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Role of Adipose-Derived Stem Cells in Wound Healing: An Update from Isolation to Transplantation: Stem Cells Repair and Restorations, Basic and Clinical Aspects
Abstract
Adipose tissue serves as an alternative mesenchymal cell source with comparable proliferation, growth factor expression, and differentiation capacity. A large body of work has suggested that adipose-derived stem cells (ADSCs) would be used in various regeneration applications. Due to having remarkable therapeutic potential, ADSCs would also be a putative therapy for non-healing wounds, which is a growing clinical concern for especially aged populations with systemic diseases such as obesity and diabetes. This chapter aims to highlight beneficial effects of ADSCs on acute and chronic wound healing by reviewing preclinical studies and clinical trials from the perspective of direct differentiation, paracrine signaling, cell recruitment, scaffold combination, and preconditioning. In addition, possible action of a mechanism for wound healing promoting activity of ADSCs will be explained in detail along with addressing efficacy and safety issues.
... ADMSCs are pluripotent stem cells which can differentiate into different lineages and secrete paracrine factors for inducing tissue regeneration (Hassan et al. 2014). In fact, ADMSCs play a major role in three main phases of a wound healing process, which are inflammatory, proliferative, and remodeling phase through paracrine signalling (Demirci et al. 2018). Thus, the advances in understanding the immunosuppression and secretion of pro-angiogenic factors by ADMSCs, have inspired the scientists and researchers across the world to explore the potential of ADMSCs alone or in combination with matrix scaffold for both acute and chronic wound repairs. ...
The naturally obtained protein Bombyxmori silk is a biocompatible polymer with excellent mechanical properties and have the potential in controlled drug delivery applications. In this work, we have demonstrated dielectric barrier discharge (DBD) oxygen (O2) plasma surface modified electrospun Bombyxmori silk/Amoxicillin hydrochloride trihydrate (AMOX)/polyvinyl alcohol (PVA) nanofibers for drug release applications with controlled plasma treatment duration (1–10 min). The findings indicate that plasma treated electrospun nanofibers for 1–3 min exhibited significant enhancement in tensile strength, Young’s modulus, wettability and surface energy. The plasma treated electrospun nanofibers for 1–5 min showed remarkable increase in AMOX released rate, whereas the electrospun nanofibers treated with plasma irradiation beyond 5 min showed only marginal increase. Moreover, the plasma treated nanofibers also exhibited good antibacterial activity against both E. coli (gram negative) and S. aureus (gram positive) bacteria. The untreated and the plasma treated silk/AMOX/PVA electrospun nanofibers for 1–3 min showed enhanced viability of primary adipose derived mesenchymal stem cells (ADMSCs) growth on them and much less hemolysis activity (< 5%). The in vitro biocompatibility of various electrospun nanofibers were further corroborated by live/dead imaging and cytoskeletal architecture assessment demonstrating enhanced cell adhesion and spreading on the plasma treated nanofibers for 1–3 min. The findings of the present study suggest that the silk/AMOX/PVA electrospun nanofibers with plasma treatment (1–3 min) due to their enhanced drug release ability and biocompatibility can be used as potential wound dressing applications.Graphic abstract
Unlabelled:
Three-dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose-derived stem/stromal cells (hASCs) in a non-cross-linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20-50 µm) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX-2), and 40% of the cells were SSEA-3-positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia-reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate-buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage.
Significance:
This study shows the therapeutic value of human adipose-derived stem cell spheroids prepared in hyarulonic acid gel. The spheroids have various benefits as an injectable cellular product and show therapeutic potential to the stem cell-depleted conditions such as diabetic chronic skin ulcer.
Mesenchymal stem cells (MSCs) have been shown to improve tissue regeneration in several preclinical and clinical trials. These cells have been used in combination with three-dimensional scaffolds as a promising approach in the field of regenerative medicine. We compare the behavior of human adipose-derived MSCs (AdMSCs) on four different biomaterials that are awaiting or have already received FDA approval to determine a suitable regenerative scaffold for delivering these cells to dermal wounds and increasing healing potential. AdMSCs were isolated, characterized, and seeded onto scaffolds based on chitosan, fibrin, bovine collagen, and decellularized porcine dermis.
In vitro
results demonstrated that the scaffolds strongly influence key parameters, such as seeding efficiency, cellular distribution, attachment, survival, metabolic activity, and paracrine release. Chick chorioallantoic membrane assays revealed that the scaffold composition similarly influences the angiogenic potential of AdMSCs
in vivo
. The wound healing potential of scaffolds increases by means of a synergistic relationship between AdMSCs and biomaterial resulting in the release of proangiogenic and cytokine factors, which is currently lacking when a scaffold alone is utilized. Furthermore, the methods used herein can be utilized to test other scaffold materials to increase their wound healing potential with AdMSCs.
Introduction Liver disease mortality and morbidity are rapidly rising and liver transplantation is limited by organ availability. Small scale human studies have shown that stem cell therapy is safe and feasible and has suggested clinical benefit. No published studies have yet examined the effect of stem cell therapy in a randomised controlled trial and evaluated the effect of repeated therapy.
Methods and analysis Patients with liver cirrhosis will be randomised to one of three trial groups: group 1: Control group, Standard conservative management; group 2 treatment: granulocyte colony-stimulating factor (G-CSF; lenograstim) 15 µg/kg body weight daily on days 1–5; group 3 treatment: G-CSF 15 µg/kg body weight daily on days 1–5 followed by leukapheresis, isolation and aliquoting of CD133+ cells. Patients will receive an infusion of freshly isolated CD133+ cells immediately and frozen doses at days 30 and 60 via peripheral vein (0.2×106 cells/kg for each of the three doses). Primary objective is to demonstrate an improvement in the severity of liver disease over 3 months using either G-CSF alone or G-CSF followed by repeated infusions of haematopoietic stem cells compared with standard conservative management. The trial is powered to answer two hypotheses of each treatment compared to control but not powered to detect smaller expected differences between the two treatment groups. As such, the overall α=0.05 for the trial is split equally between the two hypotheses. Conventionally, to detect a relevant standardised effect size of 0.8 point reduction in Model for End-stage Liver Disease score using two-sided α=0.05(overall α=0.1 split equally between the two hypotheses) and 80% power requires 27 participants to be randomised per group (81 participants in total).
Ethics and dissemination The trial is registered at Current Controlled Trials on 18 November 2009 (ISRCTN number 91288089, EuDRACT number 2009-010335-41). The findings of this trial will be disseminated to patients and through peer-reviewed publications and international presentations.
Macrophage-driven inflammation, a key feature of the early period following tendon repair, but excessive inflammation has been associated with poor clinical outcomes. Modulation of the inflammatory environment using molecular or cellular treatments may provide a means to enhance tendon healing.
To examine the effect of pro-inflammatory cytokines secreted by macrophages on tendon fibroblasts (TF), we established in vitro models of cytokine and macrophage-induced inflammation. Gene expression, protein expression, and cell viability assays were used to examine TF responses. In an effort to reduce the negative effects of inflammatory cytokines on TFs, adipose-derived mesenchymal stromal cells (ASCs) were incorporated into the model and their ability to modulate inflammation was investigated.
