Application of Autological Adipose Tissue and its Products in the Treatment of Infected Wounds of Different Genesis

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Autologous fat grafting (lipografting) and its products to stimulate the wound process is a rapidly developing area of ​​regenerative medicine. Although some preclinical studies on the efficacy of lipografting in wound healing, both in animal and in vitro models, are encouraging, it remains to be definitively established whether transplantation of autologous adipose tissue and its products improves clinical wound healing outcomes [1]. In addition, lipografting is increasingly being studied as a component of the treatment of other surgical diseases associated with tissue trauma and infection: open fractures, damage to peripheral nerves, external intestinal fistulas, Crohn's disease [2-5]. The technology of lipografting is changing and developing along with the expansion of the range of its application. It is necessary to systematize the data on the adaptation of the traditional operation of adipose tissue transplantation to the new conditions of its application.

Purpose. By systematic analysis of the literature, to determine the most effective and safe technologies for obtaining, processing and transplanting autologous adipose tissue used to treat infected wounds.

Material and methods. Search methods. In electronic databases PubMed. eLIBRARY, Embase, Cyberleninka, Web of Science searched for relevant articles from 2011 to 2021. The keywords for the search were: “autologous fat grafting”, “lipografting”, “stromal vascular fraction”, “adipose derived stem cells”, “wounds”, “wound treatment”, “burns”, “burn treatment”, “chronic ulcers”, “treatment of chronic ulcers” in Russian and English. Search algorithms: 1) “transplantation of autologous adipose tissue” or “lipografting” or “fat grafting” or “stromal vascular fraction” or “adipose derived stem cells” + “wounds” or “wound treatment”; 2) “ autologous fat grafting ” or “lipografting” or “fat grafting” or “stromal vascular fraction” or “adipose derived stem cells” + “burns” or “burn treatment”; 3) " autologous fat grafting " or "lipografting" or "fat grafting" or "stromal vascular fraction" or " adipose derived stem cells " + " chronic ulcers " or "treatment of chronic ulcers ". Based on the specified keywords, 2292 sources were included in the preliminary analysis.

Study inclusion  criteria. 1. Treatment of infected soft tissue wounds against the background of chronic circulatory disorders, diabetes mellitus; burns; fistulas; 2. Clinical presense of wound infection; 3. The use of autologous adipose tissue or its low-manipulation products in local treatment. Study exclusion  criteria: 1. Experimental studies on animals; 2. Cosmetic surgery; 3. Surgical treatment of scars. After applying inclusion and exclusion criteria, combining database results and removing duplicate entries, checking titles and abstracts, the remaining articles were read in full.

Results and their discussion.

As a result, 30 sources published over the past 10 years were analyzed. Of these: 5 RCTs, 16 clinical trials, 3 case reports, 1 systematic review, 2 literature reviews. 4 sources published earlier than 10 years are included, due to their fundamental and practical significance for the issue under consideration (Table 1),

Table 1. Summary data.

