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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 28  |  Issue : 2  |  Page : 83-88

Evaluation the effect of isogenically prepared platelet-rich plasma on the viability of composite graft in the rabbit model (Experimental study)


1 Department of Plastic, Reconstructive and Aesthetic Surgery, Bahçelievler State Hospital, Istanbul, Turkey
2 Department of Plastic, Reconstructive and Aesthetic Surgery, Medical Faculty, Istanbul Medeniyet University, Istanbul, Turkey
3 Plastic, Reconstructive and Aesthetic Surgeon, Private Practice, Istanbul, Turkey

Date of Submission19-Jul-2019
Date of Acceptance31-Aug-2019
Date of Web Publication18-Mar-2020

Correspondence Address:
Dr. Tolga Aksan
Egitim Mah. Dr. Erkin Cad. Istanbul Medeniyet University Goztepe Education and Research Hospital, Department of Plastic, Rekonstructive and Aesthetic Surgery, Kadikoy, Istanbul
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tjps.tjps_68_19

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  Abstract 


Aim: Necrosis in composite grafts used for the repair of tissue defects in the nasal wings, columella, and nasal sidewalls after tumor resection or trauma is an important problem related to the size of the graft. Success in the surgical or medical interventions on the recipient field or graft in order to enhance composite graft survival is clinically limited. The aim of this study is to research the effect of platelet-rich plasma (PRP) on composite graft viability during graft operation. Materials and Methods: One of the eight adults New Zealand white rabbits was used isogenically to obtain PRP. Circular full-thickness chondrocutaneous composite grafts in 2 cm diameter were excised from the both ears of each remaining seven rabbits. Right ears were separated as experimental group and left ears were selected as control group. In the experimental group, PRP was applied subcutaneously to the four different points on inner and outer surfaces at the graft donor and topically to the graft sides. The composite graft was turned 180°, and it is sutured back to the donor area. PRP was also applied on suture lines. Composite grafts formed in control group were turned for 180° and sutured without any application. At the end of the 21st day, histological evaluation was performed following measurement and photodocumentation of living and necrosis areas. Results: In the control group, except one total necrosis, partial necrosis and viable areas were identified at the other composite grafts. Mean viable area measurements in the experimental group were 33.62% and 21.15% in the control group (P < 0.05). Histological evaluation was based on the appearance of chondrocyte nuclei in the lacuna in cartilage tissue and the living areas were confirmed histologically. Angiogenesis, fibroblast proliferation, and inflammatory cell proliferation were not significantly different between the groups. Conclusion: Although the viability ratio in the experiment group was found significantly higher than that of the control group, 100% of viability ratio was not identified in any of the composite grafts. However, our findings support that PRP is positively effective on the enhancement of the composite graft viability as well.

Keywords: Composite graft, experimental, necrosis, platelet-rich plasma


How to cite this article:
Karahangil M, Aksan T, Ozturk MB, Mutlu OO. Evaluation the effect of isogenically prepared platelet-rich plasma on the viability of composite graft in the rabbit model (Experimental study). Turk J Plast Surg 2020;28:83-8

How to cite this URL:
Karahangil M, Aksan T, Ozturk MB, Mutlu OO. Evaluation the effect of isogenically prepared platelet-rich plasma on the viability of composite graft in the rabbit model (Experimental study). Turk J Plast Surg [serial online] 2020 [cited 2020 Mar 28];28:83-8. Available from: http://www.turkjplastsurg.org/text.asp?2020/28/2/83/280993




  Introduction Top


Reconstruction of especially of nasal defects following malignant tumor excisions or traumas can be challenging in terms of plastic surgery. While various techniques are used in the reconstruction of these regions, defects, especially in cartilaginous sites, such as the nasal wings and the columella require sufficiently thin and color-matching composite tissues containing the skin, cartilage, and mucosa.[1]

Composite grafts taken from the ear come forth, especially with their suitable three-dimensional structure, similarity to the defects in the facial region, sufficient cartilage support, low donor-site morbidity, and availability of autologous tissue.[2]

