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![]() Surgical Science, 2010, 1, 1-6 doi:10.4236/ss.2010.11001 Published Online July 2010 (http://www.SciRP.org/journal/ss) Copyright © 2010 SciRes. SS Experimental Results of the Fibrin Clot Use to Accelerate the Regeneration of Damaged Bone in the Rat Lower Jaw I. V. Maiborodin, A. I. Shevela, T. V. Perrin, I. S. Kolesnikov, V. A. Matveeva, A. A. Shevela, B. V. Sheplev, I. A. Kolmakova Center of New Medical Technologies, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia E-mail: imai@mail.ru Received June 8, 2010; accepted July 13, 2010 Abstract Morphological and radiological methods were used to study regeneration of the damaged bone of rat mandi- bles after application of platelet-enriched fibrin clot. A bone hole was artificially created, and in the natural course of regeneration, the hole was immediately filled with blood and there a blood clot formed. After one week of healing, separate islands of young bone tissue appeared. After two to three weeks, the opening in the mandible was completely replaced by the young bone tissue. When a similar bone hole was filled with autological fibrin clot, the blood clot did not form. But after one week the entire hole was filed with newly-formed fused bone tissue. By the second week after the use of fibrin clot, the bone hole had further healed and bone callus was formed. Keywords: Fibrin Clot, Regeneration of Bone, Density of Bone Tissue 1. Introduction Tissue damage leads to the rupture of blood vessels, which, in turn, is the first step of platelet activation after contact with collagen. Platelets initiate the formation of thrombus through the activation of the coagulation sys- tem. After the formation of thrombin, fibrinogen is trans- formed into fibrin, and this is the first step of wound healing. The use of fibrin preparation recreates this pro- cess and accelerates healing [1,2]. Initially, fibrin preparation was used in dentistry to accelerate hemostasis after tooth extraction, especially with patients with blood clotting problems. It was also used to close defects in the bone tissue in the maxillofa- cial region [3,4]. Then, fibrin glue was used in lieu of sutures to attach tissue during operations, and to improve the grafting of implants from artificial and synthetic materials [5,6]. Platlet-rich plasma is a modification of the fibrin glue prepared from autologous blood and containing a set of cytokines, which causes the migration and division of all mesenchymal (including chondrocytes and mesenchymal stem cells) and epithelial cells, and stimulates the syn- thesis of collagen and the matrix of connective tissue [7,8]. When fibrin degrades, it causes a migration of osteo- genic cells and gingival fibroblasts in vitro and more rapid regeneration of surgical bone defects in the ex- periment in vivo. Fibrin glues and films can serve as a substrate to support the growth of fibroblasts and their functions. Thus, the adhesive material containing fibrin and fibronectin, their monomers or degradation products, accelerate the healing of periodontal tissue, including bone tissue [9,10]. Compared to the natural course of healing, applying of platelet-enriched fibrin clot (PEFC) results in less pro- nounced acute and chronic inflammation in damaged tissues. The alteration phase very rapidly replaced by re- generative-reparative processes. Therefore, application of fibrin materials can be used to accelerate tissue regen- eration and to facilitate the grafting of implants in ex- periments and in the clinic [11-13]. It should be noted that along with the positive results of the use of preparations of fibrin, there is evidence of the ineffectiveness of these therapies in dentistry [14-16]. Thus, the literature contains many contradictory and mutually exclusive data on the effectiveness of the use of fibrin preparations in surgery and dentistry. However, these studies do not reflect the use of fibrin for regener- ating bone tissue, and in particular, PEFC prepared from autologous blood plasma with platelets. ![]() 2 I. V. MAIBORODIN ET AL. 2. Aim By using morphological and radiological methods, the natural course of healing and the result of PEFC applica- tion to the regeneration of the damaged rat lower jaw were compare in experiment. 