Effects of platelet rich plasma on the gastric serosal surface neomucosa formation: an experimental rodent model

Introduction

Patients with short bowel syndrome (SBS) has reduced intestinal absorption according to malabsorption and subsequent malnutrition, which often occurs after due to congenital defects, surgical bowel resection (volvulus, etc.) or loss of absorption associated with the disease [1], [2]. SBS may arise after the resection of more than 50% of small intestine and is certainly emerges after resection of more than 70% or when the less than 100 cm of small bowel left [3]. Increasing nutrient and fluid absorption by slowing intestinal transit time or increase the area of absorption via development of new intestinal mucosa, called neomucosa, must be purpose of the patients’ surgical treatments [3], [4]. Neomucosa formation is a potential technique for increasing the intestinal surface area including the growth of new intestinal mucosa, which takes advantage of the regenerative capability of the intestine. The regenerated intestine develops by lateral ingrowth from the surrounding mucosa and is functionally similar to normal intestinal mucosa [5].

Autologous platelet rich plasma (PRP) is the platelet concentration obtained by gradient centrifugation from thrombocytes in the plasma. During the healing process, the platelets are activated and aggregate together and then release the granules which stimulate the inflammatory cascade and healing process [6], [7]. Today, PRP injections have been safely used in many fields including dermatology, sports medicine, orthopedics, cosmetics, fasciomaxillary treatments, urology and neurophysiology and even in treatment of ulcers on extremities [6], [8], [9], [10], [11]. In surgery, some growth factor such as platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor β (TGFβ), epidermal growth factor (EGF) and fibroblast growth factor (FGF) are valuable markers used for cell generation or tissue regeneration [12], [13], [14], [15], [16], [17], [18].

So far, various animal models have been used to study the growth of intestinal neomucosa in full thickness defects patched with a variety of surfaces, including colonic serosa and abdominal wall [1], [5], [12], [19]. One of the most popular technique is the serosal patch technique. However, because of the limited serosal surface and anatomic factors, in many cases, only short segments of small intestine can be patched [5] and all procedures are still experimental. On the other hand, PRP has never been used in intestinal neomucosa formation with serosal patch technique. Among properties of PRP, there is increase tissue vascularity through increased angiogenesis. Increased angiogenesis leads to increased tissue vascularization, increased collagen synthesis, increased epithelial and granulation tissue production rates, postoperative leaks prevention and pain relief by serving as hemostasis and lymphatic band in the wound. Thus, PRP may help as an ideal paste-like agent in reconstructing the tissue and neomucosa formation since it is biologically compatible, effective and reliable [13].

In this experimental study, our aim was to investigate the potential biochemical and histopathological effects of PRP treatment on the neomucosa formation of the serosal surface of the stomach that was used as serosal patching in terminal ileal defect on rats to grow new intestinal mucosa. Our hypothesis was that PRP application promotes the healing process of intestinal anastomosis and neomucosa formation probably by accelerating tissue regeneration and remodeling through the pathways of VEGF, EGF, FGF, TGFβ factors.

Materials and methods

Surgical procedure and PRP treatment

Rats were anesthetized by a ketamine/xylazine anesthesia with dosage of 10 mg/kg xylazine and 60 mg/kg ketamine (Ketalar^®, Parke-Davis, Istanbul, Turkey, and xylazine 10 mg/kg; Rompun^®, Bayer, Istanbul, Turkey) and their abdomens were shaved. Antisepsis and asepsis was provided by 10% povidone-iodine solution and three cm midline abdominal incision was done under aseptic conditions.

Only laparotomy was applied to the Group 1 (sham group) under anesthesia.

Stomach and jejunum of the Group 2 (the control group) were manipulated and exposed.

In the Group 3, 1 cm longitudinal incision was applied on the jejunum. 0.5 mL PRP was injected to submucosal jejunum. The anastomosis of the jejunal defect with the serosal surface of stomach was performed with continuous 6.0 polypropylene sutures after the PRP injection (Figure 1A).

Figure 1:

Macroscopic findings of neomucosa formation.

(A) Application of platelet rich plasma (PRP) to the anastomotic line (along the staple line). (B) Anastomotic line between the gastric surface and jejunum. (C) Neomucosa formation on the gastric surface area of the fixed tissue.

The hearts of rat in the Group 4 were exposed to subcostal incision in order to obtain the PRP. About 5 mL blood were collected and the rats were sacrificed.

Each rat was subjected to relaparotomy under ketamine/xylazine anesthesia on postoperative day 30, and sacrification of the rats was achieved by intracardiac puncture.

