Autologous Cryopreserved Adipose Tissue Using an Innovative Technique: An In Vitro Biological Characterization.

BACKGROUND: The use of autologous adipose tissue transplantation in plastic and orthopedic surgery such as breast, reconstructions and intra-articular injection, has become an attractive surgical treatment with satisfactory clinical outcomes. Nevertheless, repeated liposuctions necessary to harvest fatty tissue normally performed with sedation or general anesthesia, may represent a noteworthy concern. OBJECTIVES: To demonstrate through an in vivo characterization, the validity of the surgical option to use cryopreserved autologous adipose tissue harvested in a single shot for repeated graft transfer, in breast reconstruction, without impairment of cell viability and sterility. METHODS: Adipose tissue is collected as a standard liposuction from patients who need numerous fat grafting procedures for breast reconstruction. According to an innovative and patented cryopreservation method, autologous adipose tissue is subsequently fractioned in a sterile bag system and frozen at RER Tissue Bank of the Emilia Romagna Region. Each graft is evaluated for sterility and cell viability immediately after harvesting and 1, 3, 6, 12 and preliminarily 18 months after cryopreservation and thawing. RESULTS: In vitro results showed that after processing, middle, and long-term cryopreservation and subsequent thawing, autologous cryopreserved adipose tissue, retains absence of bacterial contamination, high cellular viability and unmodified histomorphological properties, thereby ensuring the maintenance of the stromal vascular niche and the filling properties in multi-step different surgical procedures. CONCLUSIONS: In vitro study and sterility assessment, showed that autologous cryopreserved adipose tissue grafting is a safe procedure able to avoid multiple liposuction surgery. No impairment of either sterility, cell viability, and morphology were observed over time.

Regen Fat Code: A Standardized Protocol for Facial Volumetry and Rejuvenation.

BACKGROUND: Facial aging is a degenerative process that impairs contour and angle prominence. Rejuvenation is based on tissue replacement, volumization of the atrophic areas, and improving flaccidity and cutaneous photoaging. OBJECTIVES: The aim of this study was to apply structural fat grafting to manage volumetric deficits of the face, following a new systematic protocol called "Regen Fat Code" (RF Code) that was created to standardize structural lipotransfer methods. METHODS: This is a prospective clinical trial involving 80 healthy candidates for facial rejuvenation who were split into 2 groups. Group A underwent only structural lipotransfer; Group B underwent replacement of deep facial structures by face-lifting plus structural lipotransfer. Structural lipotransfer followed the protocol "RF Code" and 3 clinical tools were adopted for pre- and postoperative facial volumetric analysis. RESULTS: Total volume (mL) of lipotransfer in Groups A and B ranged between 1 and 20 mL (mean [standard deviation], 12 [5] mL), distributed to the different areas as follows: nasolabial fold, 3.32 [0.92] mL; superior lip, 2.0 [0.62] mL; inferior lip, 2.76 [0.71] mL; malar, 8.51 [5.25] mL; inferior eyelid, 1.2 [0.54] mL; and chin, 7.18 [1.99] mL. Areas with less mobility showed a lower absorption index than dynamic areas. CONCLUSIONS: The development of the RF Code protocol demonstrated the potential of grouping many parameters based on the lipotransfer method used to volumize and regenerate atrophic areas of the face. The protocol is easy to apply, and allows different volumizing and regenerative effects to be proposed, according to the demands of each surgical area.

Negative Pressure From an Internal Spiral Tissue Expander Generates New Subcutaneous Adipose Tissue in an In Vivo Animal Model.

BACKGROUND: Tissue expanders are widely utilized in plastic surgery. Traditional expanders usually are "inflatable balloons," which are planned to grow additional skin and/or to create space to be filled, for example, with an implant. In very recent years, reports suggest that negative pressure created by an external device (ie, Brava) induces both skin expansion and adipogenesis. OBJECTIVES: The authors evaluated and assessed the adipogenetic potential of a novel internal tissue expander in an in vivo animal model. METHODS: New Zealand female rabbits were enrolled in the study. A prototype spiral inner tissue expander was employed. It consisted of a-dynamic conic expander (DCE) with a valve at the end: when empty, it is flat (Archimedean spiral), whereas when filled with a fluid, it takes a conic shape. Inside the conic spiral, a negative pressure is therefore created. DCE is implanted flat under the latissimus dorsi muscle in experimental animals (rabbit) and then filled to reach the conical shape. Animals were investigated with magnetic resonance imaging, histology, and transmission electronic microscopy at 3, 6, and 12 months. RESULTS: Magnetic resonance imaging revealed a marked increase in newly formed adipose tissue, reaching its highest amount at 12 months after the DCE implantation. Histology confirmed the existence of new adipocytes, whereas transmission electronic microscopy ultrastructure confirmed that most of these new cells were mature adipocytes. CONCLUSIONS: Tensile stress, associated with negative-pressure expanders, generated newly white subcutaneous adipose tissue.