According to the literature, composite grafts ranging between 0.75 and 1.5 cm can be confidently used, whereas viability issues are encountered in grafts larger than 1.5 cm.[3] Among the different methods used for increasing the viability of composite grafts, some have achieved successful outcomes; however, these were not adequately adopted in clinical practice.[4],[5] Platelet-rich plasma (PRP), through growth factors resulting from the activation and degranulation of platelets, has stimulating effect on cell proliferation and differentiation in favor of neoangiogenesis and tissue regeneration.[6] While PRP is used in many medical fields, successful results are reported in the literature for its use in nonhealing wounds, bone and soft-tissue defects, degenerative diseases, in flap and graft procedures (hair, skin, bone, cartilage, etc.), and esthetic surgery and cosmetic applications.[7],[8],[9]

In the present study, we investigated the effect of PRP application performed simultaneously with graft transfer procedures on composite graft survival in grafting procedures, in regards to the effects of the growth factors for neoangiogenesis and tissue regeneration on cell proliferation and differentiation.


  Materials and Methods Top


The study was conducted with the approval (no. 2012-126) of the Animal Experimentation Ethics Committee of the Bezmialem Vakif University and used eight isogeneic New Zealand white rabbits. All eight rabbits were adult females and weighed 3–4 kg. Originally, the study was planned with 11 subjects; however, the Ethics Committee revised this number on the grounds that eight would be significant and sufficient both experimentally and statistically. Anesthesia was started with 5 mg/kg intramuscular xylazine and maintained with 40 mg/kg intramuscular ketamine.

Since all the subjects had the same blood type and antigens, blood was taken from one of the subjects for obtaining PRP and the subject was sacrificed. Of the total 63 ml blood taken from this subject, 9 ml was processed with an Autologous Thrombin Kit (Harvest Technologies Corporation, Plymouth, MA, USA), and the remaining 54 ml with a PRP kit (SmartPReP® 2 APC+™ Harvest Technologies Corporation, Plymouth, MA, USA). A four-fold increase in platelet count in the obtained PRP (from 190.000/μl to 800.000/μl) was confirmed with blood count. In total, 7 ml PRP and 2.1 ml concentration of autologous thrombin to be used for PRP activation were obtained as a result of the process [Figure 1]a. This provided the 1 ml PRP application and the 0.3 ml autologous thrombin for PRP activation for each subject immediately before the application. To ensure maximum efficacy PRP was applied to all subjects within the 4 h after preparation.
Figure 1: (a) (left) Platelet-rich plasma (PRP); (right) thrombin solution. (b) Two centimeter diameter circle area marked in the middle 1/3 part of the ear to harvest the graft. (c) Excised composite graft and ear defect. (d) Activated PRP injection was performed on all 4 sides of the recipient site. (e) The graft rotated 180 degrees and PRP was applied to the wound lips of the graft. (f) View of the ear after the operation is completed

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A full-thickness chondrocutaneous composite graft of 2-cm diameter was excised on the mid-one-thirds section of both ears in each of the remaining seven rabbits. Bleeding areas were not cauterized to avoid damage in the surrounding tissues; instead, tamponade was applied and after some waiting time the vessel was ligated to stop the bleeding. In each rabbit, PRP was topically applied to the wound lips of the graft donor site on the right ear, and a total of 0.8 ml (8 ml × 0.1 ml) was subcutaneously injected into the four edges of the defect from both sides. Then, the composite graft was rotated 180° and sutured into place with 6-0 polypropylene on both sides, and the remaining PRP (0.5 ml) was applied topically along the wound junction. The left ear was rotated 180° and sutured into place without PRP application [Figure 1]. The right ear was evaluated as the study ear and the left ear as the control. Having both a study and a control ear on the same subject ensured to maintain the same physiological conditions and helped to reduce the number of subjects to a minimum.

All control and study ears were followed up for 21 days for graft integrity, healing, and necrosis after the composite graft was adapted to the defect. Recovery of the grafts was documented with weekly photographs. On the 21st day, the ratio of living and necrotic areas to the total surface area was calculated manually using transparent millimetric paper and digital analysis program (FreeHand MX) [Figure 2]. At the end of the 21st day, the grafts were excised with 0.5 cm intact margins. Cartilage viability, vascular proliferation in the dermis, fibroblast proliferation, lymphocyte, polymorphonuclear (PMNL) leukocyte, and histiocyte infiltration were histologically evaluated. All subjects were sacrificed at the end of the process.
Figure 2: (a) Shows necrotic and viable areas of the graft on the twenty- first day in experimental group. (b) Shows necrotic and viable areas of the graft on the twenty- first day in control group. (c) Marking of viable and necrotic areas with transparent millimetric paper. (d) Marking of viable and necrotic areas by using computer software (Freehand MX)

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Statistical analyses were performed with Number Cruncher Statistical System 2007 and Power Analysis and Sample Size 2008 Statistical Software (Utah, USA). The Mann-Whitney U-test was used for intergroup comparisons.


