Efeito do Concentrado de Plasma em Falhas Ósseas Provocadas em Fêmures de Camundongos como Estimulação de Formação Óssea. Estudo Experimental Effect of Plasma Concentrate on Bone Defects Induced in Mice Femurs via Stimulation of Bone Formation: An Experimental Study

Os autores estudam experimentalmente o efeito do concentrado de plasma na estimulação óssea em camundongos. Foram utilizados dez camundongos isogênicos de linhagem gioto, onde após a coleta do sangue periférico de um camundongo, foi centrifugado este sangue e obtido um concentrado de plasma; foi utilizado o concentrado de plasma em falhas ósseas na região distal do fêmur, alternando-se os lados direito e esquerdo, sendo um lado com falha óssea isolada e o outro com falha óssea mais o concentrado de plasma e analisados quantitativamente e qualitativamente. Observou-se que o plasma não leva à estimulação da formação do calo ósseo, não ocorrendo aumento do processo inflamatório, e havendo apenas uma tendência a formar mais matriz óssea com o seu uso.


INTRODUCTION
The same as with other musculoskeletal tissues, the bone tissue is formed by mesenchymal cells inserted into an abundant extracellular matrix (1) .Unlike other tissues, the bone matrix contains minerals that provide the tissue with high strength and rigidity under load conditions (1) .
The main organic component of the bone matrix is type 1 collagen, which provides the bone with high strength to tensional forces (2) .Besides nerves, blood vessels and lymphatic vessels, bones present a complex blood supply.The periosteum consists of two layers, one external fibrous layer and one internal, more fibrous and vascular layer; the periosteum coats the external bone surfaces and participates in the fracture healing.The
Em uma fratura o sangue extravasa através dos vasos rotos e forma-se um hematoma entre as áreas da superfície fraturada ao seu redor.O hematoma fica quase totalmente contido no periosteum is thicker, and cellular in character.In breast-fed babies, as well as in small children, it presents a more complex blood supply as compared with adults.Perhaps due to these differences, in children the periosteum is more active as regards the healing of many types of fractures (1) .
There are two types of human bones, namely the cortical (compact) bone and the spongy (trabecular) bone.Long bones diaphyses consist virtually of cortical bone only.Metaphyses, on their turn, as well as most short and plane bones, have relatively thin-walled cortical bones, with large volumes of spongy bone.These differences in the distribution of cortical and spongy bones will cause differences in bone healing (1) .
According to their mechanical and biological properties, bones may be classified into two types, namely, the woven or immature bone and the lamellar or mature bone.The woven bone forms the embryonal bone and is replaced by lamellar bone as the skeleton develops (2) .Also, the woven bone forms the first tissue of the fracture repair and is replaced by lamellar bone as remodeling occurs.As compared to the lamellar bone, the woven bone has a higher deposition and resorption speed.The bone matrix has a woven bone with an irregular pattern of collagen fibrils containing approximately four times more osteocytes per volume unit than normal, as well as an irregular matrix mineralization pattern.The frequent formation of a sort of patchwork in the woven bone, plus the mineralization pattern in the form of focuses create an irregular radiographic appearance that distinguishes the woven irregular bone found in the fracture callus from the lamellar bone.Due to the lack of orientation of its collagen fibrils, the irregular mineralization and a relatively high cell concentration and water content, the woven bone is less rigid and more easily deformed than the lamellar bone (3).
A fracture will start its healing process as soon as the bone is broken and, under favorable conditions, this process follows a series of stages until the bone is completely healed.Histological characteristics of fracture repair in various time points after the injury were well described and referred mainly to fractures of several ribs in rabbits; such evidences suggest that the repairment process is similar to that found in humans.However, it should be taken into account that the type of healing is not the same for all bones under all circumstances, besides not being constant.The repair of a tubular bone is very different from that of a spongy bone and the type of healing of a segment is likely to be influenced by factors such as the rigid fixation of the fragments and the perfection of the coaptation (7,8). .The healing process of a fractured tubular bone occurs in five stages: 1. hematoma; 2. subperiosteal and endosteal proliferation; 3. callus formation; 4. healing; and 5. remodeling.However, it should be emphasized that these stages have no sharp limits and that two or more healing stages may occur simultaneously in different portions of the bone (3) .
In any fracture, blood will overflow through the broken vessels as a hematoma is formed between the areas of the broken surface around it.The hematoma is almost totally contained in the surrounding periosteum and may be shifted or removed from the ends of the bone to different extents.As the periosteum is removed, the hematoma may overflow into the surrounding soft tissues and ultimately, will be contained by muscles, fascias, and skin (7) .periósteo circundante, e pode ser deslocado ou retirado das extremidades do osso em menor ou maior intensidade.Quando o periósteo é retirado, o hematoma pode extravasar para os tecidos moles adjacentes ficando em última instância, contido por músculos, fáscias e pele (7) .
Quando a consolidação está completa, o osso recém formado forma em geral, um colar bulboso que circunda o osso e oblitera o canal medular.O tamanho do calo varia de caso para caso.Tende a ser grande nos casos onde tenha ocorrido um deslocamento periosteal extenso, um hematoma grande, ou ainda um desvio grande dos fragmentos ósseos.É em geral pequeno quando os fragmentos ósseos estiverem em posição Inevitably, a fracture will break the most part of the capillaries running along the compact bone, and the bone ring immediately adjacent to each side of the fracture will become ischemic to a certain extent, usually a few millimeters.With no blood supply, the osteocytes located near the fractured surface will die.
The most obvious characteristic of the early stages of a repair is the proliferation of cells in the deep surface of the periosteum close to the fracture.Such cells are the precursors of the osteoblasts that later must sediment the intercellular substance.The cells form a collar of active tissue that surrounds each fragment and grows towards another fragment.It is worth noticing that this cellular tissue is not the result of organization of the fracture's coagulate hematoma.In fact, the coagulate blood has little or no participation at all in the process of repairment and is replaced by the proliferation tissue, to be finally reabsorbed (5) .

