Adipose tissue extract (ATE)

Inspired by the need for a simpler method to obtain the adipose tissue secretome, adipose tissue extract (ATE) was developed, a protein-rich extract with the potential of inducing angiogenesis and adipogenesis in vitro (Sarkanen et al. 2011).

Originally, ATE production involved preparing the fat by mixing it with PBS, followed by incubation, centrifugation and filtration. Total protein content and VEGF, bFGF and IGF-1 were quantified with ELISA and cytokine array analysis at different incubation timepoints. The highest protein and IGF-1 concentrations were found within the first two hours, compared to 24 hr. While bFGF demonstrated similar concentrations at all timepoints, VEGF had higher values at 24 hr. Adding ATE to ADSC cultures provided a stable, dose-dependent cell proliferation with concentrations >200µm/ml. In ADSC and endothelial cell co-cultures, ATE at 450 µg/ml induced the formation of a capillary-like endothelial network. mRNA studies found the expression of adipogenic markers in ATE. This study discovered an adipose-derived cell-free extract rich in GFs and adipokines endowed with adipogenic and angiogenic properties. (Sarkanen et al. 2011)

Soft tissue volume defects are challenging to treat, and a popular method includes employing fillers like adipose tissue or hyaluronic acid (HA). However, these fillers undergo resorption due to a lack of neovascularization and enzymatic/non-enzymatic degradation (Papakonstantinou et al. 2012). In the pursuit to contribute to these challenges, an experimental rat model of HA and ATE implants was developed. A series of biopsies from 1 to 40 weeks post implantation demonstrated earlier and greater capillary proliferation, richer vessel density and increased adipose deposits after ATE treatment. By week 40, all control implants had degraded, whereas only 2 ATE implants partially degraded. (Sarkanen et al.

2012b). Furthermore, ATE has shown enhanced cell proliferation and angiogenic properties (Sarkanen et al. 2012 a-d). These findings opened the doors to ATE’s tissue engineering potential.

Experimental investigation by Lu et al. investigated the effect of ATE on rabbit vascularized flaps. Two adipose flaps were raised with their pedicles and sutured over silicone hemispheric porous chambers. After a week, saline or ATE

was injected into the chamber. The flaps were followed weekly for seven weeks and pathology samples obtained. They found that ATE-treated chambers formed softer and thinner capsules with a higher capillary density. In this study ATE significantly promoted adipogenesis and angiogenesis in vitro and in vivo (Lu et al. 2016).

Although ATE results were clearly reproducible in promoting angiogenesis and adipogenesis, a further step was required to validate its applicability in a clinical wound repair setting. First, a new ATE preparation method in an operating room setting would endow the clinician with the tools required for its immediate use.

Second, simulated wound assays and cell proliferation studies would explain ATE’s effect on wound repair. Third, clinical studies on human wound healing would confirm its efficacy. Finally, the best ATE delivery systems tailored to specific wound requirements would help the clinician make the best use of ATE.

A study by Fu et al. described the use of an adipose treatment obtained from the subcutaneous tissue of pigs that was formed by cutting the tissue into 3x3 mm pieces, then washed and filtered through 4 layers of gauze and made into a paste.

The application of the paste over full-thickness porcine wounds demonstrated accelerated healing and biopsies showed mature vascular networks and greater numbers of proliferation and differentiation markers, like Factor VIII-related antigen and proliferation cell nuclear antigen, compared with untreated wound biopsies (Fu et al. 2007). Compared to ATE, this paste contained adipose cells along with vascular stromal cells.

In 2017, Na et al. reported the effects of an adipose stem cell secretome on wound healing. The application of this cell-free extract promoted wound healing in vivo, and its addition to human dermal fibroblasts promoted cell proliferation and migration. They also observed that matrix metalloproteinases, as well as collagen type I were up-regulated by this extract (Na et al. 2017).

Recently, He et al. reported the use of an ALE (adipose liquid extract), prepared by incubating the lipoaspirated tissue in ice water for 10 min, followed by separation, washing in PBS, centrifugation and finally combining it with 50% PBS.