3. Material and Methods The experiment were used 6 month-old Wag male-rats weighing 180-200g. All procedures on the rats were per- formed under general anesthesia of ether inhalation in a sanitary operating environment in compliance with the “Regulations of the work using experimental animals.” At every point of the study, at least 6 rats were used. It was decided to create holes in bone tissue, which has few individual differences (especially blood vessels and nerves), and does not move when muscles are mov- ing. The lower jaw was chosen due to the fact that there is enough strength and width of the bone combined with ease of access. In addition, the rats cannot prematurely tear out their stitches. Preparation of PEFC: Several rats of the same breed were decapitated, and 2-7 ml of blood was collected in sterile glass tubes. This blood was centrifuged at 2800 rpm for 12 minutes [11,13]. Then the upper part (plate- let-rich fibrin clot or fibrin clot with platelet) was placed in sterile Petri dishes and maintained for several hours in an incubator at 37°C until use. Then, immediately before use, sterile scissors were used to cut the PEFC fragments to the correct sizes. Creation of the Bone Defect and PEFC Application in the Experiment: Under general inhalant ether anesthesia, in a clean operating room, while respecting the rules of asepsis and antisepsis, after treating the skin with 70% alcohol, a skin incision was made using a scalpel. The incision was 1.5-2 cm along the bottom edge of the man- dible. Retractors were used to detach the masticatory muscles and expose the lower surface of the lower jaw- bone. A dental drill was used at a specific manner (same size, even edges, depth control, the same rotational speed and, consequently, heating of tissues and the possibility of cooling) to create a 2 mm in diameter round hole through the bone in region of mandibular angle, the hole did not connect with the oral cavity. In the control group of rats (natural healing), the bone defect was covered with masticatory muscle and then simple running sutures were used on the skin, and it was treated with alcohol. In the other group, forceps were used to fill the holes with PEFC. The size of the PEFC was slightly larger than the holes. After packing the bones with PEFC, the bone de- fects were also covered with the masticatory muscle, the skin was sutured with continuous sutures, and alcohol was applied to the wound. All implant materials were sterile. Animals were withdrawn from the experiment after 1, 2, 3, 4 and 5 weeks after surgery. The bone tissue with defects in the mandible was studied. Fragments of the mandible were preserved in a 4% paraformaldehyde on biphosphate buffer (pH 7.4) for at least 24 hours. After preservation, the skin, subcutaneous tissue and chewing muscles were removed. The frag- ments of mandibles were decalcified in solution “Biodek R” (Bio Optica Milano, Italy) for 24 hours, dehydrated in a gradien of ethanol, lightened in xylene, and embedded in paraffin. Sections of 5-7 microns thick were stained with hematoxylin and eosin, and studied under a light microscope Axioimager M1 (Carl Zeiss, Germany) with a magnification of up to 1200 times. Radiological studies were performed to observe the reparative processes in the mandibles of experimental animals at various healing intervals (Figures 1 and 2). The tissue density was estimated in the hole itself, and in the contralateral part of the mandible. Statistical analysis was performed using applied statis- tical program of MS Excel 7.0 (Microsoft, USA). The arithmetic mean and standard deviation were determined, the differentiation between means was considered sig- nificant at p ≤ 0.05, used the Student’s criterion. 4. Results At 1 week after injury to the bones of the rat lower jaw with natural recovery, it was found that the hole was par- tially filled with blood and the hole contained some fragments of connective tissue and granulation (Figure 3). This marked the beginning of bone formation in the defect (formation of separate islands of young bones and cartilage among granulation) (Figure 3). After 2 weeks the hole was completely closed by the young bone tissue with a large number of blood vessels on the edge of the defect. Cartilage tissue was also pre- sent among the newly formed bone structures. At 3 weeks the hole was completely closed by the newly formed bone tissue. The only evidence that the defect existed were the remaining large vessels and ran- domly located bone trabeculae (bone callus). At this point, a fully formed cavity with bone marrow appeared. After 4 and 5 weeks, in most cases, the only remaining trace of the operation was bone callus (Figure 4). One week after injury to the bone with use of PEFC, the hole was completely filled with fused islets of newly formed bone (Figure 5). In other words, after the appli- cation of PEFC, bone regeneration resulted in the com- plete filling of the artificial hole after one week. In most cases, after two weeks, the injury to the bone was filled, regardless of whether PEFC was applied. The holes were closed with newly-formed bone tissue with a large number of blood vessels at the periphery of the defect and cartilage tissue in the center. Copyright © 2010 SciRes. SS ![]() A. HIGGINS ET AL. 3 Figure 1. Macroscopic appearance of rat mandible with remote masticatory muscles 1 week after injury and natural regeneration. There is no evidence of purulent inflamma- tory process. An arrow indicates the artificially created opening filled with blood clot. The two arrows—the root of the central incisor.