Results

Histopathological findings

Microscopic evaluation of the control and PRP treatment groups were presented in Figure 2. In the control group, gastric mucosa on the upper part of full-thickness of stomach wall had anastomotic regenerative intestinal epithelium on both sides. Neomucosa formation was minimal and granulation tissues were covering the most of the serosal surface (Figure 2A). On the other hand, PRP treatment group had an enlarged gastric mucosa on full-thickness of the stomach wall, and new mucosa formation (neomucosa) on serosa (Figure 2B). The epithelium with their villi were continuously shown on the intestinal neomucosa, with goblet cells and absorptive cells surrounded the intestinal villi.

Figure 2:

Histopathological and morphological evaluation of control (A1, A2) and PRP treatment groups (B1, B2), Hematoxylin and eosin.

(A1) The control group (×100). (A2) Magnified figure of A1 (×200). (B1) The PRP treatment group (×100). (B2) Magnified figure of B1 (×200).

None of the histopathological processes including inflammatory process, fibrosis, granulation tissue and neomucosa formation were observed in the sham group, coming with zero score (Table 2, Figure 3). Histomorphological evaluations showed similar intensity of chronic inflammation and fibrosis in both control and PRP treated groups (Figure 3). However, average scores of granulation tissue formation showed significant differences between all groups. The mean of granulation increased gradually in the control group and then in PRP treatment group, compared to the sham group (p<0.01 and 0.001, respectively). As the main outcome of the study, neomucosa formation was observed in all rats of control and PRP treated groups but the percentage of the total neomucosa area was 85.63% in PRP treated rats whereas the percentage in the control group remained as 39.2% (Table 2, Figure 4). Thus, the mean ratio of neomucosa area increased significantly in PRP group, compared to the control and sham group (p<0.001).

Table 2:

Histopathological findings for inflammatory process, fibrosis, granulation tissue and neomucosa formation of the three groups.

		Group 1 (sham) (n=8)	Group 2 (control) (n=8)	Group 3 (PRP) (n=8)
Chronic inflammation	X±SD	0±0a	1.38±0.18	1.5±0.19
Fibrosis	X±SD	0±0a	1.25±0.25	1.25±0.16
Granulation tissue formation	X±SD	0±0	0.63±0.18b	1.38±0.18a
Neomucosa formation	%±SD	0±0a	39.25±6.04a	85.63±4.17a

1. X±SD, Mean±standard deviation; PRP, platelet rich plasma.

2. ^ap<0.001 vs. all groups. ^bp<0.01 vs. all group.

Figure 3:

A graphic for the histopathological scores of the sham (Group 1), control (Group 2) and platelet rich plasma (PRP) treatment group (Group 3). **p<0.01 and ***p<0.001 vs. all groups.

Figure 4:

A graphic for the percentage of neomucosa formation showing increased neomucosa ratio both in the sham (Group 1), control (Group 2) and platelet rich plasma (PRP) treatment group (Group 3). ***p<0.001 vs. all groups.

Discussion

In the present study, the main outcome is the anastomotic healing effect of PRP in neomucosa formation in intestinal anastomosis depending on modulating four valid determinant factors, VEGF, EGF, FGF, TGFβ. We affirmed our hypothesis by revealing that PRP application to the intestinal anastomosis significantly promoted the healing process of anastomosis probably by accelerating tissue regeneration and remodeling through increasing VEGF, EGF, FGF, TGFβ factors.

The usage of stomach serosal patching to grow new intestinal mucosa is a technique used for enlargement of the mucosal surface [3], [4]. The regenerated intestinal mucosa develops by lateral ingrowth of the adjacent mucosa and has same functions as normal intestinal mucosa. Up to date, certain surgical procedures have been directed towards increasing the surface and time of absorption. The main autologous intestinal reconstruction procedures are the longitudinal intestinal lengthening and tailoring known as Bianchi Procedure and the serial transverse enteroplasty. They have serious complications like stricture, leakage and bleeding [7]. Thus, we aimed to use growing neomucosa technique as a treatment model of SBS.

Growing factors such as VEGF, PDGF, TGFβ, EGF and IGF have been discussed in several studies according to their healing and regeneration effects [21], [22]. PRP contains different concentrations of these growth factors that constitute the theoretical basis of the use of PRP in healing and neomucosa formation. It has been used in general surgery to solve one of the major problems of the surgeons: intestinal anastomotic leakage. However, the beneficial effects of PRP have not be elucidated in growing neomucosa method as a treatment model of SBS.

Local bacterial contamination in the anastomosis is caused re-epithelialization of the damaged mucosal borders and secondary inflammatory response. Furthermore, in the intestine several bacterial products, such as the endotoxin lipopolysaccharide produced by Escherichia coli bacteria, influence epithelial homeostasis and wound healing [23]. Production of proinflammatory and anti-inflammatory cytokines such as TGFβ could be observed in this process. According to this findings, the increase in TGFβ levels can be explained by anastomosis and inflammation. Moreover, TGFβ in PRP affects the proliferation of fibroblasts according to their biological features as a paracrine growth factor.

