Behavior of free omental grafts without vascular anastomosis implanted in cat’s experimental wounds. Behavior of free omental grafts without vascular anastomosis implanted in cat’s experimental wounds

: It is known that skin healing in cats is slower and has a higher complication rate than in dogs, what leads to search for new resources to enhance this process. Omentum properties in the aid of tissue repair are known and, among the forms of extraperitoneal omentalization, the use of free omental grafts without vascular anastomosis is advantageous, but little studied; there is no knowledge about its effect on skin healing, nor if it’s still viable when implanted in the subcutaneous space. Thus, the present study aimed to describe possible alterations produced by free omental graft without anastomosis inserted in the subcutaneous space, by means of macroscopic clinical analysis of the implantation region in cats. Twenty intact female cats were divided into two groups (Group 1: treated animals; Group 2: controls) of similar size. All animals were subjected to ovariosalpingohysterectomy (OSH) prior to the induction of an experimental wound for the implantation of the omental graft. Wounds were evaluated on days 1, 2, 4, 8, and 15 postsurgery and then weekly until no lesions were noted. Data regarding color, consistency, presence or absence of crusts, wound resistance, and volume measurements were recorded. There was activation of the omentum on the 4th day of evaluation with reduction of hemorrhage and an increase in the resistance to traction of the experimental wound. We concluded that the omentum without vascular anastomosis is capable of maintaining its viability and exert positive influence on the repair process without showing deleterious signs on the implantation site.


INTRODUCTION
Wound is defined as the interruption of the anatomical and functional integrity of the tissues. Wound healing is an orderly and complex process that has been conceptually divided into three overlapping and continuous phases ("the wound healing continuum"): inflammation, repair, and maturation (GUO & DIPIETRO, 2010). It has been shown that alterations during the healing process are more 1 Teixeira et al.
frequently observed in cats than in dogs especially if there is loss of subcutaneous tissue in these patients which may restrict the formation of granulation tissue (BOHLING & HENDERSON, 2006).
According to the literature, clinical and experimental studies have demonstrated the ability of the omentum to stimulate neovascularization, allow new lymphatic drainage, and potentiate the cicatricial process even in the presence of infection (SCHWARZKOPF et al., 2014). The omentum is used as an aid in the wound healing process mainly due to its vascular richness, and the presence/ synthesis of polypeptides and growth factors that have potent angiogenic properties (ZHANG et al., 1997). Due to the structure of this organ, there is great tissue availability and favorable vascularization (LIEBERMANN, 2000).
The types of omentum grafts that have been described in the literature include pediculate (LASCELLES et al., 1998), free with vascular anastomosis (ROA et al., 1999), and free without vascular anastomosis (REE et al., 2018).
Free omental grafts consist of the complete surgical removal of an omental fragment from its original site (donor region) and transfer (transplantation) to another region (recipient region). Once implanted into the recipient, omental grafts acquire new blood supply ensuring viability of the transplanted cells (PAVLETIC, 2010).
In small animals, the main advantages of the use of a free omental fragment without vascular anastomosis included minimizing the complications associated with pedicled omentum grafts (PETIT et al., 1979;BROCKMAN et al, 1996;LASCELLES et al., 1998) and facilitating the manipulation and implantation of the graft at sites distant from its origin, without requiring specific and expensive surgical materials as observed in the implantation technique of a free omental graft with vascular anastomosis (REE et al., 2018).
With regard to the complications that may be observed when the pedicled omental graft is used, albeit rare, partial necrosis of the omentum is the most frequently reported. This may occur due to a number of reasons including excessive tension on the vascular pedicle, pressure or strangulation at the site of exteriorization in the abdominal cavity or the use of an elongated and narrow subcutaneous tunnel. The presence of defects in the abdominal wall which results in hernia formation can also be observed as a complication during the use of this technique (BROCKMAN, 1996). With regard to the free omentum graft with vascular anastomosis, studies showed that there is still no effective success rate of the technique in veterinary medicine (PAP-SZEKERES et al., 2003).