The resulting liquid is emulsified between two syringes and re-centrifuged before filtering. In vitro, He et al. demonstrated ALE’s angiogenic and adipogenic properties. Furthermore, ALE was used topically on full-thickness mice wounds, which demonstrated accelerated re-epithelialization on days 7, 11 and 14 (He et al.

2019).

2.3 Wound classification and factors affecting wound healing

Wounds are defects with a variable loss of epidermis, dermis and subcutaneous tissue (Phillips 1994). They are classified by their etiology as traumatic (burns, open wounds) or non-traumatic (infections), surgical or non-surgical, or ulcers originating as a complication of a pre-existing disease, such as diabetes, arterial or venous insufficiency (Maver et al. 2018, Percival 2002). According to their timeline, they are considered acute when they are under two weeks, while chronic wounds have been exposed to the environment for longer periods. The Finnish Current Care guidelines define chronic wounds as defects that fail to heal after a period of 4 weeks (Current Care guidelines 2018). The latter differ in biomolecular composition and their response to treatments (Gould et al. 2015, Mast and Schultz 1996, Loots et al. 1998, Demidova-Rice et al. 2012). Venous ulcers are a product of deficient valves that result in edema formation and microcirculatory ischemia (Valencia et al. 2001, Smith et al. 1988, Lees and Stansby 2018). Neuropathic diabetic foot ulcers (DFU) originate from sympathetic denervation, which decreases the protective sensation to pain, making the patient susceptible to trauma or persistent pressure (Boulton et al.

2004, Margolis et al. 2000). Arterial ischemic ulcers are seen in the dorsal tips of the toes, feet and legs as a result of severe arteriosclerosis, which delays the arrival of oxygen to the distal leg (Hafner et al. 2000, Grey et al. 2006). Lower limb ulcers, commonly found in clinical practice, have a scarcity of GFs and high amounts of GF-degrading enzymes such as MMPs that perpetuate a non-healing cycle (Clinton and Carter 2015, Zhao et al. 2016, Mast and Schultz 1996, Fife et al. 2018).

Factors affecting wound healing can be classified into wound and patient causes.

The first comprise the etiology and the local wound characteristics such as size, depth, exposure vital organs (such as bone, neurovascular structures and bowel among others), presence of infection or cancer in the wound, exposure to radiation, hypoxia and previous treatment (Frykberg and Banks 2015, Powers et al. 2016, Clinton and Carter 2015, Zhao et al. 2016). Individual patient factors include advanced age, reduced mobility, diabetes mellitus, chronic renal or liver disease, the use of drugs and alcohol, nutritional deficiencies, and immunosuppressive states, and have a great impact on wound healing and help anticipate healing problems (Bryant and Nix 2015, Zarei and Soleimaninejad 2018, Guo et al. 2010, Deodhar and Rana 1997, Hunt et al. 2000, Landriscina et al. 2015, Lee et al. 2009).

2.4 Wound treatment

It is a challenge to treat wounds because there are so many factors simultaneously influencing healing (Guo and DiPietro 2010). Superficial wounds are debrided and sutured or covered with medicated creams, or simple dressings (petroleum gauze, tapes, or thin foams). If contaminated, they may be left open to heal by secondary intention or delayed secondary closure (Nicks et al. 2010). Complicated surgical or traumatic open wounds require more laborious monitoring, depending on their location, depth and the presence of infection. While some can be treated conservatively with dressing changes every 2-4 days, others routinely undergo surgical debridement(s) or negative wound pressure therapy, until conditions allow for definite closure or reconstruction (Dumville et al. 2015, Dumville et al. 2013, Dhivya et al. 2015). Direct wound closure, skin graft or flap coverage are the most common methods employed for wound coverage (McGregor and McGregor 2000, Janis et al. 2011). It is essential to address the underlying wound origin and not just treat the wound locally. In this sense, factors to manage include glycemic levels, ulcer prevention, managing sedentarism or lack of mobility, pressure offloading, toxic lifestyle management, malnutrition and promoting proper wound care (Hunt et al.

2000 Hess 2011, Campos et al. 2008). If unrecognized, wounds can become complicated and form fistulas, invade bone, and trigger septicemia (Grey et al. 2006).

