|Year : 2021 | Volume
| Issue : 3 | Page : 172-178
Viability and durability of diced cartilage graft combined and formed with autologous blood fibrin (experimental study)
Tolga Aksan1, Muhammed Besir Ozturk1, Mehmet Ozer2, Ilknur Mansuroglu3, Mithat Akan4
1 Department of Plastic and Reconstructive and Aesthetic Surgery, Faculty of Medicine, Istanbul Medeniyet University, Goztepe Training and Research Hospital, Istanbul, Turkey
2 Pathology Clinic, Bayburt State Hospital, Bayburt, Turkey
3 Department of Pathology, University of Health Sciences, Okmeydani Training and Research Hospital, Istanbul, Turkey
4 Private Clinic, Plastic and Reconstructive Surgery and Aesthetic Surgery, Ataşehir, Istanbul, Turkey
|Date of Submission||20-Dec-2020|
|Date of Acceptance||03-Jun-2021|
|Date of Web Publication||29-Jul-2021|
Dr. Tolga Aksan
Veysel Karani Mah, Gulhane Cad, No: 7/AA-14 Sancaktepe, Istanbul 34885
Source of Support: None, Conflict of Interest: None
Introduction: Rhinoplasty constitutes an example wherein the various types of filling materials are used for nasal dorsal deformities. In this study, we took the advantage of a simplified procedure and used fibrin isolated from the coagulated peripheral blood of the experimental animal. Durability and viability of diced autologous cartilage grafts adhered to each other and formed using fibrin extracts were assessed experimentally in a rabbit model. Subjects and Methods: Diced cartilage graft (Group 1), block cartilage (Group 2), and fibrin adhered 0.5 mm diced cartilage graft (Group 3) were placed into subcutaneous pockets in the frontoparietal region of the same experimental animal. The grafts were excised at the end of 16 weeks. The excised grafts were macroscopically evaluated, and the final volumes and weights were measured. These measurements were compared to the initial values before implantation. Finally, the specimens were evaluated the histopathological examination. Results: In Group 1, 165% increase in weight and 56% increase in volume; in Group 2, 131% increase in weight and 81% increase in volume; finally in Group 3, 184% increase in weight and 68% increase in volume were seen. Although histological examination did not reveal any difference with regard to chondroid matrix score, metachromasia, vascularization, fibrosis, and inflammation we observed significantly higher cartilage proliferation score in Group 3. Conclusion: The results indicate that these grafts can be used efficiently, particularly in rhinoplasty procedures to correct nasal deformities.
Keywords: Blood, cartilage, fibrin tissue adhesive, rabbits, rhinoplasty
|How to cite this article:|
Aksan T, Ozturk MB, Ozer M, Mansuroglu I, Akan M. Viability and durability of diced cartilage graft combined and formed with autologous blood fibrin (experimental study). Turk J Plast Surg 2021;29:172-8
|How to cite this URL:|
Aksan T, Ozturk MB, Ozer M, Mansuroglu I, Akan M. Viability and durability of diced cartilage graft combined and formed with autologous blood fibrin (experimental study). Turk J Plast Surg [serial online] 2021 [cited 2021 Sep 28];29:172-8. Available from: http://www.turkjplastsurg.org/text.asp?2021/29/3/172/322674
| Introduction|| |
A wide variety of implants and fillings are utilized particularly for maxillofacial and esthetic surgical procedures in plastic surgery. These materials could be classified as alloplastic, heterogenic (allograft), and autologous grafts. There are certain advantages and disadvantages associated with the use of these materials as grafts as they have distinctive, material-specific features. The surgeon usually chooses the material deemed appropriate based on personal experience and surgical needs.
Autologous cartilage grafts are generally used as a block for esthetic or functional purposes for nasal dorsal augmentation as well as to correct contour irregularities and alleviate sharp lines emerged after hump reduction or osteotomy as a single block or fragmented or crushed grafts. The surgically placed grafts are susceptible to resorption, and moreover, fragmented grafts may migrate and get distorted after surgery, resulting in emergence of various type of deformities. To resolve these issues which are more commonly observed in diced or crushed cartilage grafts, various technical modifications have been proposed. Covering the grafts with oxidized regenerating cellulose is the most popular approach described by Erol. This modification has been considered successful despite having certain disadvantages.