Biological Morphogenetic Surgery: A Minimally Invasive Procedure to Address Different Biological Mechanisms.

We present a methodology called biological morphogenetic surgery (BMS) that can recover (enlarge or reduce) the shape/volume of anatomic structures/tissues affected by congenital or acquired malformations based on a minimally invasive procedure. This emerges as a new concept in which the main task of surgery is the biological modulation of different remodeling and repair mechanisms. When applied, for example, to a tuberous breast deformity, the "enlarging BMS" expands the retracted tissue surrounding the gland through a cutting tip of a needle being inserted through small incisions percutaneously, accounting for the biological activity of the grafted fat. The obtained spaces might be spontaneously occupied and later filled with autologous grafted fat, which promotes tissue expansion by eliciting adipogenesis and preventing fibrosis. The "reducing BMS" creates an interruption of the contact between the derma and the hypoderma of the abnormally large areola and then promotes adipocytes to induce a fibrotic reaction, leading to areola reduction. Current evidence suggests that BMS might induce a bivalent mesenchymalization of the adipocyte, which promotes either new adipogenesis and angiogenesis of local fat (expanding BMS) or the granulation tissue/fibrotic response (reducing BMS), thus leading to the physiological recovery of the affected structures/tissues to normality. Level of Evidence: 4.

Effect of Use of Platelet-Rich Plasma (PRP) in Skin with Intrinsic Aging Process.

BACKGROUND: In previous papers, we demonstrated that the treatment of human photoaged skin with stromal-vascular fraction-enriched fat or expanded adipose-derived stem cells showed a decrease of elastosis and the appearance of new oxytalan elastic fibers in dermis and an increase in the vascular network. The utilization of fat plus platelet-rich plasma (PRP) led to an increase in the vascular permeability and reactivity of the nervous component. OBJECTIVES: The purpose of this study was to analyze the histologic and ultrastructural changes of human skin after the injection of only PRP in the retroauricular area that was not exposed to sun and did not present the photoaging process, in comparison with our previous results. METHODS: This study was performed in 13 patients who were candidates for facelift and whose ages ranged between 45 and 65 years. The PRP injection was performed in the mastoidea area. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron microscopy. RESULTS: After the injection of PRP, we observed an increase of reticular dermis thickness because of the deposition of elastic fibers and collagen, with a fibrotic aspect. A modified pattern of adipose tissue was also found at the dermohypodermal junction. Significative regenerative aspects were not found at histologic and ultrastructural analysis. The presence of foci of moderate inflammation and microangiopathy were observed. CONCLUSIONS: Treatment with PRP increased reticular dermis thickness with a fibrotic aspect. In the long term, the presence of inflammation and microangiopathy caused by PRP injection could lead to trophic alteration of the skin and the precocious aging process.

Mechanical Supplementation With the Stromal Vascular Fraction Yields Improved Volume Retention in Facial Lipotransfer: A 1-Year Comparative Study.

Background: Unpredictable volume maintenance in the long term is a major limitation of autologous fat grafting. Objectives: The authors compared results of autologous lipotransfer to the face with or without enrichment of fat with the stromal vascular fraction (SVF). Methods: Thirty patients with asymmetric depletion of facial volume were included in a prospective study. Patients were randomly assigned to undergo a single session of autologous fat transfer with washed adipose tissue (control group) or with washed adipose tissue combined with the pellet of centrifuged lipoaspirate, which contained the SVF (enriched group). Patients were evaluated clinically and from photographs. A subset of 5 patients in each group underwent computed tomography (CT) preoperatively and 12-months postoperatively for quantitative assessment of graft retention. Washed and fractionated lipoaspirates were evaluated histochemically and with flow cytometry to determine relative abundances of viable cells. Results: No major complications occurred. CT findings 12 months postoperatively indicated that patients who received SVF-enriched fat had significantly better volume retention (9.6% volume loss vs 24% in the control group; P = 0.013). Independent surgeons more frequently rated long-term aesthetic outcomes as "excellent" for patients in the enriched group (82.5% vs 47.6% for control group). Laboratory results indicated that each pellet contained approximately 16,000 intact adipose-derived stem cells. Conclusions: Lipotransfer with SVF-enriched adipose tissue is safe and associated with improved volume retention, compared with transplantation of unenriched fat. The SVF can be dissociated from lipoaspirate by centrifugation to yield a large quantity of viable regenerative cells, without enzymatic digestion. Level of Evidence: 2.