The inflammatory cytokine interleukin 1 beta (IL-1β) and macrophages of varying phenotypes induced up-regulation of pro-inflammatory factors and matrix degradation factors and down-regulation of factors related to extracellular matrix formation by TFs in culture. ASCs did not suppress these presumably negative effects induced by IL-1β. However, ASC co-culture with M1 (pro-inflammatory) macrophages successfully suppressed the effects of M1 macrophages on TFs by inducing a phenotypic switch from a pro-inflammatory macrophage phenotype to an anti-inflammatory macrophage phenotype, thus resulting in exposure of TFs to lower levels of pro-inflammatory cytokines (e.g., IL-1β, tumor necrosis factor alpha (TNFα)).
These findings suggest that IL-1β and M1 macrophages are detrimental to tendon healing and that ASC-mediated modulation of the post-operative inflammatory response may be beneficial for tendon healing.
Growing recognition of paracrine mechanisms in stem cell plasticity has resulted in considerable interest in stem cell-derived secretome. The aim of this study was to investigate the effects of lipopolysaccharide (LPS) preconditioning on the composition and hepatic regenerative activity of adipose-derived stem cell (ASC) secretome.
Conditioned medium (CM) and LPS-CM were obtained after culturing human ASCs without or with low-dose LPS (0.5 ng/ml) for 24 h, respectively. Untreated and thioacetamide-treated mouse AML12 hepatocytes were incubated for 24 h with the control medium, LPS (0.5 ng/ml), CM, and LPS-CM, respectively, and then cell viabilities were compared. CM and LPS-CM were also intravenously administrated to partially hepatectomized mice and their effects on liver regeneration were assessed using liver weight measurements, immunohistochemistry and western blotting.
In the in vitro experiments, LPS preconditioning of ASCs enhanced the mRNA expression levels of IL-6, TNF-α, HGF, and VEGF, which evoke inflammatory response or liver regeneration. LPS-CM significantly promoted thioacetamide-damaged AML12 cell viability compared with CM-incubated cells and the control cells (77%, 69%, and 65%, respectively; P < 0.05). In the in vivo experiment, LPS-CM infusion into the partially hepatectomized mice significantly reduced serum IL-6 and TNF-α levels compared to the other groups (P < 0.05) on 1 and 2 days after partial hepatectomy. Moreover, LPS-CM infusion enhanced liver regeneration (based on the liver weight changes at 7 days after partial hepatectomy, 3.73% vs. 3.22% in the CM group; P < 0.05) and significantly reduced the elevated serum levels of AST and ALT (at day 1, P < 0.05).
Our results suggest that LPS preconditioning effectively stimulates ASCs to produce the secretome beneficial to hepatic regeneration. Thus, optimizing ASC secretome profile by LPS preconditioning could be a promising approach to treat liver diseases using stem cells.
The overall clinical cardiac regeneration experience suggests that stem cell therapy can be safely performed, but it also underlines the need for reproducible results for their effective use in a real-world scenario. One of the significant challenges is the identification and selection of the best suited stem cell type for regeneration therapy. Bone marrow mononuclear cells, bone marrow-derived mesenchymal stem cells, resident or endogenous cardiac stem cells, endothelial progenitor cells and induced pluripotent stem cells are some of the stem cell types which have been extensively tested for their ability to regenerate the lost myocardium. While most of these cell types are being evaluated in clinical trials for their safety and efficacy, results show significant heterogeneity in terms of efficacy. The enthusiasm surrounding regenerative medicine in the heart has been dampened by the reports of poor survival, proliferation, engraftment, and differentiation of the transplanted cells. Therefore, the primary challenge is to create clearcut evidence on what actually drives the improvement of cardiac function after the administration of stem cells. In this review, we provide an overview of different types of stem cells currently being considered for cardiac regeneration and discuss why associated factors such as practicality and difficulty in cell collection should also be considered when selecting the stem cells for transplantation. Next, we discuss how the experimental variables (type of disease, marker-based selection and use of different isolation techniques) can influence the study outcome. Finally, we provide an outline of the molecular and genetic approaches to increase the functional ability of stem cells before and after transplantation.
The literature provides abundant evidence that mesenchymal stem cells (MSCs) are an attractive resource for therapeutics and have beneficial effects in regenerating injured tissues due to their self-renewal ability and broad differentiation potential. Although the therapeutic potential of MSCs has been proven in both preclinical and clinical studies, several questions have not yet been addressed. A major limitation to the use of MSCs in clinical applications is their poor viability at the site of injury due to the harsh microenvironment and to anoikis driven by the loss of cell adhesion. To improve the survival of the transplanted MSCs, strategies to regulate apoptotic signaling and enhance cell adhesion have been developed, such as pretreatment with cytokines, growth factors, and antiapoptotic molecules, genetic modifications, and hypoxic preconditioning. More appropriate animal models and a greater understanding of the therapeutic mechanisms of MSCs will be required for their successful clinical application. Nevertheless, the development of stem cell therapies using MSCs has the potential to treat degenerative diseases. This review discusses various approaches to improving MSC survival by inhibiting anoikis.
Background:
Sensory recovery of skin flaps is generally poor unless they are elevated as an innervated flap. The aim of this study was to elucidate if Schwann cell (SC)-like cells differentiated from adipose-derived stem cells (ASCs) could improve the cutaneous nerve regeneration in skin flaps.
Methods:
Microvascular island groin flaps were elevated bilaterally in 32 Lewis rats. On the right flap, the epigastric nerve was resected and ligated (noninnervated flap), and on the left flap, the nerve was crushed (innervated flap). ASCs, SC-like cells differentiated from ASCs (dASCs), SCs, or vehicle were simultaneously injected to the dermal and hypodermal layers of flap. After 20 weeks, the reinnervation pattern of flap was assessed immunohistochemically using a neuronal marker, PGP9.5.
Results:
dASC cultures produced significantly higher amount of nerve growth factor and brain-derived neurotrophic factor compared with ASC cultures (P < 0.01), and the level was comparable to that of SC cultures. Although a long-term survival of the transplanted cells was not found, dASCs and SCs significantly increased reinnervation density in the periphery of both types of flaps (P < 0.05), and this effect was more pronounced in noninnervated flaps. On the other hand, ASC transplantation showed no statistically significant effect on the peripheral reinnervation (P > 0.05). In the center of flap, there was no statistically significant increase in reinnervation density in all groups irrespective of flap innervation (P > 0.05).
Conclusions:
dASCs could improve flap reinnervation by 2 mechanisms: First, neurotrophic factors produced by dASCs facilitated regrowth of cutaneous axons from the surroundings of flap. Second, nerve growth factor released by dASCs induced the collateral sprouting of undamaged axons in adjacent tissues. In addition to the use of innervated flaps, dASC transplantation therapy could be a new approach to improve the sensory recovery of skin flaps.
In the field of tissue engineering and reconstruction, the development of efficient biomaterial is in high demand to achieve uncomplicated wound healing. Chronic wounds and excessive scarring are the major complications of tissue repair and, as this inadequate healing continues to increase, novel therapies and treatments for dysfunctional skin repair and reconstruction are important. This paper reviews the various aspects of the complications related to wound healing and focuses on chitosan because of its unique function in accelerating wound healing. The proliferation of keratinocytes is essential for wound closure, and adipose-derived stem cells play a significant role in wound healing. Thus, chitosan in combination with keratinocytes and adipose-derived stem cells may act as a vehicle for delivering cells, which would increase the proliferation of keratinocytes and help complete recovery from injuries.