  Results Top


All subjects survived until the end of the study. No local or general complications or infections that could affect the study were observed. Measurement and evaluation of viable areas revealed some live tissue in all composite control grafts, except for total necrosis in one graft.

Mean live areas were measured 33.6% (24.2%–39.9%) and 96.1 mm2 (69–114 mm2) in the study ears and 21.2% (0%–33.8%) and 21.1 mm2( 0–96 mm2) in the control ears [Table 1]. The higher rate of live areas in the study ears group was statistically significant compared to that of the control ears group (P < 0.05).
Table 1: Viable area measurements

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Distinct chondrocyte nuclei were observed in the cartilaginous lacunae in the histological evaluation of the areas that were assessed as viable in the macroscopic examination. Chondrocyte nuclei were not observed in the cartilaginous lacunae of graft tissue that was evaluated as necrotic, but intense inflammatory cell infiltration rich in polymorphic nucleus leukocytes was identified [Figure 3]. Vascular proliferation and fibroblast proliferation in the dermis were observed in all except the necrotic graft, but the difference between the two groups was not found significant. Inflammatory response cell infiltration was more intense in necrotic areas but histologically evaluated as comparable [Table 2].
Figure 3: (a) In viable grafts, the nuclei of chondrocytes are clearly selected in cartilage lacuna and fibroblast proliferation is observed in the dermis. (b) In necrotic grafts, the chondrocytes within the lacunae have lost their nuclei, and the density of inflammatory cells in the dermis is observed. (Hematoxylin and Eosin stained preparations were obtained at X100 magnification)

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Table 2: Histological evaluation results

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  Discussion Top


While cellular damage due to ischemia, hypoxia, and hypoglycemia occurs within the first 48 h after composite grafts are sutured to the recipient site, damage due to secondary ischemia occurs after new vascular structures are formed between the recipient bed and the graft. Transient ischemia occurs in this process as a result of weakened blood flow if PMNL leukocytes bind to the capillary endothelium of the graft. When PMNL migrate into the surrounding tissues, they release their destructive enzymes, and triggering reactions associated with increased free radicals, inflict cell damage on the composite graft. Success rate is thought to increase if new vessel formation is ensured before cell deterioration begins.[10] Since irreversible changes occur between the graft and the recipient bed before new vessel formation, the success of nonvascularized composite grafts decreases as the thickness of the graft increases. The success of the composite graft will increase if the changes in the tissue can be reduced. Experimental studies found that an average of 72 h is required for neovascularization under normal conditions.[11]

There are many studies in the literature that have investigated methods for increasing the survival of composite grafts, and rabbit ears, because of the likeness of their cartilaginous and soft-tissue structure to that of the human ear, were more preferred for viability assessment in experimental studies.[12],[13],[14] Using rabbits in our study allowed us to compare our results with those reported in the literature.

Similar studies have found a survival rate of 1%–13% in the 3rd week in ear models that did not receive any additional treatment.[13],[15] Various pharmacological agents were used in the studies aimed at enhancing composite graft survival.[16] Among these, methylprednisolone comes forth as a successful agent.[12] Aden and Biel[15] used methylprednisolone, dimethylsulfoxide, chlorpromazine, and indomethacin in their study comparing pharmacologic agents. This experimental study found that 30 mg/kg intramuscular methylprednisolone administered 1 h before the surgery and for 7 days postoperatively significantly reduced graft necrosis compared to the control group. Lim et al.,[4] in their prospective animal study, found that dimethyl thiourea and melatonin were effective in the various phases of ischemia-reperfusion and had favorable effects on the survival of composite grafts. Other applications that have been identified to increase the survival rate of composite grafts are heparin to prevent intravascular coagulation,[17] ice application to slow down metabolism,[18] and hyperbaric oxygen therapy.[16],[19] These methods, however, have disadvantages such as cost, compatibility, as well as side effects and unavailability of the drug.

