Comparative Analysis of Human Adipose-Derived Stromal/Stem Cells and Dermal Fibroblasts.

Dermal fibroblasts (DFs) share several qualities with mesenchymal stem cell/multipotent stromal cells (MSCs) derived from various tissues, including adipose-derived stromal/stem cells (ASCs). ASCs and DFs are morphologically comparable and both cell types can be culture expanded through the utilization of their plastic-adherence properties. Despite these similar characteristics, numerous studies indicate that ASC and DF display distinct therapeutic benefits in clinical applications. To more accurately distinguish between these cell types, human DFs and ASCs isolated from three individual donors were analyzed for multipotency and cell surface marker expressions. The detection of cell surface markers, CD29, CD34, CD44, CD73, CD90, and CD105, were used for phenotypic characterization of the DFs and ASCs. Furthermore, both cell types underwent lineage differentiation based on histochemical staining and the expression of adipogenic related genes, CCAAT/Enhancer-Binding Protein alpha (CEBPα), Peroxisome proliferator-activated receptor gamma (PPARγ), UCP1, Leptin (LEP), and Adiponectin (ADIPOQ); and osteogenic related genes, Runt related transcription factor 2 (Runx2), Alkaline phosphatase (ALPL), Osteocalcin (OCN), and Osteopontin (OPN). Evidence provided by this study demonstrates similarities between donor-matched ASC and DF with respect to morphology, surface marker expression, differentiation potential, and gene expression, although appearance of enhanced adipogenesis in the ASC based solely on spectrophotometric analyses with no significant difference in real-time polymerase chain reaction detection of adipogenic biomarkers. Thus, there is substantial overlap between the ASC and DF phenotypes based on biochemical and differentiation metrics.

Acellular dermal matrix in skin wound healing in rabbits - histological and histomorphometric analyses.

OBJECTIVES: To analyze the histology and histomorphometry of healing associated with acellular dermal matrix in skin wounds in rabbits. METHODS: Twelve male rabbits were divided into two groups: the control group (CG) and the matrix group (MG). Three skin wounds with a total area of 20 × 20 mm were created on the dorsal region of each animal. Photographic records of the lesions taken over a 21-day period and use of the ImageJ program allowed calculation of the wound contraction rate. The lesions were biopsied on days 3, 14 and 21 for histomorphometric analysis to define the thicknesses of the dermis and epidermis (hematoxylin-eosin) and calculate the densities of type I and type III collagen (picrosirius). RESULTS: No significant difference in the healing rate was found between the groups (p>0.05). The MG presented greater epidermal thickness on day 3 (p<0.05) and on days 14 and 21 (p<0.001). The MG presented greater dermal thickness throughout the study period (p<0.05). The type I collagen density was higher in the MG throughout the study period (p<0.05), and the type III collagen density was higher in the MG on days 3 and 14 (p<0.05) and on day 21 (p<0.001). CONCLUSION: The use of acellular dermal matrix increased the thickness of the dermal and epidermal layers and the amount of type I and III collagen during skin wound healing and did not alter the rate of wound contraction.

Acellular dermal matrix in skin wound healing in rabbits - histological and histomorphometric analyses

OBJECTIVES: To analyze the histology and histomorphometry of healing associated with acellular dermal matrix in skin wounds in rabbits. METHODS: Twelve male rabbits were divided into two groups: the control group (CG) and the matrix group (MG). Three skin wounds with a total area of 20 × 20 mm were created on the dorsal region of each animal. Photographic records of the lesions taken over a 21-day period and use of the ImageJ program allowed calculation of the wound contraction rate. The lesions were biopsied on days 3, 14 and 21 for histomorphometric analysis to define the thicknesses of the dermis and epidermis (hematoxylin-eosin) and calculate the densities of type I and type III collagen (picrosirius). RESULTS: No significant difference in the healing rate was found between the groups (p>0.05). The MG presented greater epidermal thickness on day 3 (p<0.05) and on days 14 and 21 (p<0.001). The MG presented greater dermal thickness throughout the study period (p<0.05). The type I collagen density was higher in the MG throughout the study period (p<0.05), and the type III collagen density was higher in the MG on days 3 and 14 (p<0.05) and on day 21 (p<0.001). CONCLUSION: The use of acellular dermal matrix increased the thickness of the dermal and epidermal layers and the amount of type I and III collagen during skin wound healing and did not alter the rate of wound contraction.