The aim of this study was to evaluate the wound healing of adipose-derived stem cells (ADSCs) seeded on electrospinning poly (L-lactide-co-ε-caprolactone)/poloxamer (PLCL/P123) scaffolds and the mechanisms using a rat skin tissue injury model. CM-Dil labeled ADSCs were seeded on PLCL/P123 scaffolds for 48 h (ADSCs- PLCL/P123). Twelve Sprague-Dawley rats were randomly divided into three groups: ADSCs-PLCL/P123 (A group); PLCL/P123 alone (B group); petrolatum gauze (C group). Two 1.5 cm diameter circular impressions were made by placement of a punch biopsy instrument. The wound closure percentages were calculated by planimetry on postoperative day 0, 3, 12 and 21. On postoperative day 21, full-thickness skins from each group were examined by H&E staining, CK10 and CD31 immunohistochemical staining, and CK10 immunofluorescence staining respectively. The wound closure percentages on postoperative day 21 in A, B and C groups were 95.7% ± 3.9%, 83.2% ± 15.4% and 64.3% ± 6.2%, respectively (p < 0.05). H&E staining and immunohistochemical staining showed that the epidermal structure of A group was complete and possessed the characteristics of epidermal cells, and the CK10 staining positive suggested that ADSCs were differentiated into epidermal-like structures. The microvessel density of A group was significantly higher than that of the B and C groups (p < 0.05), while no significant differences were observed between B group and C group (p > 0.05). The ADSCs seeded on PLCL/P123 scaffolds could promote wound healing and have the potential to become a new therapeutic alternative material for skin tissue engineering.
Objective: To evaluate the physiological effects of electrospun tropoelastin scaffolds as therapeutic adipose-derived stem cell (ADSC) delivery vehicles for the treatment of full-thickness dermal wounds. Approach: Using the process of electrospinning, several prototype microfiber scaffolds were created with tropoelastin. Initial testing of scaffold biocompatibility was performed in vitro through ADSC culture, followed by scanning electron microscopy (SEM) for assessment of ADSC attachment, morphology, and new extracellular matrix (ECM) deposition. The wound healing effects of ADSC-seeded scaffolds were then evaluated in a murine dermal excisional wound model. Results: For the in vitro study, SEM revealed exceptional biocompatibility of electrospun tropoelastin for ADSCs. In the wound-healing study, ADSC-treated groups demonstrated significantly enhanced wound closure and epithelial thickness compared to controls. Innovation: This is the first report on the use of tropoelastin-based biomaterials as delivery vehicles for therapeutic ADSCs. Conclusion: We have demonstrated that tropoelastin-based ADSC delivery vehicles significantly accelerate wound healing compared to controls that represent the current clinical standard of care. Furthermore, the unique mechanical and biochemical characteristics of tropoelastin may favor its use over other biological or synthetic scaffolds for the treatment of certain pathologies due to its unique intrinsic mechanical properties.
Lesions with great loss of skin and extensive burns are usually treated with heterologous skin grafts, which may lead rejection. Cell therapy with mesenchymal stem cells is arising as a new proposal to accelerate the healing process. We tested a new therapy consisting of sodium carboxymethylcellulose (CMC) as a biomaterial, in combination with adipose-derived stem cells (ADSCs), to treat skin lesions in an in vivo rat model. This biomaterial did not affect membrane viability and induced a small and transient genotoxicity, only at the highest concentration tested (40 mg/mL). In a rat wound model, CMC at 10 mg/mL associated with ADSCs increased the rate of cell proliferation of the granulation tissue and epithelium thickness when compared to untreated lesions (Sham), but did not increase collagen fibers nor alter the overall speed of wound closure. Taken together, the results show that the CMC is capable to allow the growth of ADSCs and is safe for this biological application up to the concentration of 20 mg/mL. These findings suggest that CMC is a promising biomaterial to be used in cell therapy.
This review highlights the critical role of transforming growth factor beta (TGF-β)1-3 within different phases of wound healing, in particular, late-stage wound healing. It is also very important to identify the TGF-β1-controlling factors involved in slowing down the healing process upon wound epithelialization.
TGF-β1, as a growth factor, is a known proponent of dermal fibrosis. Several strategies to modulate or regulate TGF's actions have been thoroughly investigated in an effort to create successful therapies. This study reviews current discourse regarding the many roles of TGF-β1 in wound healing by modulating infiltrated immune cells and the extracellular matrix.
It is well established that TGF-β1 functions as a wound-healing promoting factor, and thereby if in excess it may lead to overhealing outcomes, such as hypertrophic scarring and keloid. Thus, the regulation of TGF-β1 in the later stages of the healing process remains as critical issue of which to better understand.
One hypothesis is that cell communication is the key to regulate later stages of wound healing. To elucidate the role of keratinocyte/fibroblast cross talk in controlling the later stages of wound healing we need to: (1) identify those keratinocyte-released factors which would function as wound-healing stop signals, (2) evaluate the functionality of these factors in controlling the outcome of the healing process, and (3) formulate topical vehicles for these antifibrogenic factors to improve or even prevent the development of hypertrophic scarring and keloids as a result of deep trauma, burn injuries, and any type of surgical incision.
Current strategies of regenerative medicine are focused on the restoration of pathologically altered tissue architectures by transplantation of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable interest has been given to biologically active scaffolds which are based on similar analogs of the extracellular matrix that have induced synthesis of tissues and organs. To restore function or regenerate tissue, a scaffold is necessary that will act as a temporary matrix for cell proliferation and extracellular matrix deposition, with subsequent ingrowth until the tissues are totally restored or regenerated. Scaffolds have been used for tissue engineering such as bone, cartilage, ligament, skin, vascular tissues, neural tissues, and skeletal muscle and as vehicle for the controlled delivery of drugs, proteins, and DNA. Various technologies come together to construct porous scaffolds to regenerate the tissues/organs and also for controlled and targeted release of bioactive agents in tissue engineering applications. In this paper, an overview of the different types of scaffolds with their material properties is discussed. The fabrication technologies for tissue engineering scaffolds, including the basic and conventional techniques to the more recent ones, are tabulated.
Adipose-derived stromal cells (ASCs) are often referred to as adipose-derived stem cells due to their potential to undergo multilineage differentiation. Their promising role in tissue engineering and ability to modulate the immune system are the focus of extensive research. A number of clinical trials using ASCs are currently underway to better understand the role of such cell niche in enhancing or suppressing the immune response. If governable, such immunoregulatory role would find application in several conditions in which an immune response is present (i.e., autoimmune conditions) or feared (i.e., solid organ or reconstructive transplantation). Although allogeneic ASCs have been shown to prevent acute GvHD in both preclinical and clinical studies, their potential warrants further investigation. Well-designed and standardized clinical trials are necessary to prove the role of ASCs in the treatment of immune disorders or prevention of tissue rejection. In this paper we analyze the current literature on the role of ASCs in immunomodulation in vitro and in vivo and discuss their potential in regulating the immune system in the context of transplantation.
Skin substitutes are epidermal, dermal or complete bilayered constructs, composed by natural or synthetic scaffolds and by adherent cells such as fibroblasts, keratinocytes or mesenchymal stem cells. Silk fibroin is a promising polymer to realize scaffolds, since it is biocompatible, biodegradable, and exhibits excellent mechanical properties in terms of tensile strength. Moreover, fibroin can be added of others components in order to modify the biomaterial properties for the purpose. The aim of this work is to prepare silk fibroin films for adipose-derived stem cell (ADSCs) culture as a novel feeder layer for skin tissue engineering. Pectin has been added to promote the protein conformational transition and construct strength, while glycerol as plasticizer, providing biomaterial flexibility. Eighteen formulations were prepared by casting method using fibroin, pectin (range 1-10% w/w), and glycerol (range 0-20% w/w); films were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry assay, to select the optimal composition. A stable fibroin conformation was obtained using 6% w/w pectin, and the best mechanical properties were obtained using 12% w/w glycerol. Films were sterilized, and human ADSCs were seeded and cultured for 15 days. Cells adhere to the support assuming a fibroblastic-like shape and reaching confluence. The ultrastructural analysis evidences typical active-cell features and adhesion structures that promote cell anchorage to the film, thus developing a multilayered cell structure. This construct could be advantageously employed in cutaneous wound healing or where the use of ADSCs scaffold is indicated either in human or veterinary field.