Burn wounds are a special challenge because there may be a lack of skin replacement sources (in extensive burns) and their resulting scars are unsightly and functionally limiting (Deitch et al. 1983, Finnerty et al. 2016, Hettiaratchy and Dziewulski, DuBose and Swann 2018, Papini 2004, Hettiaratchy and Dziewulski 2004, American Burn Association Consensus Conference on Burn Sepsis and Infection Group, Greenhalgh et al. 2007). While superficial burns are treated with topical silver-based ointments and dressings, most deep burns require surgical treatment (Hettiaratchy and Papini 2004, Cancio et al. 2017, Baudoin et al. 2016, Daigeler et al. 2015, Kim et al. 2015). For very extensive and deep burns, staged skin grafting is the treatment protocol, and can be complemented with the use of cultured keratinocytes (Auxenfans et al. 2015, Sood et al. 2015, Cirodde et al. 2011).

2.4.1 The use of GFs in the clinical setting

Because of their crucial role in wound repair signaling, attempts have been made to deliver GFs in a clinical setting (Barrientos et al. 2014). The main issue with these studies is their low power or their deficient methodology, making it difficult to draw conclusions or obtain repeatable results. There is a definite need for further research

in the field. GF application is not only limited to cutaneous wounds, but also for promoting bone, tendon, ligament and blood vessel repair, and for anti-aging purposes. To date, GM-CSF, PDGFb, VEGF, bFGF and EGF have been used in human clinical studies to enhance wound repair (Lacci et al. 2010). Table 3 summarizes the RCTs employing GFs for wound healing.

Table 3. Growth factors employed in wound healing.

Growth factor

Name Indication Dosage Observations

PDGF-BB

Prolonged use demonstrated increased risk of developing malignancy (3% vs 1% in non-treated ulcers) (Smiell et al. 1999).

VEGF

Accelerated healing (32.5 days vs 43 days control), improved complete ulcer healing (41.4% vs 26.9% control). Improvement in limb perfusion index (Hanft et al.

2008, Kusumato et al. 2006, van Royen et al. 2005)

84.6% of patients achieved ≥85%

healing; no recurrence at 1-year follow-up (Payne et al. 2001)

GM-CSF and ischemia (Van Royen et al. 2005).

Adverse effects: pain, redness, swelling, fever (Gough et al 1997, Bianchi et al. 2002, Zhang et al.

In a prospective, randomized, double-blind trial, EGF was tested on split-thickness skin graft (STSG) donor sites and the rate of re-epithelialization evaluated.

The control site was treated with silver sulfadiazine cream and the experimental with a cream combining 10 µg/ml EGF. The experimental wounds showed 25-50%

healing one day before controls and complete epithelization was achieved 1.5 days sooner than controls (Brown, et al. 1989). The effect of HGF, VEGF and hypoxia inducible factor-1 (HIF-1) have been studied in human critical ischemic limb wounds with promising results (Attanasio and Snell 2009, Snell 2016). A study by Powell et al. found greater perfusion rates with increasing HGF doses, but no changes were seen in pain relief or wound healing (Powell et al. 2010).

A few recombinant GFs have achieved marketable success in the form of gels, ointments, lyophilizates or liquid forms for perilesional injections, but their costs are considerable and their effects variable. Treatment protocols range from single dose to re-application every 2-7 days (Barrientos et al. 2014). Their limitations include difficult and costly preparation processes, very short half-life (some as low as 3-50 min), low protein stability and accelerated degradation by enzymes. GFs should be at a concentration high enough to elicit a sustained effect, but at the same time low enough to avoid potential complications, such as carcinogenesis (Wang et al. 2017).

In vivo, the synergistic combination of GFs during early wound healing seem of greater benefit than any single GF (Kiritsy and Lynch 1993, Lynch et al.1987, 1989).

2.4.2 The use of GFs in in vitro and pre-clinical settings

The soft and hard tissue healing effects of GF have been thoroughly studied in vitro and in animal experimental models (Lynch et al. 1997, Barrientos et al. 2008).