Concomitantly with subperiosteal proliferation, cell activity occurs inside the medullar canal where proliferating cells seem to derive from the endosteum and tissues contained in the medulla of each fragment. This tissue also proliferates until it merges with the similar tissue that grows from the other fragment.
Islets of cartilage may be found in the cellular tissue that grows inside and outside the bone, forming a bridge between the fragments.Cartilage appears in different amounts ranging from absence to very large amounts.Obviously, it is not an essential element in the healing process (3) .
As the cellular tissue grows from each fragment, the fragment will mature and the basic cells will give origin to osteoblasts and, at some points, to chondroblasts that form the above-mentioned cartilage.Osteoblasts deposit an intercellular matrix formed by collagen and polysaccharide that soon becomes impregnated with calcium salts to form the immature bone of the fracture callus.Due to its texture, this callus was named "primary" bone (3) .
The formation of this bridge of primary bone provides the fracture with obvious rigidity, so when the injured bone is superficial, the callus may be felt as a rigid mass surrounding the fracture.The bone callus or primary bone can also be seen in radiographs and provides the first radiographic evidence that the fracture starts to heal (2) .
By the action of osteoblasts, the primary bone forming the primary callus is gradually converted into more mature bone with typical lamellar structure.
Once the healing is complete, usually the freshly made bone forms a bulbous collar that surrounds the bone and obliterates the medullar canal.The size of the callus varies from case to case, tending to be large where an extended periosteal displacement, a large hematoma or, yet, a large displacement of bone fragments has occurred.It is usually small when the bone fragments are in anatomical position, particularly if the fragments are rigidly fixed by a screwed metal plate or by intramedullar fixation (7)   .
The exuberant callus is rather common in children, since in them the periosteum is easily displaced from the bone by extravased blood, thus allowing bone to be formed in its interior.
In the months that follow the healing, the bone is gradually reinforced along the force lines at the expense of the excess bone existing outside the force lines, which is slowly removed.This unnoticeable remodeling process persists in constant activity in all bones for the rest of our lives, although it becomes specially anatômica e especialmente quando os fragmentos forem rigidamente fixados por uma placa de metal com parafusos ou por intramedular (7) .
The healing of a fractured spongy bone follows a pattern that is different from that of a cortical bone.Since the bone has a uniform spongy texture and no medullar canal, a more ample contact area exists among the fragments, that is, the trabecular weave that allows easier penetration and formation of bone tissue.The healing may occur directly between the surfaces of the bones and does not require neither the external callus nor the endosteal callus to occur, as is the case with cortical bones.
Trueta (10) emphasized this by observing that primitive nondifferentiated cells may be converted into osteoblasts under the influence of osteoinductive substances.The morphogenetic bone protein has such characteristic but, without a carrier, is not able to stimulate this response, since it will spread too quickly through heterotopic sites before osteoinduction can occur (11) .
In a 1997 literature review, Croci (3) describes studies proving that this non-collagen protein is obtained by acid demineralization of bovine bone and, currently, of bones from other animals and from humans, such as bone morphogenetic protein or BMP.This protein would act upon four cell-specific differentiating stages, namely, activation and proliferation of mesenchymal cells, cartilage formation, primitive bone formation and lamellar bone formation.The amount of newly formed bone is directly proportional to the amount of this protein, although its diffusion through the tissues is limited.It must be emphasized that the role of the BMP in bone healing was studied experimentally only; moreover, the stage during which it should be administered with the purpose of inducing a considerable osteogenic response is not proven yet.
Many studies were based on the use of BMP, including the ones that used transplantations with BMP combined with marrow bone, with quicker healing results, probably due to the presence in the bone marrow of osteogenic precursor cells or mesenchymal cells that respond to stimulation by BMP (12) .