Graft vascularization occurs through vascular anastomosis between the subdermal plexuses of the graft and the recipient bed. Given the necessity of this bridging phenomenon for revascularization, the diameter of the graft should be of a certain size. As in all grafts, protecting the composite graft from excessively damaging forces is important for ensuring revascularization.[20]

Studies conducted for enhancing the survival of composite grafts sought to increase the stability and the revascularization of the graft by increasing the contact surface area. In 1959, Davenport and Bernard[21] used the “tongue-in-groove” technique and preparing a wedge-shaped recipient bed that fit into the groove of the graft, increased the contact surface by 50%. This technique, improved graft stability, reduced the tension on the recipient bed, and increased the surface area for revascularization.

PRP is a small volume plasma, in which autologous platelets are concentrated; their count is usually 3–5 times the count of basal platelets. When platelets are activated and degranulated, they release seven basic growth factors. Wound healing process begins once these growth factors are released. These factors have a stimulating effect on cell proliferation and differentiation in favor of neoangiogenesis and tissue regeneration.[6] Given these effects, they are, both experimentally and clinically, used in many areas and investigated in studies.[22],[23],[24],[25],[26]

Normally platelets circulate for 7–10 days. Once they migrate out of the circulatory system and are activated, they degranulate within the 1st h and release granules containing high coagulation and growth factors.[27] There are no studies that have determined the precise duration of action of PRP. Degranulation of platelets and release of growth factors have been suggested to occur in the first 3–5 days and the activity of growth factors to last about 7–10 days.[28] (We believe that this timeframe favorably affects the 1st week, which is the most critical period for the survival of the composite graft).

Pires Fraga et al.[29] having investigated the effect of PRP on the survival of free fat grafts in a rabbit model, found that viable fat weight was significantly higher in the study group compared to the control group. Furthermore, the researchers report that histopathological evaluation found significantly higher counts of viable adipocytes and vessels in the study group, but significantly more necrotic and fibrotic areas in the control group. The researchers nevertheless emphasize the importance and necessity of activating PRP with thrombin for the method to be effective and indicate that 70% of the growth factors are released within the 1st h after PRP activation. Normally, active release of these growth factors begins with the blood clotting process. This natural process can be initiated or accelerated by adding calcium chloride or thrombin.[30]

In our study, PRP was activated with thrombin immediately prior to application in each subject, and given the difficulty of injection, active PRP was applied before turning to gel. Vascular proliferation was semi-quantitatively evaluated in our study. In the control group, no vascular proliferation was observed in 1 subject that was developing total necrosis, vascular proliferation was observed to be less in 2 of the control ears compared to the study ears, while no differences were identified between the remaining study and the control ears in terms of vascular proliferation.

PRP-related angiogenesis is reported to take 3–5 days.[31] In fact, Choi et al.[32] showed that PRP injection into the recipient site for 3 days before the surgery provided higher vascularization and increased survival rates up to 97% compared to peri- or post-operative PRP. They report to have left the unilateral perichondrium intact during the operation and fixed the grafts at the same donor site. As was the case in our study, PRP injections administered during the procedure were shown to increase graft survival in the referred study. Based on these results, although clinical application may be challenging, PRP injections administered into both the donor site and the recipient site for 3 days prior to the composite grafting procedure may significantly enhance the survival of the graft.

Sevim et al.[33] administered PRP injections to the donor site or to the graft in different groups 7 days prior to the grafting procedure and transferred the graft later in a second session to test graft survival in composite grafts of different sizes. The researchers found that PRP application increased survival rate, especially in grafts ≥1.5 cm in size and that this increase was more significant when PRP was administered to the recipient site. In our study, we administered PRP to the donor site and the edges of the graft in one session during the procedure and observed comparably favorable effects.


  Conclusion Top


Many clinical and experimental studies have been carried out with the aim of increasing the survival rate of composite grafts, since composite grafts were introduced in head-and-neck reconstruction procedures. Although there are recent studies on the effects of PRP on composite grafts, there are no studies in the literature reporting on the simultaneous application of PRP to the graft and to the donor site. Although we did not achieve 100% graft survival in our experimental study, our results show that PRP significantly increases and favorably affects composite graft survival and support the results of the similar studies available in the literature where PRP was combined with composite grafts.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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33.
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