In cell culture models, the main GFs promoting healing include VEGF, FGF, EGF, KGF, HGF, PDGF, TGFb and IGF-1. However, cytokines including stromal derived factor -1 (SDF-1), and proliferator-activated receptor gamma coactivator (PGC-1), have also demonstrated wound healing, and osteoblastic and angiogenic potential (Poniatowski et al. 2015). Interestingly, leptin has shown mitogenic and angiogenic properties in cultured human keratinocytes and endothelial cells (Bouloumié et al. 1998, Frank et al. 2000). While leptin-deficient mice demonstrated markedly delayed wound healing, systemic or topical supplementation of leptin accelerated re-epithelialization of experimental excisional wounds (Frank et al.

2000). The topical application of leptin on rabbit gingival wounds significantly

accelerated the time of wound closure (Umeki et al. 2014). Leptin’s proposed mechanism of action is through the activation of Janus kinases (Jak), which prompts the recruitment and phosphorylation of (Signal Transducer and Activator of Transcription (STAT) proteins, resulting in cell division (Frank et al. 2000, Ring et al. 2000, Takadoro et al. 2015,).

2.5 Wound healing research

The wound healing cascade is composed of a sequenced activation of molecular pathways resulting in cell division and migration. Understanding these concepts aids the researcher in discovering ways to regulate these pathways. Research in the field of wound healing is a crucial step towards improving and accelerating the healing and scarring processes, while avoiding potential complications that delay healing in clinical settings. These studies begin in the laboratory and are finalized in large human randomized controlled multicenter trials.

2.5.1 In vitro and preclinical

One of the most popular cell cultures assays to determine viability and/or proliferation rates is the cell proliferation assay. With the application of dyes, chemical reactants or colorimetric measurements, one is able to determine cell counts as indicators of cell health. When studying the effect of any given agent on cell cultures, this assay aids in determining cell toxicity and cell death (Pastar et al.

2014). Furthermore, the expression of cell proliferation markers, such as Ki-67, and basement membrane markers may aid in determining the effects of a given treatment on proliferation.

Growing cultured keratinocytes originating from fetal, adult or engineered human skin have become popular methods to evaluate wound repair (Kratz et al.

1998, Mathes et al. 2014, Pastar et al. 2014, Mendoza-Garcia et al. 2015). In scratch assays, keratinocytes are grown on special culture dishes, until a convergence of cells is observed. Subsequently, a scratch is made with a pipette tip and closely observed at different timepoints until the space is filled up with new cells. Measuring the gap closure is a reliable indicator of cell migration (Moll et al. 1998, Pastar et al. 2014, Werner et al. 2007). Similarly, fibroblast cultures have been used to understand complex wound healing processes (Driskell and Watt 2015, Sriram et al. 2015,

Driskell et al. 2013, Rinkevich et al. 2015). 2D and 3D culture models have been found to mimic the wound environment closely but lack a dermal-epidermal junction (Lebonvallet et al. 2010, Xu et al. 2012, Safferling et al. 2013). Models such as the human ex vivo skin culture model (Xu et al. 2012) and the linear wound model (Rizzo et al. 2012) have been introduced to better simulate this environment, but these cannot fully represent what happens in vivo (Marionnet et al. 2006, Menon et al. 2012).

Angiogenesis is an important part of wound healing, for cells require adequate amounts of oxygen and nutrients to achieve proliferation, differentiation and final wound closure. Besides measuring angiogenic GFs, such as VEGF, bFGF, TGFb, TNFα, PDGFb, angiopoetins, and other receptor markers, there is a plethora of angiogenesis assays involving 2D and 3D culture of endothelial cells in combination with stromal cells (Tonnesen et al. 2000).

Wound healing has also been extensively evaluated in mammalian animal models such as mice, rats and pigs (Fang and Mustoe 2008, Lindblad 2008, Park et al. 2015, Pastar et al. 2014). Partial (epidermis and part of dermis) or full-thickness (epidermis and total dermis) wounds have been studied to understand wound healing. Porcine models seem to best simulate human skin repair due to similarities in the thickness of dermis and epidermis (Seaton et al. 2015, Sullivan et al. 2001, Meyer et al. 1978).