Comparative proteomic analyses of human adipose extracellular matrices decellularized using alternative procedures.

Decellularized human adipose tissue has potential clinical utility as a processed biological scaffold for soft tissue cosmesis, grafting, and reconstruction. Adipose tissue decellularization has been accomplished using enzymatic-, detergent-, and/or solvent-based methods. To examine the hypothesis that distinct decellularization processes may yield scaffolds with differing compositions, the current study employed mass spectrometry to compare the proteomes of human adipose-derived matrices generated through three independent methods combining enzymatic-, detergent-, and/or solvent-based steps. In addition to protein content, bioscaffolds were evaluated for deoxyribose nucleic acid depletion, extracellular matrix composition, and physical structure using optical density, histochemical staining, and scanning electron microscopy. Mass spectrometry based proteomic analyses identified 25 proteins (having at least two peptide sequences detected) in the scaffolds generated with an enzymatic approach, 143 with the detergent approach, and 102 with the solvent approach, as compared to 155 detected in unprocessed native human fat. Immunohistochemical detection confirmed the presence of the structural proteins actin, collagen type VI, fibrillin, laminin, and vimentin. Subsequent in vivo analysis of the predominantly enzymatic- and detergent-based decellularized scaffolds following subcutaneous implantation in GFP+ transgenic mice demonstrated that the matrices generated with both approaches supported the ingrowth of host-derived adipocyte progenitors and vasculature in a time dependent manner. Together, these results determine that decellularization methods influence the protein composition of adipose tissue-derived bioscaffolds. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2481-2493, 2018.

Effect of Cryopreservation on Human Adipose Tissue and Isolated Stromal Vascular Fraction Cells: In Vitro and In Vivo Analyses.

BACKGROUND: Adipose tissue is a source of adipose-derived stromal/stem cells for tissue engineering and reconstruction and a tissue source for fat grafts. Although liposuction is a simple procedure for the harvest of adipose tissue, the repetition of this surgical intervention can cause adverse effects to the patient and can be a limiting factor for immediate use. Cryopreservation can avoid the morbidity associated with repetitive liposuction, allowing the use of stored tissue after the initial harvest procedure. This article focuses on the characterization of fresh and cryopreserved human adipose tissue. METHODS: Lipoaspirates from eight donors were processed as fresh adipose tissue or cryopreserved for 4 to 6 weeks. Fresh and cryopreserved tissues were collagenase digested and the stromal vascular fraction cells were characterized immediately or cryopreserved. Characterization was based on stromal vascular fraction cell proliferation and immunophenotype. In vivo fat grafting was performed in C57BL/6 green fluorescent protein mice to analyze morphology of the tissue and its adiposity using confocal microscopy, histochemical staining (i.e., hematoxylin and eosin and Masson trichrome), and immunohistochemistry (i.e., green fluorescent protein, perilipin, and CD31). RESULTS: Although tissue and stromal vascular fraction cell cryopreservation reduced the total cell yield, the remaining viable cells retained their adhesive and proliferative properties. The stromal vascular fraction cell immunophenotype showed a significant reduction in the hematopoietic surface markers and increased expression of stromal and adipogenic markers following cryopreservation. In vivo cryopreserved fat grafts showed morphology similar to that of freshly implanted fat grafts. CONCLUSION: In this study, the authors demonstrated that cryopreserved adipose tissue is a potential source of stromal vascular fraction cells and a suitable source for fat grafts.

Adipose Derived Cells and Tissues for Regenerative Medicine.