Tissue regeneration using progenitor cell-based therapy has the potential to aid in the healing of a diverse range of pathologies, ranging from short-gut syndrome to spinal cord lesions. However, there are numerous hurdles to be overcome prior to the widespread application of these cells in the clinical setting. One of the primary barriers to effective stem cell therapy is the hostile environment that progenitor cells encounter in the clinical injury wound setting. In order to promote cellular survival, stem cell differentiation, and participation in tissue regeneration, relevant cells and delivery scaffolds must be paired with strategies to prevent cell death to ensure that these cells can survive to form de novo tissue. The Bcl-2 protein is a prosurvival member of a family of proteins that regulate the mitochondrial pathway of apoptosis. Using several strategies to overexpress the Bcl-2 protein, we demonstrated a decrease in the mediators of apoptosis in vitro and in vivo. This was shown through the use of two different clinical tissue repair models. Cells overexpressing Bcl-2 not only survived within the wound environment at a statistically significantly higher rate than control cells, but also increased tissue regeneration. Finally, we used a nonintegrating minicircle technology to achieve this in a potentially clinically applicable strategy for stem cell therapy.
Background aims:
Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. However, a characterization of the population of adipose-derived stromal and stem cells (ASCs) with the greatest therapeutic potential remains unclear. Under the authority of International Federation of Adipose Therapeutics and International Society for Cellular Therapy, this paper sets out to establish minimal definitions of stromal cells both as uncultured stromal vascular fraction (SVF) and as an adherent stromal/stem cells population.
Methods:
Phenotypic and functional criteria for the identification of adipose-derived cells were drawn from the literature.
Results:
In the SVF, cells are identified phenotypically by the following markers: CD45-CD235a-CD31-CD34+. Added value may be provided by both a viability marker and the following surface antigens: CD13, CD73, CD90 and CD105. The fibroblastoid colony-forming unit assay permits the evaluation of progenitor frequency in the SVF population. In culture, ASCs retain markers in common with other mesenchymal stromal/stem cells (MSCs), including CD90, CD73, CD105, and CD44 and remain negative for CD45 and CD31. They can be distinguished from bone-marrow-derived MSCs by their positivity for CD36 and negativity for CD106. The CFU-F assay is recommended to calculate population doublings capacity of ASCs. The adipocytic, chondroblastic and osteoblastic differentiation assays serve to complete the cell identification and potency assessment in conjunction with a quantitative evaluation of the differentiation either biochemically or by reverse transcription polymerase chain reaction.
Conclusions:
The goal of this paper is to provide initial guidance for the scientific community working with adipose-derived cells and to facilitate development of international standards based on reproducible parameters.
Wound healing requires a coordinated interplay among cells, growth factors, and extracellular matrix proteins. Central to this process is the endogenous mesenchymal stem cell (MSC), which coordinates the repair response by recruiting other host cells and secreting growth factors and matrix proteins. MSCs are self-renewing multipotent stem cells that can differentiate into various lineages of mesenchymal origin such as bone, cartilage, tendon, and fat. In addition to multilineage differentiation capacity, MSCs regulate immune response and inflammation and possess powerful tissue protective and reparative mechanisms, making these cells attractive for treatment of different diseases. The beneficial effect of exogenous MSCs on wound healing was observed in a variety of animal models and in reported clinical cases. Specifically, they have been successfully used to treat chronic wounds and stimulate stalled healing processes. Recent studies revealed that human placental membranes are a rich source of MSCs for tissue regeneration and repair. This review provides a concise summary of current knowledge of biological properties of MSCs and describes the use of MSCs for wound healing. In particular, the scope of this review focuses on the role MSCs have in each phase of the wound-healing process. In addition, characterization of MSCs containing skin substitutes is described, demonstrating the presence of key growth factors and cytokines uniquely suited to aid in wound repair.
Angiogenesis is essential to wound repair, and vascular endothelial growth factor (VEGF) is a potent factor to stimulate angiogenesis. Here, we examine the potential of VEGF-overexpressing adipose-derived stromal cells (ASCs) for accelerating wound healing using nonviral, biodegradable polymeric vectors. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β-amino) esters (PBAE). Cells transfected using Lipofectamine 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed enhanced transfection efficiency and 12-15-fold higher VEGF production compared with cells transfected using Lipofectamine 2000 (*P < 0.05). When transplanted into a mouse wild-type excisional wound model, VEGF-overexpressing ASCs led to significantly accelerated wound healing, with full wound closure observed at 8 days compared to 10-12 days in groups treated with ASCs alone or saline control (*P < 0.05). Histology and polarized microscopy showed increased collagen deposition and more mature collagen fibers in the dermis of wound beds treated using PBAE/VEGF-modified ASCs than ASCs alone. Our results demonstrate the efficacy of using nonviral-engineered ASCs to accelerate wound healing, which may provide an alternative therapy for treating many diseases in which wound healing is impaired.Molecular Therapy (2012); doi:10.1038/mt.2012.234.
The promotion of wound healing using dermal substitutes has become increasingly widespread, but the outcomes of substitute-assisted healing remain functionally deficient. Adipose-derived stem cells (ASCs) have been investigated widely in regenerative medicine and tissue engineering, and they have the potential to enhance wound healing. In this study, we focused on investigating the effects and mechanism of ASCs combined with an acellular dermal matrix (ADM) to treat full-thickness cutaneous wounds in a murine model.
The ADM was prepared from the dorsal skin of nude mice by decellularization by treatment with trypsin followed by Triton X-100. The human ASCs were isolated and cultured from abdominal lipoaspirate. We created a rounded, 8-mm, full-thickness cutaneous wound in nude mice and divided the mice into the following 4 groups: silicon sheet cover only, silicon sheet with spreading ASCs, ADM only, and ASCs seeded on ADM. The granulation thickness was evaluated by histology after 7 days. Further comparisons between the ADM only and ASC-seeded ADM groups were undertaken by assessing the reepithelialization ratio and blood vessel density at postoperative days 9 and 14. Statistical analyses were conducted using Student 2-tailed t test. Immunofluorescent histology and ASC labeling were also performed to identify possible mechanisms.
The ADM was successfully prepared, and the cytometry analysis and differentiation assay provided the characterization of the human ASCs. A marked improvement in granulation thickness was detected in the ADM-ASC group in comparison with other 3 groups. A significantly increased rate of reepithelialization in the ADM-ASC group (80 ± 6%) compared to the ADM only group (60 ± 7%) was noted on postoperative day 9. The blood vessel density was evidently increased in the ADM-ASC group (7.79 ± 0.40 vessels per field) compared to the ADM only group (5.66 ± 0.23 vessels) on day 14. Cell tracking experiments demonstrated that labeled ASCs were colocalized with staining for VEGF or endothelial cell maker vWF after the transplantation of ADM-ASCs on postoperative day 14.