№	Author	Study design	Number of patients	Fat Processing	Cellularity index	Adipose derived product
1	Deng C[6]	Clinical trial	20	Mechanical (nanofat)	unknown	SVF-gel
2	Nilforoushzadeh MA[7]	Clinical trial	5	Enzymatic (collagenase)	1×104/ml	SVF-cells
3	Konstantinow A[8]	Controlled Clinical Trial	16	Enzymatic (collagenase, dyspase)	15x106 (6 ml)	SVF
4	Samoylov AS.[9]	Original Article	5	Enzymatic (collagenase)	from 10х106 to 50х106 /5 ml	SVF
5	Marino G[10]	Controlled Clinical Trial	20	Enzymatic (automatic)	3х105 /ml (5 ml)	ADSC
6	Bene MD[11]	Case series, prospective	9	Mechanical (centrifugation)	unknown	Purified fat (mid.layer)
7	Marangi GF[12]	Case series, prospective	42	Mechanical (centrifugation)	unknown	Fat
8	Del Papa N[13]	Case series, prospective	15	Mechanical (centrifugation)	unknown	Adipose derived tissue cells (mid.layer)
9	Piccolo NS[3]	Review	282	Mechanical (centrifugation)	unknown	SVF
10	Stasch T[14]	Prospective cohort study	26	without processing	unknown	Fat
11	Caviglia H[4]	Case series, prospective	6	Mechanical (centrifugation)	unknown	Concetrate fat cells
12	Kim JH[15]	Case series, retrospective	12	Enzymatic (collagenase)	unknown	SVF
13	Carstens MH[16]	Original Article	10	Enzymatic (collagenase)	unknown	SVF-cells
14	Chopinaud M[17]	Prospective pilot study	10	Mechanical (centrifugation)	unknown	Fat micrografts (mid.layer)
15	Cervelli V[18]	Case control study	10	Enzymatic (automatic)	5х104 /ml	SVF
16	Mizushima T[5]	Clinical Trial	6	Enzymatic (automatic)	3,5 ×106	Adipose derived regenerative cells
17	Giuggioli D[19]	Case Reports	1	without processing	unknown	Fat
18	Smith OJ[20]	Randomized Controlled Trial	18	without processing	unknown	Isolated lipoaspirate
19	Piccolo NS[21]	Review	27	Mechanical (centrifugation)	unknown	Fat
20	Williams EA[22]	Original Article	20	without processing	unknown	SVF
21	Biyao Z[23]	Case Reports	1	Mechanical (centrifugation)	unknown	------
22	Rangaswamy M[24]	Case Reports	3	without processing	unknown	Fat
23	Abouzaid AM[25]	Randomized control clinical trial	100	Mechanical (nanofat)	unknown	Nanofat
24	Carstens MH[26]	Сlinical trial	63	Enzymatic (automatic)	30 × 106	SVF
25	Tanios E[27]	Randomized Controlled Trial	100	Enzymatic (collagenase)	unknown	SVF-cells
26	Ryabkov MG.[28]	Prospective clinical trial	34	Mechanical (sedimentation)	unknown	Lipoaspirate (mid.layer)

 

Physiological basis of the regenerative potential of autologous adipose tissue. Lipografting has been used as an aesthetic procedure in plastic surgery and cosmetology for more than 20 years. At the beginning of the period of surgical application, adipose tissue was used as a "filler", a plastic material to change defects in body contours. A new therapeutic potential of adipose tissue was discovered in 2001 by Zuk, Hedrick et al., when mesenchymal stem cells were isolated from it, later called adipose derived stem cells (ADSCs) [29]. As a result of intensive research, it was found that ADSCs are multipotent mesenchymal stem cells and are able to differentiate into various cells, including keratinocytes, fibroblasts, endotheliocytes. In addition, ADSCs have a wide variety of regenerative and metabolic properties, and are also able to secrete various paracrine factors: epidermal growth factor, transforming growth factor-β, hepatocyte growth factor, platelet-derived growth factor, basic fibroblast growth factor, placental growth factor, vascular endothelial growth factor, which initiate tissue repair and accelerate the process of wound healing due to the activation of neovasculogenesis [2]. It was these data that subsequently became the basis for the use of ADSCs in practical wound surgery, since they made it possible to more clearly define the goal and expected physiological effects of therapy.

Features of fat harvesting in patients with infected wounds. Adipose tissue sampling in most of the sources included in the review was described as a standard procedure performed according to the methodology proposed by Coleman [30]. This technique involves the use of a 10 ml Luer Lok syringe attached to a 3-3.5 mm cannula with two 3 mm side distal holes. The length of the cannula used is 10, 15 or 20 cm, depending on the sampling site. The most common lipoaspiration procedure was performed in the area of ​​the anterior abdominal wall. Only in 4 cases, adipose tissue sampling was performed from other areas: internal thigh [3, 5, 12] and from the buttocks [18]. Lipoaspiration was performed under sterile conditions of the operating room or procedure room, usually under local tumescent anesthesia, and in one case under spinal anesthesia [3] and under general anesthesia [18]. During lipoaspiration, standard or modified Klein's solution (physiological saline or Ringer's lactate and/or local anesthetic and/or adrenaline) [19] was used, which was necessary to reduce the trauma of the procedure for extracting adipose tissue, for local hemostasis, and for pain contol. A 3-5 mm long incision was made with a scalpel, and a blunt cannula was inserted to collect fat. The intake of adipose tissue was carried out by creating a slight negative pressure while retracting the syringe plunger and simultaneously performing reciprocating hand movements. The amount of adipose tissue obtained in one manipulation, in the analyzed literature, was indicated in 8 works and ranged from 30 ml (min) [8] to 400 ml (max) [18]. In 2 sources, to calculate the required amount of adipose tissue, the formula was proposed: 1 cm3 of adipose tissue per 1 cm2 of the wound surface [14, 19].

