Systematic review: Advances of fat tissue engineering as bioactive scaffold, bioactive material, and source for adipose-derived mesenchymal stem cells in wound and scar treatment

Fat tissue (FT) has been used for many years in regenerative surgery as a bioactive material through the lipofilling/fat graft (F-GRF)–nano-fat technique, as a bioactive scaffold when it was enriched with adipose-derived mesenchymal stem cells (AD-MSCs) contained in the stromal vascular fraction (SVF), and as a direct source of AD-MSCs used in wound healing (WH) and scar treatment (ST). This systematic review aims to describe the advances in FT engineering applied to regenerative surgery (from bench to clinic), through the use of AD-MSCs, SVF contained in F-GRF in WH and ST. The work has been performed by assessing in the selected studies autologous graft of AD-MSCs, SVF, and F-GRF compared to any control for ST and WH. The protocol was developed following the Preferred Reporting for Items for Systematic Reviews and Meta-Analyses-Protocols (PRISMA-P) guidelines. A multistep search of the PubMed, MEDLINE, Embase, PreMEDLINE, Ebase, CINAHL, PsycINFO, Clinicaltrials.gov, Scopus database, and Cochrane databases has been performed to identify papers on AD-MSCs, SVF, and F-GRF use in WH and ST in which FT was used as bioactive material–scaffold and source of AD-MSCs. Of the 714 articles initially identified, 453 articles focusing on regenerative strategies in WH and ST were selected and, consequently, only 84 articles that apparently related to AD-MSC, SVF, and F-GRF were analyzed. Of these, 61 articles identified as pre-clinical, experimental, and in vitro, and 5 articles identified as a comment and systematic review were excluded. Only 18 original articles which strictly and exclusively focused on autologous AD-MSCs, SVF, and F-GRF in ST and WH were analyzed. The included studies had to match predetermined criteria according to the PICOS (patients, intervention, comparator, outcomes, and study design) approach. The identified studies described microscopic and clinical outcomes in patients treated with AD-MSCs, SVF, and F-GRF. Collected data confirmed the safety and efficacy of FT both as bioactive material–scaffold and source of AD-MSCs in WH and ST without major side effects.


