Surgical Science, 2010, 1, 1-6
doi:10.4236/ss.2010.11001 Published Online July 2010 (
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
Received June 8, 2010; accepted July 13, 2010
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
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. 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
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
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 arrowsthe 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 arrowsthe 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
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
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-
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
Copyright © 2010 SciRes. SS
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
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
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
Copyright © 2010 SciRes. SS
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. Carter, A. Goss, J. Lloyd and R. Tocchetti, “Tranex-
amic Acid Mouthwash Versus Autologous Fibrin Glue in
Patients Taking Warfarin Undergoing Dental Extractions:
A Randomized Prospective Clinical Study,” Journal of
Oral and Maxillofacial Surgery, Vol. 61, No. 12, 2003,
pp. 1432-1435.
[4] W. D. Spotnitz and R. Prabhu, “Fibrin Sealant Tissue
Adhesive—Review and Update,” Journal of Long-Term
Effects of Medical Implants, Vol. 15, No. 3, 2005, pp.
[5] W. Becker, “Fibrin Sealants in Implant and Periodontal
Treatment: Case Presentations,” Compend Contin Educ
Dent, Vol. 26, No. 8, 2005, pp. 539-545.
[6] J. Choukroun, A. Diss, A. Simonpieri, M. O. Girard, C.
Schoeffler, S. L. Dohan, A. J. Dohan, J. Mouhyi and D.
M. Dohan, “Platelet-Rich Fibrin (PRF): A Second-Ge-
neration Platelet Concentrate. Part IV: Clinical Effects on
Tissue Healing,” Oral Surgery, Oral Medicine, Oral Pa-
thology, Oral Radiology, Vol. 101, No. 3, 2006, pp. e56-
[7] M. B. Schmidt, E. H. Chen and S. E. Lynch, “A Review
of the Effects of Insulin-Like Growth Factor and Platelet
Derived Growth Factor on in Vivo Cartilage Healing and
Repair,” Osteoarthritis Cartilage, Vol. 14, No. 5, 2006,
pp. 403-412.
[8] D. Schwartz-Arad, L. Levin and M. Aba, “The Use of
Platelet Rich Plasma (PRP) and Platelet Rich Fibrin (PRF)
Extracts in Dental Implantology and Oral Surgery,” Re-
fuat Hapeh Vehashinayim, Vol. 24, No. 1, 2007, pp.
51-55, 84.
[9] E. L. Kaijzel, P. Koolwijk, M. G. vanErck, V. W. van-
Hinsbergh and M. P. deMaat, “Molecular Weight Fi-
brinogen Variants Determine Angiogenesis Rate in a Fi-
brin Matrix in Vitro And in Vivo,” Journal of Thrombo-
sis and Haemostasis, Vol. 4, No. 9, 2006, pp. 1975-1981.
[10] S. McDougall, J. Dallon, J. Sherratt and P. Maini, “Fi-
broblast Migration and Collagen Deposition During
Dermal Wound Healing: Mathematical Modelling and
Clinical Implications,” Philosophical Transactions Mat he-
matical Physical and Engineering Sciences, Vol. 364, No.
1843, 2006, pp. 1385-1405.
[11] I. V. Maiborodin, I. S. Kolesnikov, B. V. Sheplev, T. M.
Ragimova, A. I. Shevela, A. N. Kovyntsev, I. A. Ko-
makova, I. A. Pritchina, E. V. Kozlova, A. B. Voitovich
and D. N. Kovyntsev, “Granulomatous Inflammation af-
ter Use Fibrin Preparation,” Morphological Letters, No.
3-4, 2007, pp. 116-118.
[12] I. V. Maiborodin, I. S. Kolesnikov, B. V. Sheplev and T.
M. Ragimova, “Application of Fibrin and its Preparations
in Stomatology,” Stomatologiia (Mosk ), Vol. 87, No. 6,
2008, pp. 75-77.
[13] I. V. Maiborodin, I. S. Kolesnikov, B. V. Sheplev, T. M.
Ragimova, A. N. Kovyntsev, D. N. Kovyntsev and A. I.
Shevela, “Adjusting Gingival Tissues Morphology After
Dental Implantation with Fibrin Use,” Stomatologiia
(Mosk), Vol. 88, No. 1, 2009, pp. 9-13.
[14] S. J. Froum, S. S. Wallace, D. P. Tarnow and S. C. Cho,
“Effect of Platelet-Rich Plasma on Bone Growth and Os-
seointegration in Human Maxillary Sinus Grafts: Three
Bilateral Case Reports,” The International Journal of Pe-
riodontics & Restorative Dentistry, Vol. 22, No. 1, 2002,
pp. 45-53.
[15] G. Fuerst, R. Gruber, S. Tangl, F. Sanroman and G.
Watzek, “Effects of Fibrin Sealant Protein Concentrate
with and Without Platelet-Released Growth Factors on
Bony Healing of Cortical Mandibular Defects, an Ex-
perimental Study in Minipigs,” Clinical Oral Implants
Research, Vol. 15, No. 3, 2004, pp. 301-307.
[16] R. M. London, F. A. Roberts, D. A. Baker, M. D. Rohrer
and R. B. O’Neal, “Histologic Comparison of a Thermal
Dual-Etched Implant Surface to Machined, TPS, and HA
Surfaces: Bone Contact in Vivo in Rabbits,” The Interna-
tional Journal of Oral & Maxillofacial Implants, Vol. 17,
No. 3, 2002, pp. 369-376.
[17] J. Choukroun, A. Diss, A. Simonpieri, M. O. Girard, C.
Schoeffler, S. L. Dohan, A. J. Dohan, J. Mouhyi and D.
M. Dohan, “Platelet-Rich Fibrin (PRF): A Second-Gen-
eration Platelet Concentrate. Part V: Histologic Evalua-
tions of PRF Effects on Bone Allograft Maturation in Si-
nus Lift,” Oral Surgery, Oral Medicine, Oral Pathology,
Oral Radiology & Endodontics, Vol. 101, No. 3, 2006, pp.
[18] D. M. Dohan, J. Choukroun, A. Diss, S. L. Dohan, A. J.
Dohan, J. Mouhyi and B. Gogly, “Platelet-Rich Fibrin
(PRF): A Second-Generation Platelet Concentrate. Part
III: Leucocyte Activation: A New Feature for Platelet
Concentrates?” Oral Surgery, Oral Medicine, Oral Pa-
thology, Oral Radiology & Endodontics, Vol. 101, No. 3,
2006, pp. e51-e55.
[19] S. Kellouche, S. Mourah, A. Bonnefoy, D. Schoevaert, M.
P. Podgorniak, F. Calvo, M. F. Hoylaerts, C. Legrand and
C. Dosquet, “Platelets, Thrombospondin-1 and Human
Dermal Fibroblasts Cooperate for Stimulation of Endo-
thelial Cell Tubulogenesis through VEGF and PAI-1
Regulation,” Experimental Cell Research, Vol. 313, No.
3, 2007, pp. 486-499.
[20] K. Ito, Y. Yamada, T. Naiki and M. Ueda, “Simultaneous
Implant Placement and Bone Regeneration Around Den-
tal Implants Using Tissue-Engineered Bone with Fibrin
Glue, Mesenchymal Stem Cells and Platelet-Rich Plasma,”
Clinical Oral Implants Research, Vol. 17, No. 5, 2006,
pp. 579-586.