Figure 2. Macroscopic fragment of mandible of rat 1 week after a bone defect is then filled with PEFC. The artificially created hole has no macroscopic signs of inflammation, is filled, and is located at the level of the surrounding tissues. An arrow indicates the artificially created hole filled with PEFC. The two arrows—the root of the central incisor. After 3, 4 and 5 weeks of healing, the hole was com- pletely covered by newly formed bone tissue with ran- domly arranged bone trabeculae formed callus and cavi- ties with bone marrow (Figure 6). This was true in the mandibles that healed naturally and the ones with PEFC applied. After statistically controlling for data of densitometry of the rats’ mandibles’ bone defect regeneration in natu- ral healing and after applying PEFC, no significant dif- ferences in the density of tissue between the compared groups of animals was found at each point of the study. However, the density of tissue in the natural reparative processes was statistically significantly different from healthy bone on the contralateral side during the first 3 weeks. In contrast, the density of tissue in the PEFC as- sisted process was statistically significantly different from healthy bone on the contralateral side only during the first and second weeks. (Figures 7 and 8) (Table 1). That is, the bone tissue with PEFC applied became dense faster than with natural healing. In addition, it should be noted that all periods of ob- servation the density of tissue in the affected area after applying PEFC was slightly higher than during the natu- ral course of repair (Figures 7 and 8), although this dif- ference was not significant. The maximum difference in tissue density was noted from 2 to 4 weeks. By week 5, the differences were smoothed out somewhat (Table 1). Figure 3. The formation of bone structures at the periphery of the damaged part of the mandible in natural healing 1 week after surgery. Hematoxylin and eosin. Figure 4. Bone defect of the mandible with self-regeneration 4 weeks after surgery. Bone tissue struts in the callus are unstructured. Hematoxylin and eosin. Copyright © 2010 SciRes. SS ![]() 4 I. V. MAIBORODIN ET AL. Figure 5. Healing area of damaged bone of the mandible 1 week after surgery using PEFC. Bone defect filled with fused islands of the young bone tissue with a large number of vessels. Hematoxylin and eosin.
Figure 6. Structures of callus on the spot of the holes in the bones of the mandible 4 weeks after the operation and use PEFC. Hematoxylin and eosin.
5. Discussions In the experiment, when the bone tissue was damaged, an acute inflammation of the tissues occurred. This process occurs in response to direct tissue damage as a result of surgical intervention. Over time, an inflammatory reac- tion due to the operation subsides, and the process of restoration of damaged tissues begins. During the natural healing process, when the mandi- bles were damaged, the holes immediately filled with blood, and a clot formed with a large number of red blood cells. Gradually, this clot was dissolved by phagocytes (first neutrophils, then macrophages), and was gradually replaced by migrating osteogenic cells. Due to the func- tioning of osteoblasts, the young bone tissue began to take shape from the edges of the defect. These islands of young bone become wider and merge. In almost all cases, 2-3 weeks in rats, a complete bone regeneration took place in the artificially created defects. It should be noted that the morphological data on bone regeneration by the dates specified were confirmed by the results of densi- tometry. Fibrin in tissue, according to published data, reduces the severity of the inflammatory process [11-13] and limits the spread of infection [17,18]. That is, the intro- duction of PEFC in the cavity of the wound, apparently, can protect the surrounding tissues from the dissemina- tion of microorganisms, and from excessive exposure of lysosomal enzymes of phagocytes. This limits tissue de- struction and, therefore, earlier starting the regenerative processes in tissues, there is less antigen and detritus, and a more rapid cleansing of the wound. Figure 7. Bone defect of the mandible during the natural course of recovery 3 weeks after the operation, according to radiological study, artificially created hole (indicated by arrow) is preserved.
Figure 8. Artificially created opening in the bones of the lower jaw (indicated by arrows) is retained according ra- diological study 3 weeks after surgery using PEFC. The density of tissue in the defect after the application PEFC above.