Introduction
A scientific-clinical need exists for the development of biotechnologies to improve wound healing (WH) during scar treatment (ST). The number of investigations evaluating the efficacy of autologous adipose-derived mesenchymal stem cells (AD-MSCs), contained in stromal vascular fraction (SVF) of fat grafting (F-GRF) in WH and ST, has exponentially increased, during the last 15 years (2005-2020). Autologous F-GRF is an interesting procedure in regenerative plastic surgery eagerly used in a growing number of indications, from skin rejuvenation and lipofilling to wound treatment [1]. Most of fat grafts' regenerative capacity is attributed to AD-MSCs, suspended in a fatty tissue cellular matrix-SVF [2]. It consists of a mixture of endothelial, smooth muscle cells, pericytes, and leukocytes [3]. Thanks to this regenerative activity, the F-GRF, a biological autologous tissue containing several important cellular components (adipocytes and mesenchymal stem cells), extracellular matrix (ECM), vessels, and nerves [3], may be considered both as "bioactive material" when it is grafted in the damaged tissue aiming to improve the scar quality (SQ) and the WH and as "bioactive scaffold" when it is enriched with AD-MSCs. In this last case, F-GRF acts also as a scaffold for the AD-MSCs, representing an autologous biological matrix (cellular and extracellular) in which these cells may be incorporated and transported with the aim to improve the healing time and SQ, via an autologous regenerative approach. The percentage of AD-MSCs in SVF varies depending on the isolation method but is greater than in classic F-GRF [4].
There are two main types of fat tissue' (FT) processing: mechanical and enzymatic.
Mechanical techniques are appealing because they are simple and quick [5].
Enzymatic SVF isolation is more expensive but can produce higher cell counts and progenitor cell percentage than mechanical methods [6]. In the last 15 years, new approaches to FT processing have emerged. In fact, FT has been used in regenerative plastic surgery as a bioactive material through the lipofilling/micro-fat/ nano-fat/lipostructure techniques, as a bioactive scaffold when it was engineered and/or enriched with SVF, and as a direct AD-MSCs' source. Micro-fat and nano-fat are F-GRF derivates, and they are considered promising methods of lipofilling, especially in the treatment of scars' interesting superficial skin layers [4,7]. Several techniques based on centrifugation, emulsification, and filtration procedures have been described to obtain nano-fat and micro-fat [8].
The aim of regenerative strategies in ST and WH must be the development of new autologous-biotechnologies to involve AD-MSCs and SVF by ex vivo and in vitro culture or by in vivo regeneration and bio-stimulation. Autologous F-GRF as a bioactive material and scaffold has been of great interest for application in soft tissue defects. Moreover, several early efforts in the field focused on isolating AD-MSCs and SVF via enzymatic or mechanical procedures ex vivo for subsequent introduction back into the patient.
However, a major limitation encountered in this area has been the difficulty in obtaining cells to sufficient numbers for human use and the necessity to perform as cell expansion only in Good Manufacturing Practices (GMP) laboratories [8].
SVF and AD-MSCs improve adipogenesis, vascularization, and growth factor (GF) production; hence, some have tried using them in ST [9][10][11]. The scientific data on this topic has not been properly collected and summarized. Few systematic reviews have been conducted regarding autologous fat transplantation/lipofilling in the treatment of scars, but none has focused also on the use of isolated AD-MSCs/SVF [12][13][14] or on the use of F-GRF as a bioactive scaffold for them.
In this systematic review, data from investigations reporting the use of AD-MSCs, SVF, and related F-GRF, in ST, analyzing WH, to evaluate such interventions' efficacy, were gathered.

European rules and international guidelines on AD-MSCs and SVF manipulation
The AD-MSCs and SVF isolation procedures are regulated by the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The European rules are represented by Regulation no. 1394/ 2007 of the European Parliament for Advanced Therapies (http://www.trovanorme.salute.gov.it/norme/ renderNormsanPdf?anno=0&codLeg=47245&parte= 1%20&serie=), which introduces the definition of "tissue engineering products" (TEP). Cells and tissues should only be considered TEP if they undergo "significant manipulation." The same regulation defines the difference between minimal and extensive manipulation.
To complete the description of the rules available in the European legislative panorama and the translation of extracts related to the subject matter, the authors note the "Reflection paper on classification of advanced therapy medicinal products" of May 21, 2015, EMA/CAT/ 600280/2010 rev.1 Committee for Advanced Therapies (CAT) (http://www.ema.europa.eu/docs/en_GB/ document_library/Scientific_guideline/2015/06/WC5001 87744.pdf).
Briefly, here were reported the Criteria for Somatic Cell Therapy Medicines (sCTMP) and TEP: 1. sCTMP and TEP contain or consist of engineered tissues and/or cells. To be considered "engineered," tissues or cells must meet at least one of the following criteria: a. Extensive manipulation (cell culture based on cell expansion, genetic modification of these, their differentiation/stimulation with growth factors (GFs)) b. Various essential functions (not homologous use) Enzymatic digestion of a tissue to release cells is also considered to be substantial manipulation when the aim is to dissociate cell-cell contacts and the released cells are administered into patients with or without subsequent manipulation. An example would be keratinocytes from the skin, for which enzymatic digestion would destroy the tissue architecture and functional interactions of the cells, which cannot be regained in the cell suspension: this would be considered as substantial manipulation. If the enzymatic digestion leads to isolation of functionally intact tissue units (e.g., pancreatic islets), or there is scientific evidence that the original structural and functional characteristics are maintained, the procedure is not considered substantial manipulation. In case a tissue is treated to remove cells and to be used without any cellular components (e.g., amniotic membrane, bone), the product is not an advanced therapy medicinal product (ATMPs) because it does not contain cells or tissue.
If the number of certain cells (e.g., Mesenchymal stem Cells (MSCs) in fat grafts) is enriched by selection and the processing does not change the characteristics of the cells, this is not considered a substantial manipulation (http://www.ema.europa. eu/docs/en_GB/document_library/Scientific_ guideline/2015/06/WC500187744.pdf), chapter 2.2.4 comma a).
For all the above-mentioned reasons, cells or tissues employed for the same essential function, not extensively or substantially manipulated (including fat enzymatic digestion-if the number of AD-MSCs is enriched by selection and the processing does not change the characteristics of the cells), are not considered a substantial manipulation and must not be considered ATMPs.
The present investigation has been developed in agreement with research contract #1467/2017-associate professor contract #13489/2021 between the first investigator, P.G., and the University "Tor Vergata," Rome, Italy.