More recently, studies using free omental grafts without vascular anastomosis have demonstrated promising results in aiding fracture repair, reducing mean bone healing time (BALTZER et al., 2015) and stimulating osteogenesis in hypertrophic nonunion (SAIFZADEH et al., 2009). BALTZER et al. (2015 used free omental grafts in the osteosynthesis of radial and ulnar fractures of dogs. These authors reported that the main complications reported in the surgical site were the difficulty in suturing the skin due to the presence of graft, edema, swelling, and erythema. These complications resolved 3 to 5 days following the surgery. Complete resolution of these complications occurred at day 7 postsurgery.
To the authors' knowledge, there are no published studies to date on the use of free omental graft as an aid in skin wound healing, and its behavior when implanted in the subcutaneous space has not been described. For this reason, the aim of this study was to evaluate possible alterations produced by free omental graft without anastomosis in the implantation region through macroscopic clinical analysis.

MATERIALS AND METHODS
Twenty female cats aged between 1 and 7 years old, originated from the Experimental Chemistry unit of the Veterinary Parasitology Laboratory (LQEPV) of the Federal Rural University of Rio de Janeiro (UFRRJ) that were subjected to elective ovariosalpingohysterectomy (OSH) were used in this study.
The animals were subjected to a full physical examination and laboratory evaluation that consisted of complete blood chemistry and CBC analysis including renal and hepatic function assessment and FIV/FeLV testing. Cats that presented any abnormalities in these laboratory tests were removed from the study.
All patients were subjected to the evaluation of the body condition scoring (BCS) on a previously published scale of 1 to 5 points according to EDNEY & SMITH (1986).
The animals were allocated into 2 groups of 10 cats each which differed in the use of free omentum. In the Treated Group (TG), a free omental graft without vascular anastomosis was placed under the experimental surgical wound of the skin whereas in the Control Group (CG) the procedure of synthesis of the experimental wound was performed without using the omentum.
Surgeries were performed at the Surgical Research and Extension Center (CCPE) of the Veterinary Institute of UFRRJ. Animals were put under general inhalation anesthesia, placed in dorsal recumbency, and median retroumbilical celiotomy and OSH surgery were performed. At the end of the OSH and prior to the celiorrhaphy, a fragment of the greater omentum was removed from the animals of the TG. The omentum was folded in order to expose its ventral aspect and, on the right side of the bursal portion, two blood vessels of the right gastroepiploic arcade were identified to limit the width of the fragment. The extension was obtained 2.5 cm to 3 cm from the caudal border of the omental pouch. After vascular ligation with 3-0 polyglactin 910, the omental fragment was excised and stored in sterile lactated Ringer's solution.
The abdominal wall musculature was sutured in standard X with 3-0 polyamide and subcutaneous cellular tissue with continuous suture using 3-0 absorbable polyglactin 910.
Prior to skin suturing, another incision of approximately 2 cm long located 1 cm cranial to the incision for OSH was made and was considered as the experimental wound. Such incision involved skin and subcutaneous tissue. For the production of a receptor bed, the subcutaneous tissue was drawn on each side of the incision in order to remove a fragment approximately 0.5 cm wide over the entire length of the incised skin. These procedures were performed in cats of both groups (TREATED and CONTROL).
The omental fragment maintained in sterile lactated Ringer's solution was then manipulated into a metal vat, and a portion of the omentum measuring approximately 2.0cm x 1.0cm was surgically removed. This tissue fragment was fixed to the muscular fascia with single stitches using 3-0 polyamide suture at each vertex of the experimental surgical wound on animals of the TG (Figure 1). Separate single points were used for skin raffia with 3 sutures made in Postoperative treatment consisted of broad-spectrum antibiotic therapy with benzathine penicillin in single dose (40.000U/Kg; Intramuscular route) right after the surgery, anti-inflammatory therapy with meloxicam (0.1mg/kg; Oral route; SID; 4 days), and analgesia with dipyrone (25mg/kg; Oral route; BID; 5 days) and tramadol hydrochloride (1mg/kg; Oral route; BID; 5 days). The animals wore a protective plastic collar until the stitches were removed.