The phases of wound healing are inflammatory, proliferation and maturation phases. TGFβ, an anti-inflammatory/prohealing cytokine, is major regulator of wound healing. In some studies, it was shown TGF presented high levels in the proliferative phase. Inflammation is an supplementary part of wound healing, and the pro-inflammatory cytokines are significant in this treatment. Injury begin an acute inflammatory reaction that enclose the discharge of a number of cytokines including IL-1β, TNFα and IFNγ. Following the intervention after the early injury, there is a replacement in the cytokine profile from pro-inflammatory to anti-inflammatory/prohealing cytokines, including IL-10, IL-13 and TGFβ. The anti-inflammatory cytokines restrict the period and greatness of the inflammatory response that provides the wound accesing to proliferative phase. This shift from an inflammatory environment to a healing one is coordinated by macrophages, likely under the direction of cytokine signals. Redstones et al. showed that, in the dynamic milieu of the healing process, glucagon-like-peptid-2 increased wound IL-1β, IFNγ levels but not TGFβ. Therefore, patients receiving GLP-2 agonist therapeutically who require surgery should not have impaired anastomotic healing [24].

TGFβ impact on the equilibrium between synthesis and breakdown of the ECM, inducing an elevation in production of matrix components and a reduction in proteolytic activity of ECM, resulting in fibrogenesis and differences in the ECM. Also, regulatory T cells are tethered by TGFβ. Therefore, TGF suppression may decrease fibrosis but rises inflammation and tissue damage in the intestinal wall. On the other hand, some studies concern that a common TGF suppression might be followed by inadequate wound healing.

However, in some models, it appears that low concentrations of TGFβ stimulate fibrosis, while high concentrations of TGFβ inhibit fibroblast proliferation and collagen synthesis [25]. In our study, TGFβ was found as significantly increased in PRP treated group compared to non-treated groups. It is suggested that there should be a balance between TGFβ levels and inflammatory factors to accelerate serosal neomucosa formations, as in the case of fibrosis and surgical wound healings.

In our study, EGF and FGF levels were also found significantly increased in PRP treated group compared to control and sham groups. This is based on the characteristic of PRP, initiating the healing process via the degranulation of the α-granules in platelets after their activation; these granules contain synthesized and prepackaged growth factors including PDGF, TGFβ, VEGF and EGF. These growth factors are active polypeptides which facilitate regeneration of injured tissue through acceleration of cell proliferation and matrix formation [26] thus, it is natural to show increased levels of these factors in PRP treatment. Moreover, FGF is known to regulate the tissue homeostasis and vascular branching morphogenesis and also increases the levels of TGFβ [19], so we observed increased levels of FGF in accompany with the elevated levels of TGFβ.

The reason for the treatment of PRP is to reduce the amount of red blood cells that are less useful in the healing process and to increase the rate of platelets amount of to 94% to stimulate healing [27]. Leukocytes produce metalloproteinase, free radicals, reactive oxygen species, and nitrogen, which can cause damage to healing [28]. Limitation of our study may be the presence of small amount leukocytes in PRP.

In years, the number of relevant articles have been published concerning PRP treatment, but comparing the all findings is arduous, because of the differences in processing platelets and using the distinct techniques. Most of them in accordance with positive effect of these substances on anastomosis repair, while the one article showed that applying of PRP could only enhanced fibrosis and granulation tissue, without restoration the breaking strength of anastomotic areas [29]. In our study, the fibrosis degree increased independently of PRP treatment but the level of granulation on the tissue was significantly different between non-treated and treated rats.

Inflammatory, proliferative, and the maturation phases which occurred with overlapping form intestinal healing process. And they take respectively, 0–4, 3–14 and 10–180 day after the surgery [30]. Usually, during the first phase, fibrin contributes to wound healing and strength, but the major strain is allocated to the sutures. Fibrosis which is the finding of secondary wound healing is composed of the chronic phase of healing. In our study, it was detected at similar density in all tissues, with no significant difference in the histopathological examination that was performed at a late stage. Therefore, fibrosis levels became similar between our non-treated and treated rats since FGF levels in PRP treated rats were also not as high as the levels of other biochemical parameters.

The main outcome of the study is that neomucosa formation in PRP treatment group was observed as approximately two-fold larger than the area of the control group. The use of autologous PRP has been considered a promising advance for new surgical and clinical approaches. Furthermore, recent advances in PRP usage should eliminate the risk of immunological reactions. This type of biological treatment mimics natural tissue healing, while optimizing and reducing the time required for therapy [5]. Our histopathological and biochemical findings reveal that the PRP therapy in gastrointestinal anastomoses is truly a beneficial and surgically applicable treatment.

It is important to point out that PRP therapy can be excellent preventive surgical technique against the anastomotic leakage. Furthermore, treatment with PRP might potentially find their place in a health system situation that is characterized by an increasing shortage of financial resources.