Adipose-derived stem cells seeded on an ADM can enhance wound healing, promote angiogenesis, and contribute to newly formed vasculature, and VEGF-expressing ASCs can be detected after transplantation. This model could be used to improve the other clinical applications of ASCs and to decipher the detailed mechanism by which ASCs interact with wound tissue.
Cell-based therapeutic intervention has emerged as a new approach to accelerate wound closure. Adipose-derived stem cells (ASCs), as a fascinating cell source, have received much attention in tissue repair and regeneration. In this study we evaluated the potential of acellular dermal matrix (ADM) scaffold serving as a carrier for the delivery of ASCs and investigated its therapeutic effects on wound healing. First, ASCs were isolated and characterized for multidifferentiation potential. ASCs-ADM grafts were then prepared, and ADM scaffold was shown to support the in vitro growth and proliferation of ASCs. Next, we analysed paracrine factors in conditioned medium and found that ASCs-ADM grafts secreted various cytokines, including VEGF, HGF, TGFβ and bFGF. Moreover, ASCs-ADM conditioned medium notably stimulated the migration and proliferation of fibroblasts. In vivo, we established an excisional wound model in diabetic rats which received phosphate-buffered saline (PBS), ADM or ASCs-ADM grafts, respectively. Our results demonstrated that implantation of ASCs-ADM significantly enhanced tissue regeneration and increased epithelialization, resulting in accelerated wound closure. Immunofluorescence analysis further indicated that capillary density was evidently increased in the ASCs-ADM group compared with the control or ADM group. In addition, western blot analysis showed that ASCs-ADM significantly increased the expression of angiogenic factors, which was consistent with in vitro data. Taken together, our results suggest that targeted delivery of ASCs via ADM scaffold accelerate diabetic wound healing through a paracrine mechanism, with enhanced granulation tissue formation and increased re-epithelialization and neovascularization. Copyright © 2012 John Wiley & Sons, Ltd.
Effective therapy for chronic radiation injuries, such as ulcers, is prone to infection. Stiffness is expected since the therapeutic radiation often involves wider and deeper tissues and often requires extensive debridement and reconstruction, which are not sometimes appropriate for elderly and compromised hosts. Autologous adipose-derived regenerative cells (ADRCs) are highly yielding, forming relatively elderly aged consecutive 10 cases, 63.6±14.9 y (52-89 y), with mean radiation dose of 75.0±35.4 Gy (50-120 Gy) were included with at least 10-month follow-up. Minimal debridement and ADRC injection in the wound bed and margin along with the injection of mixture of fat and ADRCs in the periphery were tested for efficacy and regenerated tissue quality by clinically as well as imaging by computed tomography and magnetic resonance imaging. Uncultured ADRCs of 1.6±1.3×10(7) cells were obtained. All cases healed uneventfully after 6.6±3.2 weeks (2-10 weeks) post-operatively. The done site morbidity was negligible and without major complications, such as paralysis or massive haematoma. The regenerated tissue quality was significantly superior to the pre-operative one and the mixture of fat and ADRCs connected to the intact tissue was very soft and pliable. Mean follow-up at 1.9±0.8 y (0.9-2.9 y) revealed no recurrence or new ulceration after treatment. Thus, the ADRCs treatment for decades-long radiation injuries is effective, safe and improves the quality of wounds.
Wound healing of burned skin remains a major goal in public health. Previous reports showed that the bone marrow stem cells were potent in keratinization and vascularization of full thickness skin wounds.
In this study, mesenchymal stem cells were derived from rat adipose tissues and characterized by flowcytometry. Staining methods were used to evaluate their differentiation ability. A collagen-chitosan scaffold was prepared by freeze-drying method and crosslinked by carbodiimide-based crosslinker.
The results of immunecytochemistry and PCR experiments confirmed the adipose-derived stem cells (ASC) in differentiation to the keratinocytes under the treatment of keratinocyte growth factor. The isolated ASC were seeded on the scaffolds and implanted at the prepared wounds. The scaffolds without cells were considered as a control and implanted on the other side of the rat. Histopathological analyses confirmed the formation of new tissue on the scaffold-cell side after 14 days with the formation of dermis and epidermis.
These results indicated the capacity of ASC in differentiation to keratinocytes and also wound healing in vivo.
Werner, Sabine, and Richard Grose. Regulation of Wound Healing by Growth Factors and Cytokines. Physiol Rev 83: 835–870, 2003; 10.1152/physrev.00032.2002.—Cutaneous wound healing is a complex process involving blood clotting, inflammation, new tissue formation, and finally tissue remodeling. It is well described at the histological level, but the genes that regulate skin repair have only partially been identified. Many experimental and clinical studies have demonstrated varied, but in most cases beneficial, effects of exogenous growth factors on the healing process. However, the roles played by endogenous growth factors have remained largely unclear. Initial approaches at addressing this question focused on the expression analysis of various growth factors, cytokines, and their receptors in different wound models, with first functional data being obtained by applying neutralizing antibodies to wounds. During the past few years, the availability of genetically modified mice has allowed elucidation of the function of various genes in the healing process, and these studies have shed light onto the role of growth factors, cytokines, and their downstream effectors in wound repair. This review summarizes the results of expression studies that have been performed in rodents, pigs, and humans to localize growth factors and their receptors in skin wounds. Most importantly, we also report on genetic studies addressing the functions of endogenous growth factors in the wound repair process.
Editing a book of this nature was a simultaneously exhilarating and frightening experience. It was exhilarating to draw from cell biologists, biochemists, and molecular biologists, as well as those dermatologists, pathologists, and pul monologists who are cell biologists at heart, to author chapters. At the same time, it was frightening to ask such busy investigators to devote their precious time to writing chapters that summarize not just their own endeavors but their entire area of expertise. However, the authors assuaged our fears by enthusi astically accepting the proposal to write on specific topics despite the time burden, and to update and willingly accept our editorial comments. In the editors' view, the authors have captured the important scientific data in their respective fields, have organized the data into an understandable outline, and have applied the information to elucidating wound repair processes. The explosion of new, important discoveries in the field of wound repair and related areas as our book was developing has been very unsettling. This observation predicts obsolescence. In response to this possibility, the authors and the editors have attempted to build fundamental concepts upon existing data. Hopefully, these concepts will help provoke further experimentation to unravel the complex, interwoven processes of wound repair. The book has been organized into three parts: Inflammation, Granulation Tissue Formation, and Extracellular Matrix Production and Remodeling.
In this chapter, essential aspects of cutaneous wound repair/scar formation, including the early response and the cellular phase, will be covered. Fundamental differences between wound repair and regeneration, with emphasis on fibroblast and endothelial cell contribution to respective dermal architecture, will be discussed. 'Essential ingredients' for cutaneous regeneration will be explored in detail. Lastly, issues regarding stem cells, progenitors and cellular 'plasticity', as they relate to cutaneous wound healing, will be examined.
Adipose tissue has long been considered an energy storage and endocrine organ; however, in recent decades, this tissue has also been considered an abundant source of mesenchymal cells. Adipose-derived stem cells are easily obtained, show a strong capacity for ex vivo expansion and differentiation to other cell types, release a large variety of angiogenic factors, and have immunomodulatory properties. Thus, adipose tissue is currently the focus of considerable interest in the field of regenerative medicine. In the context of coronary heart disease, numerous experimental studies have supported the safety and efficacy of adipose-derived stem cells in the setting of myocardial infarction. These results have encouraged the clinical use of these stem cells, possibly prematurely. Indeed, the presence of cardiovascular risk factors, such as hypertension, coronary disease, diabetes mellitus, and obesity, alter and reduce the functionality of adipose-derived stem cells, putting in doubt the efficacy of their autologous implantation. In the present article, white adipose tissue is described, the stem cells found in this tissue are characterized, and the use of these cells is discussed according to the preclinical and clinical trials performed so far.