In this study, we obtained diced cartilage graft from experimental animals (rabbit) and adhered to each other and reshaped using fibrin obtained following autologous blood coagulation. The durability, viability, and ability to maintain the reformed shape were assessed using cartilage grafts prepared in the manner we described.
| Subjects and Methods|| |
A total of eight New Zealand rabbits (4 month old) weighing 3000–3500 g were used in this study. General anesthesia was induced using Xylazine (3–5 mg/kg) and Ketamine (30–40 mg/kg). Autologous cartilage grafts were obtaining by amputating 2/3 distal region of the left ear of the experimental animal, followed by excision off the skin and perichondrium. A single, 25 mm × 7 mm in size cartilage was cut off from the thickest region of the smooth surface, which was in close proximity to anterior border, of isolated cartilage block. The remaining cartilage was then diced into 1 mm × 1 mm fragments. The diced cartilages were prepared in 0.5 ml volume using a syringe [Figure 1].
|Figure 1: Marking the location of the block cartilage (1 mm thickness) from the cartilage graft obtained from the left ear (a), preparing 1x1mm cartilage pieces (b), diced cartilage graft in a volume of 0.5ml (c)|
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These prepared 0.5 ml in volume cartilage grafts was mixed with 2.5 ml of blood drawn from the right ear of the rabbit in a metal container. For 5 min, the metal container was subjected to the rotation motion to enable adhering all cartilage fragments to fibrin glue. Plasma and erythrocytes were removed from these clot-cartilage complexes using gauze. The fibrin-sliced cartilage mixture was shaped into the cylindrical form without disturbing adherence maintained by fibrin glue and placed again into 1 ml syringe. To prevent detachment of sliced cartilages from each other during placing the cartilage-fibrin complex into the syringe and to facilitate the procedure a syringe with a 2.5 mm slit on the side was used [Figure 2]. We observed that this mixture had a volume around 0.55–0.60 ml and a 0.1–0.2 g increase in total weight.
|Figure 2: Diced grafts of 0.5ml of cartilage (a) were glued with autogenous fibrin using 2.5ml of blood (b to d) and placed in the syringe(e,f)|
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In accordance with the study plan, we created three 1 cm × 4 cm pockets with no connection with each other in the frontal region of each experimental animal and each pocket created for the placement of grafts was considered as a single experimental condition.
Group 1 (first pocket): Cartilage grafts diced into the smaller pieces of equal size (1 mm × 1 mm) were shaped into the cylindrical form of 0.5 ml volume using a syringe and placed into the pocket.
Group 2 (second pocket): A single block of 25 mm × 7 mm cartilage graft having a thickness of 1 mm was placed into the pocket.
Group 3 (third pocket): 0.5 ml volume of cartilage grafts diced into smaller pieces of equal size, measured using a syringe. Cartilage grafts were adhered to each other using fibrin isolated from the same experimental animal, shaped into a cylindrical form and was placed into the pocket [Figure 3] and [Figure 4].
|Figure 3: Prepared grafts(a), and placed the grafts in the subperiosteal pockets prepared on the rabbit's head(b to d).|
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At the end of 16 weeks, all experimental animals underwent euthanasia after clinical evaluation, weight and volume of surgically excised grafts, which were placed during the prior operation, were measured. Moreover, macroscopic examination was performed to evaluate whether the grafts preserved their shape at the time of first surgical operation [Figure 5].
|Figure 5: Sixteen weeks later, the skin on the grafts was excised and then the grafts were excised from their places and examined|
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To evaluate the cartilage differentiation and density of excised cartilages, histopathological examination was performed.