Before cats were restrained for wound evaluation, the behavior of these animals while they were still inside individual cages was analyzed and videotaped. The animals' interest in the environment and in their surroundings were considered during this assessment. The cage's door was opened and the animal's reaction was observed. The physical interaction between the animal and the researcher consisted of gentle, smooth, slow movements until he or she was allowed to touch the animal's flank and the wounds for local pain assessment. In doing so, interest in moist food and appetite were also assessed.
Experimentally induced wounds were evaluated on days 1, 2, 4, 8 and 15 when the stitches were removed. After this period of time (time span), the evaluations were performed weekly in animals from the TG only until the volume of the omentum implantation region could no longer be noticed or measured.
Data on the gross aspect of the wounds (TATARUNAS et al., 1998) were recorded on written form and scored as: color (0 -no changes were noted in the surgical wound; the wound was compared with the integrated skin in the animal's abdomen, 1pinkish wound, 2 -reddish/purple wound); presence or absence of crusts on the surgical wound (0absence of crusts, 1 -mild crusting of the surgical wound, 2 -moderate crusting of the surgical wound, 3 -severe crusting of the surgical wound); wound strength (1 -suture dehiscence, 2 -spontaneous separation of the wound edge, 3 -separation of the wound edge under slight tension -when slight traction is made on the surgical wound, 4 -healing without the possibility of separation of the wound edge); palpation of the omentum implantation site (1 -the omentum implantation site was soft to the touch; the consistency of the omental graft site was similar to the consistency of the omentum; 2 -the omentum implantation site was firm to the touch -the consistency of the omental graft site was similar to the consistency of the abdominal wall).
A pachymeter was used to measure length, width and height of the omentum implantation site. These measurements were then recorded in written form.
The difference between the moments of the measurements of length, width, and height of the experimentally induced wound site was analyzed using the Kruskal-Wallis test and measured with the Dunn's multiple comparison test considering a significant difference when P<0.05.

RESULTS AND DISCUSSION
The most striking and significant change observed in the omental graft implantation site was the marked increase in volume ( Figure 2) which was noted 72h postsurgery as shown in table 1. Prior to this, no increase in volume was felt on palpation of the experimental wound. It is suggested that this is the time span required for the graft to form adhesions and vascular branches (THOMPSON, 1945) and releases its growth factors at the implantation site. This change in volume, which is referred to as "activated omentum", is possibly related to the release of endothelial growth factor (VEGF), since the omentum is considered a primary and excellent source of VEGF compared to other tissues of the body (ZHANG et al., 1997;LITBARG et al., 2007).
Until the 15 th day this volume could be noticed by palpation and measured with the aid of a pachymeter in all animals from the TG. The volume of the experimentally induced wound site could still be detected by palpation even several days after surgery. However, it was no longer possible to clearly delineate the extent and depth of the surgical wound and to measure it in the animals evaluated. The period of time in which the volume of the omental implant site could still be noticed on visual inspection and palpation and measured varied between 15 and 35 days, with an average of 25 days. After this period of time, weekly visits were done in order to assess these wounds until it was no longer possible to feel any volume in the omental grafting at the implant site. Few studies have described the behavior of a free omental at the graft fixation site. BALTZER et al. (2015) noted a local increase in volume with resolution of all cases on day 7 postsurgery. Our results are not in agreement with those previously published by these researchers as in the present study a reduction in volume of the omental graft was noticed on day 15 postsurgery. One hypothesis that may explain the slow graft regression would be differences in the vascularization of the implantation site. In the study carried out by BALTZER et al. (2015), free omentum was implanted on bone fractures surrounded by muscular tissue which is richer in vascularization than the subcutaneous tissue which is the site of implantation in the present study. Therefore, we may infer that the inflammation processes and repair have occurred more slowly.