Copyright © 2015 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.
Resumen
Se ha considerado al tejido adiposo como de almacenamiento energético y como un órgano endocrino; sin embargo, en las últimas décadas se lo ha considerado como una fuente abundante de células mesenquimales. Las células madre derivadas del tejido adiposo son de fácil obtención, presentan una gran capacidad de expansión ex vivo y gran plasticidad a otros tipos celulares, liberan gran variedad de factores angiogénicos y presentan propiedades inmunomoduladoras. Por ello, actualmente constituyen un foco de gran interés en la medicina regenerativa. En el contexto de enfermedad cardiaca coronaria, múltiples estudios experimentales han avalado la seguridad y la eficacia del uso de las células madre derivadas del tejido adiposo en el contexto de infarto de miocardio. Todo ello ha promovido, quizá precozmente, su uso clínico. De hecho, se ha demostrado que la presencia de factores de riesgo cardiovascular como hipertensión, enfermedad coronaria, diabetes mellitus u obesidad, altera y merma la funcionalidad de las células madre derivadas del tejido adiposo, lo que deja en entredicho la eficacia basada en el implante de células madre derivadas del tejido adiposo autólogas. En el siguiente artículo se describe el tejido adiposo blanco, se caracterizan las células madre que lo componen y se discute sobre su uso según los estudios preclínicos y clínicos realizados hasta el momento.
Human adipose-derived mesenchymal stem cells (hASCs) are attractive cell source for skin tissue engineering. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted cluster hASC in a skin wound animal model. The hASCs were cultured in monolayer or clusters. The LLLT, hASCs, hASC clusters, and hASC clusters transplantation with LLLT (cluster + LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining, and elastin van gieson histochemistry. The survival, differentiation, and secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) of the cluster ASC were evaluated by immunohistochemistry and Western blotting. The cluster + LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the cluster group. The secretion of growth factors was stimulated in the cluster + LLLT group compared with the ASCs and cluster group. These data suggest that LLLT is an effective biostimulator of cluster hASCs in wound healing that enhances the survival of hASCs and stimulates the secretion of growth factors in the wound bed.
Copyright © 2015. Published by Elsevier Inc.
Mononuclear cells (MNCs) and mesenchymal stem cells (MSCs) derived from the bone marrow and other sources have received significant attention as donor cells for treating various neurological disorders due to their robust neuroprotective and antiinflammatory effects. Moreover, it is relatively easy to procure these cells from both autogenic and allogenic sources. Currently, there is considerable interest in examining the usefulness of these cells for conditions such as status epilepticus (SE) and chronic epilepsy. A prolonged seizure activity in SE triggers neurodegeneration in the limbic brain areas, which elicits epileptogenesis and evolves into a chronic epileptic state. Because of their potential for providing neuroprotection, diminishing inflammation and curbing epileptogenesis, early intervention with MNCs or MSCs appears attractive for treating SE as such effects may restrain the development of chronic epilepsy typified by spontaneous seizures and learning and memory impairments. Delayed administration of these cells after SE may also be useful for easing spontaneous seizures and cognitive dysfunction in chronic epilepsy. This concise review evaluates the current knowledge and outlook pertaining to MNC and MSC therapies for SE and chronic epilepsy. In the first section, the behavior of these cells in animal models of SE and their efficacy to restrain neurodegeneration, inflammation and epileptogenesis are discussed. The competence of these cells for suppressing seizures and improving cognitive function in chronic epilepsy are conferred in the next section. The final segment ponders issues that need to be addressed to pave the way for clinical application of these cells for SE and chronic epilepsy. This article is protected by copyright. All rights reserved.
© 2015 AlphaMed Press.
Infection is the likeliest single cause of delayed healing in healing of chronic open wounds by secondary intention. If neglected it can progress from contamination to colonization and local infection through to systemic infection, sepsis and multiple organ dysfunction syndrome, and it can be life-threatening. Infection in chronic wounds is not as easy to define as in acute wounds, and is complicated by the presence of biofilms. There is, as yet, no diagnostic for biofilm presence, but it contributes to excessive inflammation - through excessive and prolonged stimulation of nitric oxide, inflammatory cytokines and free radicals - and activation of immune complexes and complement, leading to a delay in healing. Control of biofilm is a key part of chronic wound management. Maintenance debridement and use of topical antimicrobials (antiseptics) are more effective than antibiotics, which should be reserved for treating spreading local and systemic infection. The continuing rise of antimicrobial resistance to antibiotics should lead us to reserve their use for these indications, as no new effective antibiotics are in the research pipeline. Antiseptics are effective through many mechanisms of action, unlike antibiotics, which makes the development of resistance to them unlikely. There is little evidence to support the theoretical risk that antiseptics select resistant pathogens. However, the use of antiseptic dressings for preventing and managing biofilm and infection progression needs further research involving well-designed, randomized controlled trials.
© 2015 British Association of Dermatologists.
Currently available skin substitutes are still associated with a range of problems including poor engraftment resulting from deficient vascularization, and excessive scar formation, among others. Trying to overcome these issues, this work proposes the combination of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) structures with adipose-derived stem cells (ASCs) to offer biomechanical and biochemical signaling cues necessary to improve wound healing in a full-thickness model. PHBV scaffold maintained the wound moisture and demonstrated enough mechanical properties to withstand wound contraction. Also, exudate and inflammatory cell infiltration enhanced the degradation of the structure, and thus healing progression. After 28days all the wounds were closed and the PHBV scaffold was completely degraded. The transplanted ASCs were detected in the wound area only at day 7, correlating with an up-regulation of VEGF and bFGF at this time point that consequently led to a significant higher vessel density in the group that received the PHBV loaded with ASCs. Subsequently, the dermis formed in the presence of the PHBV loaded with ASCs possesses a more complex collagen structure. Additionally, an anti-scarring effect was observed in the presence of the PHBV scaffold indicated by a down-regulation of TGF-β1 and α-SMA together with an increase of TGF-β3, when associated with ASCs. These results indicate that although PHBV scaffold was able to guide the wound healing process with reduced scarring, the presence of ASCs was crucial to enhance vascularization and provide a better quality neo-skin. Therefore, we can conclude that PHBV loaded with ASCs possesses the necessary bioactive cues to improve wound healing with reduced scarring.
Copyright © 2015. Published by Elsevier Ltd.
Older adults are more likely to have chronic wounds than younger people, and the effect of chronic wounds on quality of life is particularly profound in this population. Wound healing slows with age, but the basic biology underlying chronic wounds and the influence of age-associated changes on wound healing are poorly understood. Most studies have used in vitro approaches and various animal models, but observed changes translate poorly to human healing conditions. The effect of age and accompanying multimorbidity on the effectiveness of existing and emerging treatment approaches for chronic wounds is also unknown, and older adults tend to be excluded from randomized clinical trials. Poorly defined outcomes and variables; lack of standardization in data collection; and variations in the definition, measurement, and treatment of wounds also hamper clinical studies. The Association of Specialty Professors, in conjunction with the National Institute on Aging and the Wound Healing Society, held a workshop, summarized in this article, to explore the current state of knowledge and research challenges, engage investigators across disciplines, and identify research questions to guide future study of age-associated changes in chronic wound healing.