Chondroid matrix (collagen content using Masson Trichrome), metachromasia (by using Toluidine Blue) and chondrocyte number in lacunae, vascularization, fibrosis, inflammation (by using H and E), and chondrocyte proliferation (by using Ki 67 immunohistochemistry) were assessed. All specimens were evaluated by the same blinded pathologist. Matrix metacromasia, inflammation, vascularization, fibrosis, and chondroid matrix collagen contents were scored semiquantitatively, including “+”: low, “++”: moderate, “+++”: high, “++++”: very high. The number of chondrocyte nuclei in the lacunae was calculated at × 400 magnification area. Ki 67 proliferation index was calculated and scored by proportioning the number of chondrocyte nuclei showing nuclear staining in the areas where proliferation was most intense, to the total number of nuclei in the same area. The ratio of nuclear staining cells to the total number of nuclei in the same area was scored as “+” if 1%–2%, “++” if 3%–4%, “+++” if 4%–5%.
| Results|| |
Upon excision of skin covering the grafts, we observed that all the grafts were in place, none of cartilage pieces in any group migrated and there was slight vascularization in tissue in close proximity to grafts in all groups. After the isolation of implanted grafts through excision, grafts in the second group (block cartilage) preserved the structure and natural cartilage elasticity. Cylindric structure of grafts in Groups 1 and 3 was deformed with blunted corner edges and transformed slightly to have a ellipsoid-discoid share. However, the grafts were readily detachable from the surrounding tissue. Grafts in Groups 1 and 3 had significant hypertrophy. Close inspection revealed that tiny cartilage pieces were clearly and unequivocally visible. The texture of grafts in Group 1 was harder than that of grafts in Group 2, whereas grafts in Group 3 had the hardest texture among all the groups.
Assessment of weight and volume measurements
We observed an increase in weight and volume in all groups. Groups 1 and 3 had the most significant increment [Table 1].
|Table 1: Volume and weight measurements of cartilage grafts before and at the end of the experiment|
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Average of initial and final weight measurements in diced cartilage graft group was 0.31 ± 0.05 g and 0.82 ± 0.27 g (165% increase), respectively (P = 0.012). In block cartilage graft group, average initial weight was 0.13 + 0.01 and average final weight was 0.30 ± 0.11 g (131% increase) (P = 0.012). On the other hand, in fibrin-adhered cartilage group, graft weights were measured before and after the application of fibrin. Average of initial weight of grafts before and after fibrin application was 0.31 + 0.04 g and 0.41 + 0.03 g, respectively, whereas average of final measurement was 0.89 + 0.3 g. Based on these results, we observed 184% and 117% increase in weight when final measurements were compared to initial measurements conducted before and after the application of fibrin, respectively [Graph 1].
Average of initial and final volume measurements in sliced cartilage graft was 0.50 ± 0.00 ml and 0.78 ± 0.21 ml (56% increase), respectively (P = 0.017). In block cartilage graft group, average initial volume was 0.21 + 0.03 ml and average final volume was 0.38 ± 0.08 ml (81% increase) (P = 0.011). In autologous fibrin-adhered cartilage group, graft volumes were measured before and after the application of fibrin. Average of initial volume of grafts before the fibrin application was 0.50 + 0.00 ml while average of final measurement was 0.84 + 0.17 ml. Based on these results, we observed 68% increase in volume when the final volume was compared to initial volume prior fibrin application whereas when compared to the measurement after fibrin application, there was 44% increase in volume (P = 0.029 and P = 0.017) [Graph 2].
The findings obtained after the histopathological examination to assess cartilage differentiation and density are summarized in [Table 2].
Statistical analysis of histological findings revealed that there was no statistical significance among groups with regard to metachromasia, inflammation, and chondrocyte fraction (P > 0.05). In diced cartilage and autologous fibrin-adhered diced cartilage groups compared to block cartilage group, we observed a trend toward increase in fibrosis scores (P = 0.393). Proliferation scores in autologous fibrin-adhered diced cartilage group were significantly higher compared to diced and block cartilage groups (P = 0.043).
| Discussion|| |
A variety of materials including alloplastic, heterogenic (allograft-homograft), and autogenic grafts in rhinoplasty operations, particularly to correct or prevent dorsal irregularities in cases underwent osseocartilaginous dorsum reduction procedures have been described and utilized. Each of this material has certain advantages and disadvantages. Varying degree of success has been described with the use of alloplastic materials, although they are not associated with donor region morbidity. However, infection and foreign body reaction are commonly encountered potential issues. Graft resorption and potential infections are the main issues observed with the use of allogeneic grafts. The use of autogenic grafts is the most popular approach, although the use of this type of graft is associated with partial resorption and donor region morbidity.