2.5.2 Clinical

Initial wound evaluation relies heavily on the eye of the clinician. Undoubtedly, adequate clinical history and physical examination help in determining a diagnostic and treatment plan. Certain wound characteristics like its anatomic location assists a clinician in probable etiology. For example, spontaneous ulcers located in the dorsum of the foot usually relate to arterial deficiency. The features of a wound bed and edges, such as color, smell, edge eversion and rolling also aid in determining a cause (Grey et al. 2006, Robson and Barbul 2006). Nonetheless, once the diagnosis is made, it is essential to have an objective method to assess the wound during every visit to determine its evolution and response to treatment.

Determining the size and depth of the wound are key characteristics in registering their progression (Kantor and Margolis 1998, Flanagan 2003). Because most wounds are irregular in shape, their measurement can be difficult. Planimetry, is the estimation of wound surface area and its progress until complete closure. It can be measured manually using transparent acetate film or through

photo/videography by tracing the edges of the wound (Bohannon and Pfaller 1983, Gethin and Cowman 2006, Kim et al. 1987, Rogers et al. 2010, Wunderlich et al.

2000). The measurement is then made manually from a millimetric grid or analyzed by computer software (Gorin 1996, Wendland and Taylor 2017, Wendelken et al.

2011, Lagan et al. 2000), but this method is considered imprecise (Mayrovitz and Soontupe 2009, van Zuijlen et al. 2004, Grey et al. 2006, Robson and Barbul 2006, Bloemen et al. 2012). Even though there has been advancement in the area of digital imaging and software processing, ideal image analysis software is still lacking. There are software tools that aid in analyzing biomedical imaging; however, these are costly, inaccessible, and require extensive training to obtain adequate analyses (Eliceiri et al.

2012, Meijering et al. 2016). The Image J “project” is an open source piece of software that includes advanced tools allowing the user to process and analyze images in a simple manner (Rasband 1997-2018, Abramoff, et al. 2004, Schneider et al. 2012, Rueden et al. 2017). However, most image software products have their limitations, and do not replace the precision of an experienced clinician. A completely re-epithelialized wound is one that is dry (free from secretions), and the patient able to return to normal activities.

2.5.3 Split-thickness skin graft donor sites as a wound healing model

After direct wound opposition, skin graft closure is considered the simplest form of wound coverage in reconstructive surgery. Skin grafts are thin dermo-epidermal components that can be split or full thickness (STSG and FTSG, respectively) (Ratner 1998). STSGs are harvested with an electric or manual dermatome, and classified according to their thickness as ultrathin, thin, intermediate, and thick (Andreassi et al. 2005). These wounds are so superficial that they are left to re-epithelialize on their own, which normally occurs within 15 days under optimal conditions (Voineskos et al. 2009, Demirtas et al. 2010, Masella et al. 2014, Wiechula 2003). STSG donor sites are great models to evaluate wound healing, because wounds are controlled for size, depth, and location and can test different treatments and predict healing patterns (Nuutila et al. 2012, Nuutila et al. 2013, Serebrakian et al. 2018, Kazanavičius et al. 2017). In donor site wound healing studies, the primary endpoint is usually complete epithelialization (Still et al. 2003, Ottomann et al. 2010, Innes et al. 2001, Gao et al. 1992, Demirtas et al. 2010, Dornseifer et al. 2011). In spite of the fact that donor sites heal spontaneously, a frequent problem is the resultant discoloration and scar (Rakel et al. 1998). This is why areas normally

covered by clothing are chosen for STSG harvest i.e. inner thighs, abdomen and gluteal area. (Neligan 2013, Osman and Emara 2018, Park et al. 2012).

Brown et al. studied the epidermal regeneration of partial thickness donor site wounds employing daily photographic planimetry for surface area analysis (Brown et al.1989). Although they are a good model to evaluate wound healing, skin graft donor sites are difficult to evaluate. This is because, although most heal by day

Brown et al. studied the epidermal regeneration of partial thickness donor site wounds employing daily photographic planimetry for surface area analysis (Brown et al.1989). Although they are a good model to evaluate wound healing, skin graft donor sites are difficult to evaluate. This is because, although most heal by day

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