© 2015 by the American Geriatrics Society and the Wound Healing Society.
Wound healing is a complex and dynamic biological process that involves the coordinated efforts of multiple cell types and is executed and regulated by numerous growth factors and cytokines. There has been a drivein the past two decades to study the therapeutic effects of various growth factors in the clinical management of non-healing wounds (e.g. pressure ulcers, chronic venous ulcers, diabetic foot ulcers). For this review, we conducted anonline search of Medline and Pub Medicaland critically analyzed the literature regarding the role of growth factors and cytokines in the management of these wounds. Wefocused on currently approved therapies, emerging therapies and future research possibilities. In this reviewwe discuss four growth factors and cytokines currently being used on and off label for the healing of wounds. These include: granulocyte-macrophage colony stimulating factor (GM-CSF), platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF). While the clinical results of using growth factors and cytokines are encouraging, many studies involved a small sample size and are disparate in measured endpoints. Therefore, further research is required to provide definitive evidence of efficacy.
Objective: The relationship between the wound contraction and levels of α-smooth muscle actin (α-SMA) has been revealed in different studies. We aimed to investigate the effects of mesenchymal stem cells (MSCs), mainly bone-marrow-derived stem cells (BSCs) and adipose-derived stem cells (ASCs), and find out the α-SMA, fibroblast growth factor (FGF), transforming growth factor beta, and vascular endothelial growth factor (VEGF) levels on an in vivo acute wound healing model after the application of MSCs. Approach: Four circular skin defects were formed on the dorsum of Fisher rats (n=20). The defects were applied phosphate-buffered saline (PBS), ASCs, BSCs, and patchy skin graft, respectively. The healing time and scar area were noted. Results: There was a statistical decrease in the healing time in ASC, BSC, and skin graft groups (p<0.05). However, the scar was smaller in the PBS group (p<0.05). The α-SMA levels were statistically lower in ASC, BSC, and graft groups (p<0.05). The FGF levels were statistically higher in ASC and BSC groups (p<0.05). The differentiation of the injected MSCs to endothelial cells and keratinocytes was observed. Innovation and Conclusion: MSCs decrease the healing time and contraction of the wound while increasing the epithelization rate by increasing angiogenesis.
Emerging evidence has shown that adipose tissue is the richest and most accessible source of mesenchymal stem cells. Many different therapies for chronic wounds exist with varying success rates. The capacity of adipose-derived stem cells (ASCs) to promote angiogenesis, secrete growth factors, regulate the inflammatory process, and differentiate into multiple cell types makes them a potential ideal therapy for chronic wounds. The aim of this article was to review all preclinical trials using ASCs in problem wound models. A systematic search was performed and 12 studies were found where different chronic wound models across different animals were treated with ASCs. Different ASC sources and delivery methods were used in the described studies. Studies demonstrated improved wound healing with utilization of ASC, and this treatment modality has so far shown great potential. However, more preclinical studies and large-scale clinical trials are needed to show if the emerging therapy can satisfy expectations.
Actually, autologous fat grafts have many clinical applications in breast surgery, facial rejuvenation, buttock augmentation, and Romberg syndrome as well as a treatment of liposuction sequelae.
The aim of this article was to describe the preparation and isolation procedures for stromal vascular fraction (SVF), the preparation of platelet-rich plasma (PRP), and the clinical application in the treatment of the scar on the face.
Ten patients with burns sequelae (n = 6) and post-traumatic scars (n = 4) were treated with SVF-enhanced autologous fat grafts obtained by the Celution System. Another 10 patients with burns sequelae (n = 5) and post-traumatic scars (n = 5) were treated with fat grafting based on the Coleman technique mixed with 0.5 mL of PRP.To assess the effects of their treatment, the authors compared their results with those of a control group consisting of 10 patients treated with centrifuged fat.
In the patients treated with SVF-enhanced autologous fat grafts, we observed a 63% maintenance of contour restoring after 1 year compared with only 39% of the control group (n = 10) treated with centrifuged fat graft (P < 0.0001). In the patients treated with fat grafting and PRP, we observed a 69% maintenance of contour restoring after 1 year compared with that of the control group (n = 10).
Autologous fat grafting is a good method for the correction of scars on the face instead of the traditional scar surgical excision.
There is a clear clinical need for cell therapies to repair or regenerate tissue lost to disease or trauma. Adipose tissue is a renewable source of stem cells, called adipose-derived stem cells (ASCs), that release important growth factors for wound healing, modulate the immune system, decrease inflammation, and home in on injured tissues. Therefore, ASCs may offer great clinical utility in regenerative therapies for afflictions such as Parkinson's disease and Alzheimer's disease, spinal cord injury, heart disease, and rheumatoid arthritis, or for replacing lost tissue from trauma or tumor removal. This article discusses the regenerative properties of ASCs that can be harnessed for clinical applications, and explores current and future challenges for ASC clinical use. Such challenges include knowledge-based deficiencies, hurdles for translating research to the clinic, and barriers to establishing a new paradigm of medical care. Clinical experience with ASCs, ASCs as a portion of the heterogeneous stromal cell population extracted enzymatically from adipose tissue, and stromal vascular fraction are also described.
Introduction:
In the past decade human adipose tissue has been identified as a source of multipotent stem cells. Adipose tissue derived stem cells (ASCs) are characterised by immunosuppressive properties and low immunogenicity. Therefore, they can be used in regenerative medicine, as well as applied to induce graft tolerance or prevent autoimmunity. ASCs can be easily harvested with low morbidity, which is their main advantage over mesenchymal stem cells (MSCs) derived from other sources.
Areas covered:
The review focuses on reported clinical applications of ASCs and discusses technical approaches of their isolation and processing. The differences in phenotype and differentiation preferences between ASCs and other MSCs that may affect the choice of a particular cell type for the future therapy are also described.
Expert opinion:
ASCs seem to be the perfect tool for regenerative medicine and immunosuppressive cellular therapies. Nevertheless, there are some tasks that should be addressed by the future studies: i) ASCs require better characterisation; a set of markers determining ASCs should be clearly defined; ii) there is need for more studies on safety of reconstructive therapies with ASCs in cancer patients (e.g., after mastectomy); iii) release criteria should be determined for freshly isolated and ex vivo expanded ASCs designed for clinical applications.
Preexisting cirrhosis usually leads to an inadequate and delayed regeneration of the future liver remnant (FLR) after portal vein embolization (PVE). Bone marrow-derived mesenchymal stem cells (BMSC) are promising candidates for therapeutic applications in liver diseases. In this study, the efficacy of autologous BMSCs transplantation to promote FLR regeneration was investigated in a rat cirrhotic model.
Autologous BMSCs were expanded and labeled with PKH26, and then were injected immediately into nonembolized lobes after PVE through portal vein in cirrhotic rat. At 7, 14, and 28 d after this, liver weight and Ki-67 labeling index were measured, and blood analysis was performed. Cirrhotic degree of FLR was assessed by hydroxyproline content assay and histopathology. Gene expressions of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-10 (IL-10), and matrix metalloproteinase-9 (MMP-9) were detected with real-time reverse transcriptase-polymerase chain reaction. Distribution and hepatocyte differentiation of BMSCs in FLR were determined by confocal microscopy.