In 2 animals from the TG, bilateral increase in volume of the mammary glands was observed. Such increase size was noted in the thoracic mammary glands and in the abdominal mammary glands as well. We may attribute such enlargement of the mammary glands to the angiogenic effect of the omental tissue in view of the marked increase from the intimate contact between the omental fragment and the abdominal mammary glands. The paracrine capacity and signaling of the omental adipose tissue (ZHANG et al., 1997) has been reported in the literature in conjunction with the aforementioned richness in the production of VEGF and mammary gland stimulation may be attributed to omentum activation and release of these factors. For this reason, care should be taken in the selection of animals subjected to omental grafting. Omental grafting should be discouraged in those animals with a diagnosis or suspicion of neoplasia.
The resistance of the experimental wound presented a significant advantage in the TG in the 2 days of evaluation of this parameter, as shown in figure 3. Such advantage was attributed to the presence of the omental graft in the subcutaneous space fixed in the muscular fascia due to its scarring potential (ZHANG et al, 1997). Although, in the CG the wound had presented a small dead space due to the removal of the subcutaneous tissues, that is primordial for feline wound healing, it is considered that there was no negative influence on the cicatricial process since an extensive tissue loss is necessary for impairment of wound healing . In the first 48h, both groups of animals showed differences in the color of the surgical wounds. The TG had a low staining score compared to the CG which had a higher staining score in the first couple of days (Figure 4). This fact may be related to the action of the graft as a natural hemostatic agent (THOMPSON, 1945;HOSGOOD, 2006;KOPPE et al., 2014) and also by the modulation of the local inflammatory reaction due to its presence in the dead space left by the withdrawal of the subcutaneous tissue. From day 4 onwards it was possible to notice an increase in the staining score in the TG which persisted for a longer period of time when compared to the CG. This may have occurred due to the fact that this is the time when the omental graft is activated generating a more pronounced metabolic process.
Most of the cats had a body condition scoring (BCS) 3 corresponding to the normal condition. Two animals of the TG were classified as  obese (BCS 5) and in the CG 1 animal had a body condition score 2. The two animals classified with BCS 5 presented a more severe inflammatory process and wound color changes persisted after the 10th day postsurgery. The omental implant in an obese animal should be evaluated with greater care as the omentum is equally greasy. These animals showed marked exudate/discharge during the wound healing process, increased crust formation, and changes in wound color for a longer period of time when compared to the other animals of the same group. This may be related to the prolongation of the inflammatory process (PAZ-FILHO, 2012).
With regard to the consistency of the lesion, in the first 4 days the omentum graft was soft and slightly mobile. At the end of 1 week, it was mostly firm and non-mobile. Our findings do not corroborate those from Thompson's report published in 1945 in which, at the end of 72 h, the free omentum graft applied to the lung lesion was firmer. It was also reported that at the end of 1 week there was absorption of the adipose tissue, and the graft could be separated with difficulty. In an experimental study in which the rat tibia was used, the free omental graft showed changes in consistency and became a firm tissue attached to the bone within 14 days postsurgery (OLOUMI et al., 2006). This change in consistency may be related to the absorption of the adipose tissue and fixation at the implanted site emphasizing its adherence ability.
There was no difficulty in skin suturing of the experimental wound with the presence of the omentum graft as previously reported by BALTZER et al. (2015). This is probably due to the fact that part of the subcutaneous tissue at the implant site was removed. In addition, BALTZER et al. (2015) implanted the graft on fractures with a metal implant in bones with little muscle covering.
The literature on use of a free omental graft without vascular anastomosis is scarce as there are few published studies on the subject to date. To the authors' knowledge, there are no articles on the patency of the free omental grafts in soft tissue and skin wounds which are of paramount importance in clinical studies.

CONCLUSION
Based on our findings, we concluded that the free omentum without vascular anastomosis is able to maintain its viability when applied in the subcutaneous tissue of cats without deleterious effects of the graft over the implanted region.