Search strategy and literature screening
This systematic review was registered in the International Prospective Register of Systematic Reviews (PROSPERO, https://www.crd.york.ac.uk/ prospero/#myprospero) with ID code number: CRD42021230163.
This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews (PRISMA) and Meta-Analysis (http://www. prisma-statement.org) [15].
The research was conducted by two investigators (P.G. and S.G.) in accordance with the PRISMA guidelines and the Cochrane handbook [16]. A multistep search of the PubMed, MEDLINE, Embase, PreMEDLINE, Ebase, CINAHL, PsycINFO, Clinicaltrials.gov, Scopus, and Cochrane databases was performed to identify studies, published before December 1, 2020, on WH and ST treatment with AD-MSCs, SVF, and F-GRF, searching without a language or publishing-time restriction.
One hundred thirty-five articles using the keyword "stromal vascular fraction wound healing," 265 articles using the keyword "adipose-derived mesenchymal stem cells wound healing and scars," 301 using the keyword "fat grafting wound healing and scars," and 13 articles using the keyword "adipose-derived mesenchymal stem cells and stromal vascular fraction scars and wound healing" were found, as reported in Fig. 1.

Study assessment
The aim of this systematic review has been to assess the selected articles comparing local injections-grafting of autologous AD-MSCs, SVF, and F-GRF compared to any control for WH and ST.

Study selection
Seven hundred fourteen articles focused on WH and ST were initially identified and selected using PRISMA flow (www.prisma-statement.org) (Fig. 2). A total of 261 articles were excluded. Of this amount, 189 were duplicates and/or not adequate, while 72 articles were considered bias (not correctly match with the treatment and keywords used). Four hundred fifty-three articles were initially selected. Consequently, it was decided to include only clinical trials on autologous use of AD-MSCs, SVF, and F-GRF also referred to as regenerative strategies in WH and ST. For this reason, 369 articles not correctly matched with the topic (abstract/title not suitable, and allogeneic use) were excluded.
Eighty-four articles apparently related to the use of AD-MSCs, SVF, and F-GRF in ST and WH were selected. Of these, 61 articles were identified as preclinical, experimental, or in vitro studies. Additionally, of the 23 articles selected, 5 articles were excluded (3 comments and 2 systematic reviews).
Consequently, only 18 articles that strictly and exclusively focused on autologous use of AD-MSCs, SVF, and F-GRF in ST and WH were analyzed and included in this systematic review.
These 18 studies were evaluated and summarized by their study characteristics and study outcomes ( Table 2).

Data extraction
Data were independently extracted by the first investigator (P.G.) and checked by the last investigator (S.G.) only from the retrieved articles. Any disagreement on the extracted data has been settled by a consensus among P.G. and S.G. including also the co-authors (C.C. and A.S.). The following data have been extracted: first author, year of publication, study design, number of patients, type of procedure, and primary and secondary outcomes. The quality of the included investigations was independently assessed using two investigators (P.G. and S.G.) using the Cochrane Collaboration's Risk of Bias Assessment tool for randomized controlled trials (RCTs) [16] while using the Newcastle-Ottawa Scale to evaluate the individual non-randomized studies [35].

