special importance within the complex cellular interactions ongoing in bone remodeling. Previously, we analyzed the role of CYR61/CCN1 as an extracellular signaling molecule in human osteoblasts. Since mesenchymal stem cells of bone marrow are important progenitors for various differentiation pathways in bone and possess increasing potential for regenerative medicine, here we aimed to analyze the expression of CCN family members in bone marrow-derived human mesenchymal stem cells and along the osteogenic, the adipogenic and the chondrogenic differentiation. RESULTS: Primary cultures of human mesenchymal stem cells were obtained from the femoral head of patients undergoing total hip arthroplasty. Differentiation into adipocytes and osteoblasts was done in monolayer culture, differentiation into chondrocytes was induced in high density cell pellet cultures. For either pathway, established differentiation markers and CCN-members were analyzed at the mRNA level by RT-PCR and the CYR61/CCN1 protein was analyzed by immunocytochemistry.RT-PCR and histochemical analysis revealed the appropriate phenotype of differentiated cells (Alizarin-red S, Oil Red O, Alcian blue, alkaline phosphatase; osteocalcin, collagen types I, II, IX, X, cbfa1, PPARgamma, aggrecan). Mesenchymal stem cells expressed CYR61/CCN1, CTGF/CCN2, CTGF-L/WISP2/CCN5 and WISP3/CCN6. The CYR61/CCN1 expression decreased markedly during osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation. These results were confirmed by immuncytochemical analyses. WISP2/CCN5 RNA expression declined during adipogenic differentiation and WISP3/CCN6 RNA expression was markedly reduced in chondrogenic differentiation. CONCLUSION: The decrease in CYR61/CCN1 expression during the differentiation pathways of mesenchymal stem cells into osteoblasts, adipocytes and chondrocytes suggests a specific role of CYR61/CCN1 for maintenance of the stem cell phenotype. The differential expression of CTGF/CCN2, WISP2/CCN5, WISP3/CCN6 and mainly CYR61/CCN1 indicates, that these members of the CCN-family might be important regulators for bone marrow-derived mesenchymal stem cells in the regulation of proliferation and initiation of specific differentiation pathways.     

Impaired fat storage capacity in adipocyte precursors of I versus C57BL mice

I mouse strain displays adipocyte hypoplasia responsible for smaller fat pad size compared with C57BL mice. We investigated possible alterations in the proliferation and/or differentiation capacity of preadipocytes from the stroma-vascular fraction of adipose tissue in the I mouse strain. Control C57BL and I mice were studied at 8 weeks of age, and both adipose and stromal cells were isolated from epididymal and inguinal adipose tissue localizations. Results showed that the lower epididymal adipose mass in I mice was accompanied by a decrease in stromal cell number compared with C57BL mice. In inguinal fat pads, total cell number in the stroma-vascular fraction was unmodified; lipoprotein lipase activity significantly increased in stromal cells from I mice compared with control mice. In this depot, further characterization of cells from the stroma-vascular fraction by separation of cells according to density showed an increased number of preadipocytes in the I mouse whole stromal cell population. These preadipocytes seemed unable to undergo terminal maturation, thus leading to a decrease in the number of mature adipocytes. These results indicated that resistance to fat accumulation in I mice is characterized by site-dependent impairment of both the proliferative rate and the differentiation capacity of adipocyte precursors.     

Sympathetic denervation of one white fat depot changes norepinephrine content and turnover in intact white and brown fat depots

It is well-established that the sympathetic nervous system (SNS) regulates adipocyte metabolism and recently it has been reported that sensory afferents from white fat overlap anatomically with sympathetic efferents to white fat. The studies described here characterize the response of intact fat pads to selective sympathectomy (local 6-hydroxydopamine (6OHDA) injections) of inguinal (ING) or epididymal (EPI) fat in male NIH Swiss mice and provide in vivo evidence for communication between individual white and brown fat depots. The contralateral ING pad, both EPI pads, perirenal (PR), and mesenteric (MES) pads were significantly enlarged 4 weeks after denervating one ING pad, but only intrascapular brown adipose tissue (IBAT) increased when both ING pads were denervated. Denervation of one or both EPI pad had no effect on fat depot weights. In an additional experiment, norepinephrine turnover (NETO) was inhibited in ING, retroperitoneal (RP), MES, and IBAT 2 days after denervation of both EPI or of both ING pads. NE content was reduced to 10-30% of control values in all fat depots. There was no relation between early changes in NETO and fat pad weight 4 weeks after denervation, even though the reduction in NE content of intact fat pads was maintained. These data demonstrate that there is communication among individual fat pads, presumably through central integration of activity of sensory afferent and sympathetic efferent fibers, that changes sympathetic drive to white adipose tissue in a unified manner. In specific situations, removal of sympathetic efferents to one pad induces a compensatory enlargement of other intact depots.     

Anatomical structure of the buccal fat pad and its clinical adaptations

Before performing plastic and aesthetic surgery around the buccal area, the authors reviewed the anatomical structures of the buccal fat pad in 11 head specimens (i.e., 22 sides of the face). The enveloping, fixed tissues and the source of the nutritional vessels to the buccal fat pad and its relationship with surrounding structures were observed in detail, with the dissection procedure described step by step. The dissection showed that the buccal fat pad can be divided into three lobes-anterior, intermediate, and posterior-according to the structure of the lobar envelopes, the formation of the ligaments, and the source of the nutritional vessels. The buccal, pterygoid, pterygopalatine, and temporal extensions (superficial and profound) are derived from the posterior lobe. The buccal fat pad is fixed by six ligaments to the maxilla, posterior zygoma, and inner and outer rim of the infraorbital fissure, temporalis tendon, or buccinator membrane. Several nutritional vessels exist in each lobe and in the subcapsular vascular plexus forms. The buccal fat pads function to fill the deep tissue spaces, to act as gliding pads when masticatory and mimetic muscles contract, and to cushion important structures from the extrusion of muscle contraction or outer force impulsion. The volume of the buccal fat pad may change throughout a person's life. Based on the findings of the dissections, the authors provide several clinical applications for the buccal fat pad, such as the mechanism of deepening the nasolabial fold and possible rhytidectomy to suspend the anterior lobe upward and backward. They suggest that relaxation, poor development of the ligaments, or rupture of the buccal fat pad capsules can make the buccal extension drop or prolapse to the mouth or subcutaneous layer. As such, the authors refined their methods and heightened their focus when using the buccal fat pad to perform a random or pedicled buccal fat pad fat flap or to correct a buccal skin protrusion or hollow.