Although the lipoaspiration in patients with infected wounds is described by the authors as standard, it still has a number of significant features that distinguish it from the standard technology of aesthetic surgery. These features include: 1. The choice of a zone for obtaining adipose tissue is determined by the clinical situation, namely, the maximum distance from the zone of damaged skin. This is especially important in patients with extensive burns. 2. The amount of adipose tissue obtained in one manipulation may be limited due to extensive damage or severe exhaustion in patients with burn wounds.

Fat processing. Adipose derived products in wounds treatment. Adipose tissue products used in the treatment of wounds. Treatment options for lipoaspirate have varied in different sources. In 10 studies, the production of the final product from adipose tissue was carried out by an enzymatic method using collagenase [5, 7-10, 15, 16, 18, 26, 27], 4 of them used automatic Celution systems [5, 10, 18, 26] , and in one case, dispase was also used as an enzyme [8]. In 14 studies, lipoaspirate was subjected to minimal mechanical processing in the form of centrifugation for 1-3 min at a speed of 920-3500 rpm [3, 4, 11-13, 17, 21, 23] or sedimentation [28] followed by removal of the middle layer containing “purified fat”, or not processed at all [14, 19, 20, 22, 24]. In only 2 studies, lipoaspirate was mechanically emulsified by moving between two 10-cc syringes connected to each other with a mother-mother Luer-Lok connector and the so-called “nanofat” was obtained [6, 25]. The resulting products in 11 cases were designated as “stromal-vascular fraction (SVF) of adipose tissue” [3, 6-9, 15, 16, 18, 22, 26, 27], and in 8 of them enzymatic methods of processing lipoaspirate were used [ 7-9, 15, 16, 18, 26, 27], in 2 of them mechanical methods [3, 6] and in one were not processed [22]. Among the enzymatic methods, only 2 products had a name other than “SVF” - “solution of adipose derived stem cells” [10] and “adipose derived regenerative cells” [5]. Among the mechanical processing methods, with the exception of the two cases mentioned above, the authors used the terms: "fat", "purified fat", "cells from adipose tissue", "nanofat", "fat micrografts". Thus, despite the fact that most authors describe the technology for obtaining and processing adipose tissue as “standard”, the stages of the technology involve the same type of manipulation, but the products used for injection into the wound are designated very differently. A unified terminology for adipose derived products used in the wounds treatment has not yet been adopted. The most common is the term "stromal-vascular fraction of adipose tissue".

Application methods. The next stage of lipografting is the introduction of the resulting product into the area of the recipient wound. Judging by the analyzed sources, two methods of delivery are most often used in the treatment of wounds: local injections or application of adipose derived products directly to the wound surface (Table 2).

 

Table 2. Application methods of adipose derived products.