Expanded Stem Cells, Stromal-Vascular Fraction, and Platelet-Rich Plasma Enriched Fat: Comparing Results of Different Facial Rejuvenation Approaches in a Clinical Trial.

BACKGROUND: In a previous study, the authors demonstrated that treatment with expanded adipose-derived stem cells or stromal vascular fraction (SVF)-enriched fat modify the pattern of the dermis in human beings, representing a skin rejuvenation effect. Considering that expanded stem cells require a cell factor, the authors wanted to assess similar results by replacing them with platelet-rich plasma (PRP), which is easier to obtain and for which an empirical regenerative effect has been already described. OBJECTIVES: To determine if PRP injection could replace the cutaneous regenerative effect of adipose-derived stem cells. METHODS: This study was performed in 13 patients who were candidates for facelift. The patients underwent sampling of fat by liposuction from the abdomen and submitted to one of three protocols: injection of SVF-enriched fat or expanded adipose-derived stem cells or fat plus PRP in the preauricular areas. Fragments of skin were removed before and 3 months after treatment and analyzed by optical and electron microscopy. RESULTS: The use of fat plus PRP led to the presence of more pronounced inflammatory infiltrates and a greater vascular reactivity, increasing in vascular permeability and a certain reactivity of the nervous component. The addition of PRP did not improve the regenerative effect. CONCLUSION: The use of PRP did not have significant advantages in skin rejuvenation over the use of expanded adipose-derived stem cells or SVF-enriched fat. The effect of increased vascular reactivity may be useful in pathological situations in which an intense angiogenesis is desirable, such as tissular ischemia.

Cryopreserved Subcutaneous Adipose Tissue for Fat Graft.

UNLABELLED: Cryopreservation of subcutaneous white adipose tissue (sWAT) avoids multiple surgeries in patients subjected to reconstructive procedure. Fat grafts were performed subcutaneously on 26 mice treated with fresh (13 mice) or cryopreserved (13 mice) human sWAT. Cytofluorometry for CD marker expression of stem cells, differentiation capability, and in vivo survival of fat grafts were evaluated. In vitro analysis evidenced that cryopreservation did not affect the stem potential of samples. In vivo MRI showed that grafts were well preserved in 13 mice treated with fresh sWAT, whereas in 13 animals treated with thawed fat, graft volumes were strongly reduced after 1 week. Ultrastructural studies performed both on fresh and thawed specimens demonstrated that grafts performed with thawed sWAT are able to store lipids more slowly with respect to grafts performed with fresh sWAT and adipocytes maintained a multilocular appearance. Collected data demonstrated that the protocol of cryopreservation could maintain the regenerative capability of the sWAT, but the rate of reabsorption after fat grafting is higher using cryopreserved sWAT. Maintaining the stem potential of sWAT after cryopreservation is a very important aspect for reconstructive and regenerative medicine. The employment of cryopreserved sWAT represents an interesting goal for surgeons. Surely there is the necessity to improve the protocol of cryopreservation. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Influence of negative pressure on the viability of adipocytes and mesenchymal stem cell, considering the device method used to harvest fat tissue.