A variety of tissues such as cartilage, bone, fascia, and dermis have been utilized as autologous tissue sources.,,,,,,, Lin and Lawson compared grafts and implants used in rhinoplasty with regard to complication and found that autologous cartilage grafts stand as a superior source for this type of procedures.
A technique developed by Erol and termed “Turkish delight” using diced cartilages wrapped in Surgicel® has gained popularity in rhinoplasty. Despite reported short- and long-term success, experimental studies revealed that grafts prepared using this approach substantially resorbed.,, Daniel and Calvert. and Calvert et al. observed that the whole graft had underwent resorption when they used Surgical-wrapped diced cartilage grafts. On the contrary, the use of diced cartilage graft wrapped in autogenic temporal fascia yielded long-term durable responses. In a similar study, Brenner et al. reported that autologous temporal fascia had a negative impact on cartilage viability and regeneration. However, the use of a different donor as source and incision scar is the limiting factors for this approach.
Diced cartilage grafts can be implanted to the nasal dorsum without wrapping in any material. Although sufficient durable responses can be achieved, this approach has not gained popularity as cartilage pieces could migrate leading to new deformities and subcutaneous irregularities.
Bracaglia et al. mixed the diced cartilage grafts with fibrin glue to keep them together and adhered them to each other, used the reshaped cartilage grafts to correct dorsal irregularities after rhinoplasty and claimed they did not observe volume loss in the long-term follow-up. Although it stands as a rational approach, additional cost associated with the use of fibrin glue is considered a disadvantage.
Öreroğlu et al. developed a novel approach that we also adopted in our study, prepared grafts by mixing bone powder and diced cartilage grafts with patient blood and used these grafts for dorsal irregularities in rhinoplasty. In a similar manner, Codazzi et al. used grafts prepared by adhering diced cartilage grafts using warmed blood for dorsal irregularities in rhinoplasty. Codazzi prepared the mixture after warming blood rather than using blood at the room temperature and claimed that this could facilitate and accelerate coagulation. Although both authors reported successful results, this study is the first study permitting in-depth analysis of the findings. In this study, by taking advantage of natural coagulation, isolated fibrin glue was used to hold together the diced cartilage grafts and the impact of this approach on graft shape and viability was assessed experimentally in a controlled fashion.
In line with multiple studies in this field, we used rabbit ear cartilage as it serves as a reliable animal model and increases the possibility to procure sufficient amount of cartilage.,,,,,, The experimental animals were followed up for 120 days, the longest reported duration in the literature, to attain more reliable results.
Blood coagulation, characterized by multiple coagulation factors playing roles in intricate coagulation cascades eventually leading to conversion of fibrinogen to fibrin, is set off by extrinsic pathway activation triggered by injury to vessel wall or surrounding tissue or intrinsic pathway initiated in the blood itself. Although initiation of clotting differs between extrinsic and intrinsic pathways, they ultimately converge on a common pathway resulting in clot formation. In this study, diced cartilages were mixed with blood to obtain a naturally-occurring clot and cartilage pieces were trapped in the clot. By this means, a single reformable soft cartilage graft was obtained, which was composed of multiple small cartilage pieces.
The cartilage graft prepared in this manner contains fibrin, platelets, and low amount of erythrocytes in addition to cartilage. Fibrin network adheres cartilages to each other as well as provides a formidable framework resistant to in vivo forces through cross-linked fibrin molecules. In histopathological evaluation, although the average number of chondrocytes in lacunae per unit in diced cartilage group (109.37/×5) was not statistically different than fibrin-glued diced cartilage group (117.5/×5), it is considered that autologous fibrin glue keeps the cartilages denser through holding the pieces together and makes them less susceptible to external forces. Moreover, it enables the nourishment of cartilage fragments without creating a physical barrier that could mitigate diffusion and is also associated with more favorable cartilage viability. In this regard, the previous studies demonstrated that dicing cartilages into smaller fragments facilitates their nourishment and results in higher chondrocyte density. In this study, as we have not placed a barrier such as Surgicel, fascia or Alloderm between diced cartilages, there was no barrier that could potentially hinder cartilage nourishment.