Copyright © 2010 SciRes. SS ![]() A. HIGGINS ET AL. 5 Table 1. The bone density in defect of the lower jaw in comparison with surrounding intact tissues (S ± σ). Regeneration Process Time after operation Natural Healing After Using PEFC Difference in Density (Fibrin- Control) in Defect 1 Week 0.892 ± 0.053* 0.913 ± 0.017* 0.021 ± 0.05 2 Weeks 0.922 ± 0.038* 0.953 ± 0.021* 0.031 ± 0.033 3 Weeks 0.914 ± 0.033* 0.949 ± 0.036 0.035 ± 0.051 4 Weeks 0.912 ± 0.059 0.942 ± 0.048 0.03 ± 0.043 5 Weeks 0.913 ± 0.064 0.924 ± 0.063 0.011 ± 0.008 Note: * - data, significantly different from the intact bone on the con- tralateral side (р ≤ 0.05). In addition, the fibrin clot acts as matrix capturing mi- grating leukocytes (neutrophils), endotheliocytes and fi- broblasts [7-10]. Thrombospondin-1 from platelets stimu- lates tubulogenez (initial stage of angiogenesis) by en- dothelial cells [10]. Migrating through fibrin [7,8], neutrophils more rap- idly reach all sections of the wounds, even wounds cov- ered with layers of pus and detritus and, thus, tissues are more rapidly cleared from the antigenic substances (mi- croorganisms and the same detritus). In addition, when moving through fibrin clot neutrophils partially dilute it with its own enzymes, so even dense fibrin clots become less dense, and similar to a net. Fibroblasts, located in the fibrin network [7,8,10], be- gin the synthesis of collagen, not only from the bottom of the wound, but also from its cavity, thus the scar tissue forms more rapidly. It should be noted that the fibrin not only facilitates the migration of fibroblasts, but it also accelerates the synthesis of connective tissue [5,7,8,11-13,20]. Fibrin also stimulates the migration of endotheliocytes [7-9], and therefore the process of angiogenesis begins more quickly [19]. The newly formed blood vessels are located not only in the granulations on the wound bottom, but also in the fibrin net. The more rapid growth of blood vessels, in turn, facilitates migration of leukocytes from the blood vessels and synthesis of components of con- nective tissue. When the bone injuries were filled with PEFC, there was no need to wait for the blood clot will be destroyed and the red blood cells will be eliminated via through phagocytosis. After one week in most cases, the bone defect was already filled with fused islets of newly- formed bone tissue. That is, when PEFC was applied, the artifical defect was almost completely filled after one week. By the second week after using PEFC there was a fur- ther gradual filling of newly formed bone tissue in the defect with a large number of blood vessels in the pe- riphery. By third week the formation of bone callus completely covered the opening of the bone, also red bone marrow was observed in the defect. These changes continued to occur to varying degrees in subsequent pe- riods of observation. Fibrin is present in both natural healing and in the PEFC enhanced process. Fibrin facilitates the migration of neutrophils, endothelial cells, macrophages, osteoblasts and other cellular elements. However, what distinguishes natural healing is the large number of red blood cells in the blood clot. The presences of these cells in the fibrin net impede the migration of the aforementioned cellular elements. In addition, some potential phagocytes will be spent not only on the intake of detritus, but also on phagocytosis of red blood cells from a clot. Thus, on the basis of the foregoing, we conclude that when PEFC is applied, the start of the repair processes is more intense than in the spontaneous healing. The hole in the bone quickly filled with islands of bone tissue, which merged earlier than in the natural process. Appar- ently, the formation of the young bones begins immedi- ately after the operation without the need to spend time for the process of lysis and the removal of red blood cells from the clot. Since application of PEFC causes a more intense re- generation of damaged bone, it appears to be advisable to use PEFC to accelerate the reparative processes of bone tissue in dentistry, surgery and traumatology. This work was financial supported by the fundamental research program of the Presidium of RAS “Fundamental Science - Medicine” (project № 21.31 “Development of technologies for process management of bone tissue re- generation using biodegradable polymers”). 6. Conclusions In the natural course of regeneration, when the mandibles of rats were damaged, the defect was filled with a blood clot with a large number of red blood cells. After 1 week of healing, the damaged area contained separate islands of young bone tissue, as well as fragments of the blood clot and granulation. After 2-3 weeks of healing, the opening in the bone of the lower jaw was completely replaced by the young bone tissue. When the side of damaged rat mandibles was filled with PEFC, no blood clot was formed. After one week, the entire bone defect was filled with newly formed islets fused bone. By the second week after using PEFC there was a further substitution of the defect with bone tissue and the formation of bone callus. 7. References [1] M. E. Laidmae, J. L. McCormick, J. J. Herod, T. Pastore, E. S. Salum, P. A. Sawyer, Janmey and R. Uibo, “Stabil- ity, Sterility, Coagulation, and Immunologic Studies of Salmon Coagulation Proteins with Potential Use for Mammalian Wound Healing and Cell Engineering,” Copyright © 2010 SciRes. SS ![]() I. V. MAIBORODIN ET AL. Copyright © 2010 SciRes. SS 6 Biomaterials, Vol. 27, No. 34, 2006, pp. 5771-5779. [2] M. Valbonesi, “Fibrin Glues of Human Origin,” Best Practice & Research Clinical Haematology, Vol. 19, No. 1, 2006, pp. 191-203. [3] G. 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