BACKGROUND: Transplanted adipose tissue has many applications in regenerative medicine. However, fat grafting yields unpredictable results because the fat that is transferred can suffer variable degrees of fat reabsorption. It is necessary to identify methods and maneuvers to minimize reabsorption rates and provide predictable long-term results. OBJECTIVES: Our study aimed to identify the optimal method of harvesting, as well as the optimal pressure regime for fat aspiration. The primary objective was to assess the degree of adipocyte and mesenchymal stem cell death that occurred with the various devices and pressure levels used to harvest fat. METHODS: This study was a prospective, randomized, comparative study in 15 healthy male and female subjects aged 25 to 60 who were undergoing abdominal cosmetic surgery. Various apparatuses and pressure regimens were used to harvest 8 samples of fat tissue. These samples (R1 = R8) underwent histological analysis in order to verify the integrity and functionality of the adipocytes and mesenchymal stem cells that had been harvested. RESULTS: A total of 14 females and 1 male underwent abdominal cosmetic surgery. Quantitative analysis revealed that the adipocytes in all 8 samples had homogeneous quantitative profiles. The adipose mesenchymal stem cell (AMSC) analysis, according to Friedman ANOVA, revealed no significant variation in the percentage of mesenchymal stem cells (P = .045) between the various samples. CONCLUSIONS: The type of device, nozzle diameter tip, and pressure regimen used in this study for harvesting fat tissue did not significantly affect the number of the adipocytes or viable AMSC harvested. LEVEL OF EVIDENCE: 3 Therapeutic.

Current Fat Grafting Practices and Preferences: A Survey from Members of ISPRES.

Fat grafting has established its niche in a wide spectrum of aesthetic and reconstructive procedures. A consensus-based method of harvest, processing, enrichment, injection, and assessment is lacking, despite the rising trends in its application. We conducted a survey among plastic surgeons to evaluate and identify trends of fat grafting practices. Methods: We conducted an electronic survey with a 30-item questionnaire of 62 members of the International Society of Plastic Regenerative Surgeons. We collected demographic information, techniques, and experiences related to large volume (100-200 ml) and small-volume (<100 ml) fat grafting. Results: The majority of the respondents worked predominantly as aesthetic surgeons. The donor area selection was based on the patient's fat availability (59.7%). For fat enrichment, platelet-rich-plasma and adipose stem cells were routinely used by 12.9% and 9.7% of respondents, respectively. A 3- to 4-mm cannula with three holes was the most preferred instrument for large-volume fat harvesting (69.5%). For small-volume fat grafting, 2-mm cannulas (75.8%) with Mercedes tip (27.3%) were used most commonly. For processing, decantation of fat was performed by 56.5% of respondents (without exclusivity). For handheld injections (without exclusivity), respondents preferred a 1- to 2-mm cannula with a 1 cm3 syringe. The most popular method of outcome assessment was photographic evaluation. Conclusions: The respondents' tendencies were similar to those reported in the previous literature, with some exceptions, such as the technique for preparing fat and enrichment. A wider cross-sectional survey, involving national delegates and global representatives of plastic surgeons, is anticipated.

Treatment of chronic anal fissures and associated stenosis by autologous adipose tissue transplant: a pilot study.

PURPOSE: A substantial body of recent evidence suggests that autologous adipose tissue transplant promotes healing in different lesions associated with local ischemia. We report the outcome of lipoaspirate transplant in hard-to-treat chronic anal fissures. METHODS: Eight patients were included in this pilot study, 5 with single and 3 with multiple fissures. All had intense anal pain and had previously undergone proctologic surgery, with internal sphincterotomy performed in 6 patients. Severe stenosis was present in 3 patients and moderate stenosis in 2. Preoperative assessment included anoscopy, anorectal manometry, colonoscopy, and microbiological tests to exclude inflammatory, neoplastic, or infectious diseases. Surgical treatment consisted of transplant of purified autologous fat retrieved from the hypogastrium. Follow-up was scheduled after 1 week, at 2, 3, 6, and 12 months, and thereafter on a yearly basis. RESULTS: All of the patients were discharged several hours after surgery. No early postoperative complication was observed. Complete healing and pain remission were achieved in 6 patients (75%), 4 of whom were treated in a single session, whereas 2 patients required 2 and 3 sessions. Anal stenosis was resolved in 4 of 5 patients (80%). The therapy was unsuccessful in 2 patients (25%). Mean follow-up was 18 months (range, 3-36 months). CONCLUSION: Perianal autologous fat transplant can be safely performed for the treatment of complex anal fissures. It is well tolerated and offers encouraging results, although further research is warranted because of the small number of patients treated and the relatively short follow-up time.

Determining the oncological risk of autologous lipoaspirate grafting for post-mastectomy breast reconstruction.