The fibrin filaments get degraded over time and cellular elements including leukocytes and fibroblasts enable the removal of cellular debris in the clot. This process leads to emergence of permanent adhesion between cartilage fragments and hence generation of a stable graft. Macroscopic and histological findings corroborated these results.
The number of platelets in rabbits ranges from 200 to 650 × 106/cm3 and as a component of clotting cascade platelets are abundant in the framework of prepared graft. In this technique, which is the subject of our study could be assumed, this condition creates an effect similar to platelet-rich plasma (PRP) or platelet-rich fibrin (PRF). The platelet harbor granules facilitating clotting and growth factors assisting in wound healing. Previous studies have shown that PRP has a significant regenerative effect on cartilage. Bullocks et al. took a different approach and used diced cartilage grafts formed using fibrin glue, which was generated by mixing plasma and PRP isolated from patient blood following centrifugation with bovine thrombin and calcium and achieved favorable clinical results. Gode et al. evaluated the effects of using PRF with diced cartilage grafts in rhinoplasty and achieved better permanence in grafts with PRF. In our study, significantly superior regeneration ratio in cartilage grafts reshaped using fibrin glue compared to other groups suggests that platelets may play an essential role.
Platelets carry a multitude of growth factors and transforming growth factor β1 (TGF-β1) 40–100 times in higher amounts than those in other type of cells and immediately release these soluble factors to surrounding milieu upon activation. TGF-β1 is pleiotropic cytokine exerting a multitude of functions under biological and pathological conditions. Recent studies demonstrated that TGF-β1 plays a pivotal role in the development of fibrosis. It has been shown that concurrent use of PRP and TGF-β1 blocking agents give rise to less fibrosis and more favorable wound healing. In a similar manner, the presence of fibrin has been linked to increased fibrosis. In our study, it could be reasoned that statistically not significant trend toward increased fibrosis and more rigid texture in fibrin-glued diced cartilage group when compared to other groups were due to this type of effects of platelets and fibrin.
Previous studies demonstrated that cartilage grafts implanted in subcutaneous plane of rabbits could have up to four times increase in weight and volume, depending on the duration of implantation. In this study, volume and weight comparison was carried out before placing the grafts in subcutaneous tissue, 16 weeks after implantation and at the end of the experiment. In all groups, more than 100% and 50% increases in volume and weight, respectively, were observed. The increases in both volume and weight are significant despite the fact that soft-tissue contribution could reflect upon these measurements as complete detachment and extraction of pure cartilage grafts from surrounding tissues was challenging. The present study shows that the extracted grafts grew larger along with rabbits as these animals were in the active growth phase of their life cycle. These results demonstrate that implanted cartilage grafts not only act as fillers but also retained their viability and regeneration capacity. However, the growth of the graft has not been reported in similar clinical applications in humans (18–20). Experimental studies in adult rabbits that have completed the growth phase may provide more precise information.
Cartilage volumes in Groups 1 and 3 were set to 0.5 ml of equal volume and autologous fibrin was then applied to diced cartilages having 0.5 ml of volume in Group 3, considering the fact that generated fibrin could disappear over time through fibrinolysis which could lead to reduction in graft volume. To have a clear view on whether measurements with or without fibrin could skew the results final measurements were compared to both measurements performed before and after fibrin application. In both groups, the final measurements were significantly higher than the initial measurements. As expected, the increase was even more pronounced when compared to measurements before fibrin application.
| Conclusion|| |
The findings obtained by macroscopic and histological assessment led us to conclude that the use of diced cartilage grafts adhered to each other by autologous naturally occurring fibrin may be use in rhinoplasty operations through provision of permanent sufficient support.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]