№	Author	Characteristic of wound	Application method
1.	Deng C[6]	Chronic wounds (>3 months)	Approximately 3/4 of the prepared SVF gel was injected directly into the base and edges of the wound, and the remaining gel covered the wound as a dressing, which was then covered with saline gauze.
2.	Nilforoushzadeh MA[7]	Fill-thickness neuropatic diabetic ulcers	A three-layer hydrogel skin substitute (inoculated with autologous keratinocytes, fibroblasts, and SVF) was transplanted onto the wounds.
3.	Konstantinow A[8]	Chronic venous and arterial ulcers from от 2 сm2 до 400 сm2.	4 ml of suspension of SVF was injected to a depth of 5-10 mm into the central area and the area adjacent to the ulcer in different directions. An additional 2.5 ml of the cell suspension was soaked in a collagen sponge cut to fit the wound and placed on the wound area.
4.	Samoylov AS.[9]	Thermal burns III–IV in TBSA from 1 to 80 %	Intradermal administration of autologous SVF AT was performed at 10–15 points around the damage to the skin.
5.	Marino G[10]	Chronic diabetic foot ulcers	Isolated cells were injected along the edges of the ulcer; cells were injected to a depth of 1 cm, trying to distribute them in all directions.
6.	Bene MD[11]	Digital ulcers	2–3 ml of fat was injected into the fingers using blunt cannulas, at the border of larger ulcers of different depths, or at the base of the fingers for smaller ulcers.
7.	Marangi GF[12]	Pressure ulcers.	1 cm3 of fat per 1 cm2 of the recipient zone was injected into the deep subcutaneous layer, where ischemic damage occurs, using a 3 mm diameter lipostructure cannula.
8.	Del Papa N[13]	Single diabetic foot ulcer (>5 months)	0.5–1 ml of autofraction was injected into the base of the affected finger by successive introduction of small aliquots in different directions from the injection site.
9.	Piccolo NS[3]	Burn wounds, diabetic foot ulcer, arterial ulcers	A 1 ml syringe is inserted through the needle puncture hole and (with force) is inserted directly under the wound bed or scar. The fat is then deposited retrograde over several "passes" until the entire area is entered. On average, 1.6–2.0 ml is injected for every 10 cm2 of area; 25–30 passes are required to inject 1 ml. Also injected through the wound and applied to the wound.
10.	Stasch T[14]	Chronic ulcers of lower extremities; wound surface more 2 сm2; and etiology, include only venous, arterial, diabetic, postoperative, posttraumatic and pressure.	2 ml of lipoaspirate per 1 cm2 of the wound, followed by infiltration of lipoaspirate into the edges of the wound and injection under the lesion into the bottom of the cleaned ulcer, if possible.
11.	Caviglia H[4]	Cutaneous fistulas	The concentrate of fat cells is injected in small portions along the edge of the lesion.
12.	Kim JH[15]	Post-injections face skin necrosis	The final solution was injected into the lesion in the subcutaneous and dermal layers near the wound.
13.	Carstens MH[16]	Arterial ulcers of lower extremities	In the presence of an ulcer, SVF (3–4 ml) was infiltrated directly under the wound and along its edges.
14.	Chopinaud M[17]	Hypertensive ulcers	Insertion of fat micrografts around and under the wound.
15.	Cervelli V[18]	Posttraumatic ulcer of lower  extremities	Direct injection into the bottom of the ulcer or injection into the perilesional area
16.	Mizushima T[5]	Cutaneous fistula no more 10 ml.	1/4 volume of the concentrated cell solution was injected percutaneously into the tissues around the fistula, and 1/4 volume was injected endoscopically into the submucosal tissue around the fistula. The remaining half was mixed with thrombin solution and injected with fibrinogen solution. Finally, the fistula was filled with fibrin glue containing ADSC and the skin was sutured.
17.	Giuggioli D[19]	Stump-skin ulcer	From 0.5 to 1 ml of fat is injected into the edges of the wound and into the subcutaneous plane around the entire perimeter of the finger stump (crown-shaped
18.	Smith OJ[20]	Single diabetic foot ulcer >25 mm2 and <10000 mm2 (>3 months)	The incision was made at least 1 cm from the edge of the wound and the fat was infiltrated using a threading technique to allow the fat to enter the natural tissue planes. Approximately 2 ml of lipoaspirate was infiltrated per 1 cm2 of the wound.
19.	Piccolo NS[21]	Face burns and wounds.	Introduced through the needle puncture hole and immediately inserted under the wound bed. The fat is then deposited retrogradely, in several "passes", until the entire area is transplanted (through the required number of punctures along the periphery of the wound). On average, 1.8–2.5 cm3 of fat is injected for every 10 cm2, and 25–30 “passes” must be made to inject 1 cm3.
20.	Williams EA[22]	Chronic venous ulcers of lower extremities	Fat is injected using microcannulas to reimplant it around the edges of the ulcer.
21.	Biyao Z[23]	Diabetic foot ulcer	unknown
22.	Rangaswamy M[24]	Flaps-needed difficult wounds.	Three-layer matrix of platelet-rich fibrin gel, condensed liposuction own fat and porous collagen dressing.
23.	Abouzaid AM[25]	Superficial and deep burns	Single injection of autologous adipose tissue, followed by coating of the wound with nanofat.
24.	Carstens MH[26]	Diabetic foot ulcer.	SVF cells in a volume of 20 cm3 were injected into the subcutaneous tissues surrounding the ulcer along the perimeter using a series of injections of a small volume (0.5 cm3)
25.	Tanios E[27]	Chronic trophic ulcers.	Autologous stem cell injections every 3 weeks.
26	Ryabkov MG.[28]	Chronic trophic and pressure ulcers. posttraumatic wound	The middle fraction of lipoaspirate was moved to the surface of the wound or filled its cavity.