Endpoint definition
The efficacy of AD-MSCs, SVF, and F-GRF, in particular of F-GRF/nano-fat as bioactive material-scaffold and AD-MSC source, was primarily evaluated by a reduction in healing time and scar area during WH and by an improvement of soft tissue volume maintenance and skin quality during ST. Secondarily, by the satisfaction of patients from the surveys, and changes of scar's outcomes compared with pictures and histological analysis taken before and after the treatment sections. Given that, various test methods were taken through the studies included, only the most widely used methods would be set at the endpoints for all pooled studies. All side effects including local injection pain and increased sensitivity in the treated area have been analyzed.

Study subjects
Eighteen articles were included in quantitative synthesis (meta-analysis). No significant side effects were reported in the analysis. The human clinical autologous use regarded the treatment of soft tissue defects, scars, acne scars, outcomes of scars, chronic wound, post-traumatic scars, burns, and outcomes of burns.

AD-MSCs, SVF isolation procedures, and fat graft preparation
The most commonly used procedures were enzymatic SVF isolation [18-21, 25, 28, 30, 31] and nano-fat [22-24, 26, 27, 29, 32-34], while Wu et al. [17] used spectroscopy for SVF isolation. The fatty tissue was harvested from the abdomen or multiple sites, including flanks, hips, or thighs. Protocols of enzymatic SVF isolation varied between studies. Most commonly (6 studies) they included FT digestion with collagenase and subsequent centrifugation. Lee et al. [12,28] condensed the fat prior to enzyme addition. Zhou et al. [21] cultured AD-MSC fraction after isolation and eventually produced a cell-free medium, rich in GFs and cytokines, later used in the study as the bioactive scaffold. Nano-fat as a bioactive material was used in 9 cases. Its production, however, differed between studies. In six cases, fat condensation was performed prior to mechanical emulsification. In two studies, nano-fat was additionally centrifuged afterward. In three cases, nano-fat was produced classically. Gentile et al. [22] produced 3 modified versions of nano-fat-enriching it with mechanically isolated SVF and performing additional mechanical processing. In this case, the obtained product was a bioactive material-scaffold ready to be injected.

Selected studies analyzed
The studies analyzed have been represented by case reports (n = 3), case series (n = 7), case-controlled studies (n = 2) [18,28], and prospective studies (n = 7), as analyzed in the supplemental material section detailing.

Outcomes and endpoints
In addition to clinical evaluation, endpoint evaluation methods included biopsy with immunochemistry stain, photographic evaluation, global photographs, physician global assessment score, and patient global assessment score. The satisfaction surveys and scales were taken        from the perspective of patients, and other observers were also used to evaluate the efficacy of AD-MSCs, SVF, and F-GRF/nano-fat in some of the recruited studies.

Critical assessment of the study design
Performing a deep analysis of the selected studies during this investigation, a lack of standardized and widely share protocol for the isolation methods of AD-MSCs and SVF has been highlighted, as well as standardized evaluation procedures. In particular, there is a lack of widely shared consensus on the preparation procedures of F-GRF/nano-fat (centrifugation, filtration, emulsification) and on the digestion method (enzymatic vs mechanical). Additionally, the difficulty in clearly interpreting results was determined by the wide range of the studies analyzed (from pilot studies to randomized trials).

Side effects
No major side effects have been displayed in the analyzed papers. Only tolerable and temporary pain during and immediately after the procedures and transient edema have been described by some patients during fat harvest.