CASES
Ten mice of the Gioto isogenic mouse strain were used, provided by the Bioterium of the Lusiadas Faculty of Medical Science of Santos.
Each animal was placed in a separate cage numbered from 1 to 10, and subjected to surgical procedures in the femur.
Ten femurs were alternated between the right and left sides, that is, five right and five left femurs as the control group (Group I), plus five right and five left femurs forming the group to receive previously collected plasma (Group II).

OBJECTIVE
The objective of this study is to experimentally evaluate bone healing in mice, using a hemoderivative (blood plasma) to stimulate bone callus formation in bone defects produced in mice femurs.

MATERIALS
1. Approximately 10 ml of peripheral blood of a mouse from the Gioto isogenic mouse strain were collected by surgical dissection of the abdominal aorta and puncture with a thick needle and a syringe, and centrifuged to enact blood phase separation.This product was then used in Group II of the trial.
2. Ten adult isogenic mice of the Goto mouse strain, weighing approximately 250 grams, provided by the Bioterium of the Lusiadas Faculty of Medical Science of Santos (Santos, SP, Brazil).After a physical evaluation, the physically able animals underwent surgical procedures and were divided into two groups (Group I and Group II); the experiments were performed on alternate sides.
Group I included the animals in whose femurs a 2-mm bone defect was induced alternating the right and left sides, so we obtained five femurs of mice that were operated in their right sides and five in their left sides, with no replacements (control group).
Group II included the animals whose bone defects were produced similarly to Group I, followed by introduction of previously obtained plasma concentrate.This gave us five operated right femurs with hemoderivative, plus five operated left femurs.
In summary, we had ten mice, therefore twenty operated femurs (ten on each side); Group I is the control group where bone defects were induced with no replacements, while in Group II the plasma concentrate was employed.

Anesthesia protocol:
Anesthesia protocol: Anesthesia protocol: Anesthesia protocol: Anesthesia protocol: All animals were intraperitoneally anesthesized using a 2% Virboxil ® (xylazine hydrochloride) solution at a dose of 0.01 ml/kg combined with Francotar ® (ketamine) at a dose of 0.50 mg/100 g.

Surgical technique: Surgical technique: Surgical technique: Surgical technique: Surgical technique: After the thighs were bilaterally trichotomized and 1% topic povidine was used as an antiseptic, sterile fields were positioned. The skin incision measured about 1 centimeter distal from the femur region, with dieresis of the soft tissues and hemostatic control.
Then a periosteal elevation was performed using a dislocator, followed by perforation of the right and left femurs one centimeter above the knee joint interline, using a drill and a titanium 2mm diameter drill bit using manual pressure and continuous irrigation with 0.9% saline (Figure 1A).This procedure was performed in the control group only, while the other group received injected plasma (Figure 1B).Soft tissues were sutured using 4/0 nylon monothread in isolated stitches.
After being confined in individual cages numbered from 1 to 10, the mice received 0.1 ml sodium dipyrone during the first 12 hours of the postoperative period.The animals were fed standard normoprotein feed and water ad libitum and maintained in the individual cages at room temperature between 19 and 20 ºC.