Autologous BMSCs significantly increased the FLR weight ratio to the total liver and the Ki-67 labeling index, and serum albumin levels were significantly higher and total bilirubin levels were significantly lower in the BMSCs group compared with the controls without BMSCs transplantation 14 and 28 d post-PVE. BMSCs significantly decreased the hydroxyproline content and collagen accumulation, up-regulated the expressions of HGF, IL-10, VEGF, and MMP-9 28 d post-PVE, and expressed hepatocyte-specific markers, such as α-fetoprotein, cytokeratin 18, and albumin in a time-dependent manner in FLR.
Autologous BMSCs can differentiate into hepatocyte and promote FLR regeneration after PVE in cirrhotic liver, which may be through improving local microenvironment by decreasing cirrhosis, up-regulating the gene expressions of VEGF, HGF, IL-10, and MMP-9.
Adult peripheral blood contains a limited number of endothelial progenitor cells that can be isolated for treatment of ischemic diseases. The adipose tissue became an interesting source of stem cells for regenerative medicine. This study aimed to investigate the phenotype of cells obtained by culturing adipose-derived mesenchymal stem cells (ad-MSCs) in the presence of endothelial growth supplements compared to endothelial cells obtained from umbilical cord blood (UCB). Passage 3 ad-MSCs and mononuclear layer from UCB were cultured in presence of endothelial growth media for 3 weeks followed by their characterization by flow cytometry and polymerase chain reaction. After culture in endothelial inductive media, ad-MSCs expressed endothelial genes and some endothelial marker proteins as CD31 and CD34, respectively. Adipose tissue could be a reliable source for easy obtaining, expanding and differentiating MSCs into endothelial-like cells for autologous cell-based therapy.
Background:
Diabetes can lead to impaired wound healing and skin grafts used surgically for diabetic wounds are often complicated with necrosis, although different therapies have been proposed. Adipose-derived stem cells (ASCs) participate in tissue repair processes and may have a role during impaired wound healing. In this study, autologous transplantation of ASCs was used to determine if it increases angiogenesis and skin graft survival and enhances wound healing in diabetic rats.
Methods:
Adipose-derived stem cells were successfully isolated and cultured. A full-thickness skin graft model was used to determine the effects of locally administered ASCs in 10 rats rendered diabetic (group 1), whereas 10 others served as controls (group 2). Histological examination of skin grafts followed after 1 week. Additionally, immunohistochemical staining intensity of vascular endothelial growth factor (VEGF) and transforming growth factor β3 (TGF-β3) was assessed in all grafts.
Results:
The gross and histological results showed significantly increased survival, angiogenesis, and epithelialization. Mean area of graft necrosis was significantly less in group 1 than in group 2 (7.49% vs 39.67%, P < 0.001). Statistically significant increase of capillary density, collagen intensity, VEGF, and TGF-β3 expression was noted in group 1 compared with group 2.
Conclusions:
These findings suggest that autologous ASC transplantation can enhance skin graft survival in diabetic rats through differentiation, vasculogenesis, and secretion of growth factors such as VEGF and TGF-β3. This might represent a novel therapeutic approach in skin graft surgery for diabetic wounds.
Diabetic wound is one of the most serious complications of diabetes mellitus. There are no significantly effective therapies for chronic non-healing diabetes ulcer so far. This study aimed to explore the feasibility of healing impaired wound using artificial dermis constructed with human adipose derived stem cells (ASCs) and poly (L-glutamic acid)/chitosan (PLGA/CS) scaffold in streptozotocin-induced diabetic mice.
ASCs were isolated from fresh human lipoaspirates and expanded ex vivo for three passages, and then cells were seeded onto PLGA/CS scaffold to form artificial dermis. Expression of VEGF and TGFβ1 by ASCs presented in artificial dermis was determined. The artificial dermis was transplanted to treat the 20 mm × 20 mm full-thickness cutaneous wound created on the back of diabetic mice. Wound treated with scaffold alone and without treatment, and wound in normal non-diabetic mice served as control.
Cells growing within scaffold showed great proliferation potential, depositing abundant collagen matrix. Meanwhile, expression of VEGF and TGF-β1 by seeded ASCs maintained at a consistent high level. After treated with ASC based artificial dermis, diabetic wounds exhibited significantly higher healing rate compared with wounds treated with scaffold alone or without treatment. Histological examination also demonstrated an improvement in cutaneous restoration with matrix deposition and organization. Further quantitative analysis showed that there was a significant increase in dermis thickness and collagen content on artificial dermis treated wounds.
ASC/PLGA artificial dermis can effectively accelerate diabetic wound healing by promoting angiogenic growth factors and dermal collagen synthesis.
Understanding the mechanisms that direct mesenchymal stem cell (MSC) self-renewal fate decisions is key to most tissue regenerative approaches. The aim of the study here was to investigate the mechanisms of action of platelet-derived growth factor receptor β (PDGFRβ) signalling on MSC proliferation and differentiation. MSC were cultured and stimulated with PDGF-BB together with inhibitors of 2(nd) messenger pathways. Cell proliferation was assessed using ethynyl-2' -deoxyuridine and phosphorylation status of signalling molecules assessed by Western Blots. To assess differentiation potentials, cells were transferred to adipogenic or osteogenic media, and differentiation assessed by expression of differentiation association genes by qRT-PCR, and by long term culture assays. Our results showed that distinct pathways with opposing actions were activated by PDGF. PI3K /Akt signalling was the main contributor to MSC proliferation in response to activation of PDGFRβ. We also demonstrate a negative feedback mechanism between PI3K /Akt and PDGFR-β expression. In addition PI3K/Akt downstream signal cascades, mTOR and its associated proteins p70S6K and 4E-BP1, were involved. These pathways induced the expression of cyclin D1, cyclin D3 and CDK6 to promote cell cycle progression and MSC proliferation. In contrast, activation of Erk by PDGFRβ signalling potently inhibited the adipocytic differentiation of MSCs by blocking PPARγ and CEBPα expression. The data suggest that PDGFRβ-induced Akt and Erk pathways regulate opposing fate decisions of proliferation and differentiation to promote MSC self-renewal. Thus, activation of multiple intracellular cascades is required for successful and sustainable MSC self-renewal strategies. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
Objective
To characterize mesenchymal stem cells (MSCs) from human synovial membrane (SM).Methods
Cell populations were enzymatically released from the SM obtained from knee joints of adult human donors and were expanded in monolayer with serial passages at confluence. Cell clones were obtained by limiting dilution. At different passages, SM-derived cells were subjected to in vitro assays to investigate their multilineage potential. Upon treatments, phenotypes of cell cultures were analyzed by histo- and immunohistochemistry and by semiquantitative reverse transcription–polymerase chain reaction for the expression of lineage-related marker genes.ResultsSM-derived cells could be expanded extensively in monolayer, with limited senescence. Under appropriate culture conditions, SM-derived cells were induced to differentiate to the chondrocyte, osteocyte, and adipocyte lineages. Sporadic myogenesis was also observed. Five independent cell clones displayed multilineage potential. Interestingly, only 1 clone was myogenic. Donor age, cell passaging, and cryopreservation did not affect the multilineage potential of SM-derived cells. In contrast, normal dermal fibroblasts under the same culture conditions did not display this potential.Conclusion
Our study demonstrates that human multipotent MSCs can be isolated from the SM of knee joints. These cells have the ability to proliferate extensively in culture, and they maintain their multilineage differentiation potential in vitro, establishing their progenitor cell nature. SM-derived MSCs may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.