This study compares the incidence of local and regional recurrence of breast cancer between two contiguous time windows in a homogeneous population of 137 patients who underwent fat tissue transplant after modified radical mastectomy. Median follow-up time was 7.6 years and the follow-up period was divided into two contiguous time windows, the first starting at the date of the radical mastectomy and ending at the first lipoaspirate grafting session and the second beginning at the time of the first lipoaspirate grafting session and ending at the end of the total follow-up time. Although this study did not employ an independent control group, the incidence of local recurrence of breast cancer was found to be comparable between the two periods and in line with data from similar patient populations enrolled in large multicenter clinical trials and who did not undergo postsurgical fat tissue grafting. Statistical comparison of disease-free survival curves revealed no significant differences in relapse rate between the two patient subgroups before fat grafting and after fat grafting. Although further confirmation is needed from multicenter randomized clinical trials, our results support the hypothesis that autologous lipoaspirate transplant combines striking regenerative properties with no or marginal effects on the probability of post-mastectomy locoregional recurrence of breast cancer.

Fat Grafting for Facial Contouring Using Mechanically Stromal Vascular Fraction-Enriched Lipotransfer.

Autologous fat graft has limitations, especially long-term unpredictability of volume maintenance. The mechanical enrichment of fat graft with adipose-derived stem cells (ADSCs) could guarantee the survival of fat grafts. After decantation, washing, and centrifugation of lipoaspirate, the authors carried out histochemical analysis and flow cytometry to determine the best layers for preparing ADSC-enriched fat. After centrifugation, the stromal vascular fraction (SVF) was separated by mechanical dissociation and mixed with another layer of intact adipocytes, which was injected into patients. All patients showed volumetric improvement after a single lipotransfer section, without overcorrection. The method is safe, has low cost, and is easily reproducible.

Photoaging Skin Therapy with PRP and ADSC: A Comparative Study.

BACKGROUND: Stem cells from adipose tissue (ADSCs) and platelet-rich plasma (PRP) are innovative modalities that arise due to their regenerative potential. OBJECTIVE: The aim of this study was to characterize possible histological changes induced by PRP and ADSC therapies in photoaged skin. METHODS: A prospective randomized study involving 20 healthy individuals, showing skin aging. They underwent two therapeutic protocols (protocol 1: PRP; protocol 2: ADSCs). Biopsies were obtained before and after treatment (4 months). RESULTS: PRP protocol showed unwanted changes in the reticular dermis, mainly due to the deposition of a horizontal layer of collagen (fibrosis) and elastic fibers tightly linked. Structural analyses revealed infiltration of mononuclear cells and depot of fibrotic material in the reticular dermis. The ADSC protocol leads to neoelastogenesis with increase of tropoelastin and fibrillin. There was an improvement of solar elastosis inducing an increment of macrophage polarization and matrix proteinases. These last effects are probably related to the increase of elastinolysis and the remodeling of the dermis. CONCLUSIONS: The PRP promoted an inflammatory process with an increase of reticular dermis thickness with a fibrotic aspect. On the other hand, ADSC therapy is a promising modality with an important antiaging effect on photoaged human skin.

Photoaged Skin Therapy with Adipose-Derived Stem Cells.

BACKGROUND: The major intrinsic cause of facial skin degeneration is age, associated with extrinsic factors such as exposure to sun. Its major pathologic causes are degeneration of the elastin matrix, with loss of oxytalan and elaunin fibers in the subepidermal region, and actinic degeneration of elastin fibers that lose their functional properties in the deep dermis. Therapy using autologous adipose mesenchymal stem cells for regeneration of extracellular matrix in patients with solar elastosis was addressed in qualitative and quantitative analyses of the dermal elastic fiber system and the associated cells. METHODS: Mesenchymal stem cells were obtained from lipoaspirates, expanded in vitro, and introduced into the facial skin of patients submitted after 3 to 4 months to a face-lift operation. In the retrieved skin, immunocytochemical analyses quantified elastic matrix components; cathepsin K; matrix metalloproteinase 12 (macrophage metalloelastase); and the macrophage M2 markers CD68, CD206, and hemeoxygenase-1. RESULTS: A full de novo formation of oxytalan and elaunin fibers was observed in the subepidermal region, with reconstitution of the papillary structure of the dermal-epidermal junction. Elastotic deposits in the deep dermis were substituted by a normal elastin fiber network. The coordinated removal of the pathologic deposits and their substitution by the normal ones was concomitant with activation of cathepsin K and matrix metalloproteinase 12, and with expansion of the M2 macrophage infiltration. CONCLUSION: The full regeneration of solar elastosis was obtained by injection of in vitro expanded autologous adipose mesenchymal stem cells, which are appropriate, competent, and sufficient to elicit the full structural regeneration of the sun-aged skin. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.