Adipose tissue is now recognized as a complex organ serving endocrine, immune, and metabolic functions. Adipose depots are composed of mature adipocytes as well as stromal vascular fraction (SVF) cells, a heterogeneous population of B and T lymphocytes, endothelial cells, macrophages, pericytes, smooth muscle, and stromal cells that can be isolated by enzymatic digestion. When placed into culture medium, a subset of the SVF cells can adhere to the plastic surface and expand in number. This latter population, known as "adipose-derived stromal/stem cells" (ASC), exhibits trilineage (adipo-, chondro-, osteo-) differentiation potential. There are currently more than 180 clinical studies underway worldwide exploring the regenerative medical application of SVF cells and ASC in a range of medical conditions. Plastic surgeons have a particular interest in the use of autologous fat and SVF enhanced fat for cosmetic and reconstructive surgical procedures. Orthopedic and craniofacial surgeons have begun to use ASC to treat bone and musculoskeletal defects with success. Furthermore, studies are underway to exploit the immunomodulatory function of ASC to treat immune-mediated disorders such as Crohn's disease. Indeed, it is postulated that adipose tissue and cells modulate tissue regeneration and inflammatory responses through their secretion of paracrine factors. Continued advances in this emerging field will require harmonization of international standards and guidelines defining the release criteria as well as the safety and efficacy of adipose-derived cells and tissues. Currently, there are no accepted standard guidelines for autologous fat harvesting technique, processing, or method of injection. Close collaboration between academia, industry, and regulatory authorities will be necessary to ensure that adipose-derived products are affordable and quality controlled throughout the globe.

Cryopreserved Adipose Tissue-Derived Stromal/Stem Cells: Potential for Applications in Clinic and Therapy.

Adipose-Derived Stromal/Stem Cells (ASC) have considerable potential for regenerative medicine due to their abilities to proliferate, differentiate into multiple cell lineages, high cell yield, relative ease of acquisition, and almost no ethical concerns since they are derived from adult tissue. Storage of ASC by cryopreservation has been well described that maintains high cell yield and viability, stable immunophenotype, and robust differentiation potential post-thaw. This ability is crucial for banking research and for clinical therapeutic purposes that avoid the morbidity related to repetitive liposuction tissue harvests. ASC secrete various biomolecules such as cytokines which are reported to have immunomodulatory properties and therapeutic potential to reverse symptoms of multiple degenerative diseases/disorders. Nevertheless, safety regarding the use of these cells clinically is still under investigation. This chapter focuses on the different aspects of cryopreserved ASC and the methods to evaluate their functionality for future clinical use.

Capsular Weakness around Breast Implant: A Non-Recognized Complication.

Capsular contraction is a frequent complication following breast augmentation. On the other hand, capsular weakness, a not widely recognized complication, may occur around the implant. A weak capsule allows the migration of the prosthesis to the lateral region of the thoracic region or inferiorly, towards the abdomen, due to gravitational forces. The cause of capsular weakness remains unresolved. Implant malposition, with lateral or downward displacement, breast asymmetry, improper contour, with implants moving in the pocket that compromise the aesthetic outcome of breast augmentation and require surgical correction may be different symptoms from the same clinical problem. Capsular weakness is a short or mid-term complication of breast augmentation. Most techniques aim to correct the malposition by making sutures to increase the resistance to the displacement of the implant, rearrange the structures using the capsule as flaps to remodel the envelope of the new pocket, obtaining a more stable and reliable result. In this article, four cases of displacement of breast prosthesis with capsular weakness are described and the surgical treatment that included a capsulotomy and capsulorraphy is described.

The Relative Functionality of Freshly Isolated and Cryopreserved Human Adipose-Derived Stromal/Stem Cells.

The capability of multipotent mesenchymal stem cells to maintain cell viability, phenotype and differentiation ability upon thawing is critical if they are to be banked and used for future therapeutic purposes. In the present study, we examined the effect of 9-10 months of cryostorage on the morphology, immunophenotype, colony-forming unit (CFU) and differentiation capacity of fresh and cryopreserved human adipose-derived stromal/stem cells (ASCs) from the same donors. Cryopreservation did not reduce the CFU frequency and the expression levels of CD29, CD73, CD90 and CD105 remained unchanged with the exception of CD34 and CD45; however, the differentiation capacity of cryopreserved ASCs relative to fresh cells was significantly reduced. While our findings suggest that future studies are warranted to improve cryopreservation methods and agents, cryopreserved ASCs retain sufficient features to ensure their practical utility for both research and clinical applications.