Discussion
The high number of intervention options to improve WH and scar-tissue quality might cause some confusion during the decision-making process. Dermal substitutes, advanced dressings, hyaluronic acid, fat graft, PRP, CO 2 and related fractional lasers, steroid injections, dermabrasion, and surgical procedures (like advancement flap, sliding flap, rotation flap, or zetaplastic) are all important elements in WH and ST procedures [36,37]. The plastic surgeons should choose an adequate technique according to the clinical examination, promoting the regenerative strategies when possible. In this systematic review, information from 18 selected studies has been harvested, reporting outcomes in a total of 665 patients. This body of evidence is significant, but EBM studies of levels I-II are few. Fiftyfive percent of the selected studies (10/18) are case reports or case series. Among RCTs, there was a significant bias risk, represented by the absence of information about allocation concealment, blinded outcome assessment, and selective outcome reporting. Additionally, only small study groups were analyzed among RCTs. Nevertheless, the authors have shown very interesting results, suggesting beneficial effects of AD-MSCs/SVF in ST and related WH. The scars' and wound healing outcomes obtained with AD-MSCs, SVF, and F-GRF/nano-fat-used as bioactive material and/or bioactive scaffold or as cellular suspensionhave been objectively assessed, reducing the risk of bias.
The outcomes analyzed suggest that esthetic results and patients' satisfaction have been better when AD-MSCs, SVF, or F-GRF/nano-fat enriched with SVFs (bioactive material-scaffold) were administered, in comparison with classic F-GRF. The F-GRF volume maintenance, besides, improves in nano-fat/SVFtreated groups [18,31]. Three studies compared PRP with AD-MSCs/SVF/F-GRF/nano-fat and suggested improved scar area reduction in the PRP group, in particular when F-GRF was enriched with PRP (bioactive material-scaffold) [18] with little differences in fat resorption, showing clinical outcomes and/or microscopic findings [18,20,33].
Gu et al. [27] visualized sebaceous and sweat glands, usually absent or scarce in scars, 6 months after nano-fat injection, used as bioactive material.
Zhou et al. [21] showed that topically administered AD-MSC-conditioned medium (bioactive material-scaffold) improves fibers' alignment. Undoubtedly, the abundance of SVF/AD-MSC isolation methods and multiple nano-fat processing protocols pose a challenge to interpreting collected results. They also reflect a great heterogeneity of clinical practices.
Some authors modified the original nano-fat production procedure by additional centrifugations before and/or after the homogenization step [22-24, 27, 33, 34] improving in some cases the outcomes in terms of cell yield [22].
Gentile et al. [18,22] showed a post hoc association between AD-MSC quantity and clinical outcomes. The significance of this observation, however, may be undermined by a relatively small study population. In vitro studies demonstrated that similar modifications increase AD-MSC output [33,[38][39][40][41] without affecting the composition of secreted proteins [41].
The data outcomes seem to suggest the higher stem cell yield translates into clinical improvement. This concept should be demonstrated through EBM level I-II studies. Factors such as surgeon's craftsmanship or postgraft care play an important role in shaping the outcome, but they are difficult to assess objectively [42]. This review has been limited by the small group of studies available and analyzed, and the exclusion of preclinical studies.

Conclusions
Analyzed data are substantial and significant, even if with average medical evidence and an EBM level of III and IV (EBM III-case-controlled studies and EBM IVcase series) for beneficial effects of AD-MSCs/F-GRF-related interventions in WH and ST-both clinically and on a microscopic level. Many evidence, suggests that SVF/nano-fat is non-inferior to common approaches, such as PRP or fractional CO 2 laser in terms of clinical outcomes. Collected data confirmed the safety and efficacy of F-GRF/nano-fat both as bioactive material/bioactive material-scaffold and source of AD-MSCs in WH and ST without major side effects. Given the current treatments differ in methodology and treatment technique, further studies are needed to define standardized protocols. Moreover, large-scale randomized trials still need to be conducted to confirm its efficacy.
For these reasons, the authors invite all the audience to improve the level of publications in this field by focusing prevalently on EBM level I-II studies.
Additional file 1. Selected studies analyzed.