 

In 17 studies, the application of i adipose derived products was local intradermal injections into the edges and under the bed of the recipient wound [3-6, 8-22, 25, 27]. In 4 cases, in addition to injections, applications were additionally used on the wound surface [5, 6, 8, 25]. Only 4 studies used isolated application of adipose tissue products to the wound surface [3, 7, 24, 28]. It should be noted that extensive wounds of the skin were the subject of treatment and study of only two studies included in the analysis [8, 9]. In the rest, there were either no mentions of the size of the wound surface [3, 4, 6, 7, 10-13, 15-19, 21-28], or patients with single wounds of small sizes were included in the study [5, 14, 20]. This may explain the predominant use of local injections as a method of delivering the drug to the wound. In the treatment of extensive and/or multiple infected wounds, local injections, as a method of introducing products of autologous adipose tissue, may be unnecessarily time-consuming, traumatic, and, in case of extensive burns, technically unfeasible.

Dosage of autologous adipose derived products. For an objective assessment of the effectiveness of the use of any drug or product introduced into the human body, it is necessary to determine its dose, which makes it possible to unify the method of its use. Most products from adipose tissue, regardless of the method of processing, contain stem cells, so the cellularity of the product can be considered an objective characteristic, i.e. the content of cells in 1 ml. It should be noted that out of 26 sources included in the review, the cellularity of the administered product was objectified and indicated only in 7 cases [5, 7-10, 26]. The cellularity index ranged from (min) 1x10⁴/ml [7] to (max) 50x10⁶/ml [9]. However, a positive effect in the form of wound healing or trophic ulcers was achieved in all the considered works. Further randomized clinical trials are needed to accurately recommend the minimum effective and maximum tolerable doses for treating wounds with adipose tissue products.

Conclusions. Systematization of literature data on the use of autologous adipose derived products for wound treatment has shown that at present this technology is safe, provided with an accessible instrumental base, has positive results and is being actively developed. The technique for sampling adipose tissue is standard, but has a number of features in the presence of infected wounds in patients. Among the products of adipose tissue, there is still no single nomenclature, therefore, in order to unify the methods for their production and processing, it is necessary to approve a common terminology for all. Further randomized clinical trials, which will also include extensive and multiple wounds, are required to determine the minimum effective and maximum allowable dose in the treatment of wounds with adipose tissue products. In addition, it is promising to develop new means and methods for delivering adipose tissue products in the treatment of infected wounds of various origins.

About the authors

Nikita Anatolyevich Koloshein

University Clinic of the Federal State Budgetary Educational Institution of Higher Education "Volga Research Medical University" of the Ministry of Health of the Russian Federation

Author for correspondence.
Email: n.koloshein@mail.ru
ORCID iD: 0000-0002-4563-5478
SPIN-code: 8542-6969

Junior Researcher of the Burn Injury Study Group

Russian Federation, 603005, Russia, 10/1 Minin and Pozharsky Square

References

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