Figure 1 A and B. A -Bone defect of 2mm in distal femur. B -Injection of concentrated plasma.
and quantitative anatomopathological evaluation of the material.
Protocol for killing mice: Protocol for killing mice: Protocol for killing mice: Protocol for killing mice: Protocol for killing mice: Mice of both Group I and Group II were killed after being intraperitoneally anesthesized with xylazine hydrochloride at a dose of 0.01 ml/kg combined with ketamine at a dose of 0.50 mg/100 g.After the anesthesia, inhalation with ether was applied by mask using a lethal dose until cardiac and respiratory arrest was achieved.

Anatomopathological exam Anatomopathological exam Anatomopathological exam Anatomopathological exam
Anatomopathological exam protocol: protocol: protocol: protocol: protocol: The specimens removed were identified according to the number of the mouse and the side that was removed, according to the study protocol.All specimens were fixed in 10% formol solution for a 12hour period, decalcified in nitric acid for 24 hours and longitudinally sectioned along their longer axes.Then they were identified as per the protocol and the tissues were processed in baths of alcohol and xylol (in that sequence) and paraffinembedded in blocks to be sliced into 4-mm thick histological sections and stained by the hematoxyline-eosine method.
Two or three sections were produced from each block, that were then examined under a Nikon Alphaphot-2 (400x) Microscope (Figures 2A and B); we used the parameters below, that were taken into account in this study: 1. Hemorrhage; 2. Polymorphonuclears; 3. Granulation tissue; 4. Osteoblast activity; 5. Bone matrix; and 6.Osteoclasts.

EVALUATION CRITERIA
The anatomopathological evaluation was performed according to the usual parameters adopted by our Pathological Anatomy service to evaluate the inflammatory process and bone callus formation and scored for presence or absence of the findings obtained, as shown in Table 1.

STATISTICAL ANALYSIS
We observed both the absolute and relative frequencies of incidence of the anatomopathological endpoints in the sections obtained from Groups I and II.We also statistically described in both groups the quantitative parameters of he-presente estudo: 1. Hemorragia, 2. Polimorfonucleares, 3. Tecido de granulação, 4. Atividade osteoblástica, 5. Matriz óssea e 6. Osteoclastos.

-Statistical analysis of anatomopathological endpoints by
Fisher's test.
For the comparisons among parametric results we used Fisher's exact test and compared the events for each finding in Group I and Group II.For all results we adopted a 5% p value (p=0.05) and a trend between 5 and 10% (0.05<p<0.10).

RESULTS
Table 2 shows the results obtained using the criteria adopted in this study for Group I (control group) and Group II (with plasma concentrate).

COMPARATIVE STUDY
Table 3 shows the results of endpoints hemorrhage, polymorphonuclears, granulation tissue, osteoblast activity, bone matrix and osteoclasts in sections obtained from Group I (control group) and Group II (plasma group).

DISCUSSION
Studies directed to bone healing deserve more and more emphasis in the orthopedics area, mainly as regards the reduction in the healing time, which remains a challenge in Orthopedics.Reduce the time and use biological syntheses in the management of pseudoarthroses are a challenge to all professionals involved, and fully justify trials such as the present one (4) .
Ham and Harris (4) used rabbitt ribs to describe a process that is similar to that of humans in terms of healing, and reported that such processes are comparable, at least as regards the aspects studied; the stages described by those authors served as a base for our study.

made a rather complete review of certain aspects of the tubular bone repair, which is quite different from the spongy bone repair, and reached the conclusion that the type of healing of a given bone is influenced by various factors such as rigid fixation and a perfect reduction of the fragments.
In rabbits with intramedu-ainda é um desafio na Ortopedia.Diminuir o tempo e usar sínteses biológicas no tratamento de pseudoartroses são um desafio para todos os profissionais envolvidos e justificam plenamente experimentos como este (4) .
Em virtude da área da fratura ser pequena o número de campos microscópicos não ultrapassou cinco campos HPF por uma llar osteosyntheses inserted in the femur in an induced bone defect, Tavares and Cafalli (9) observed that the osteosyntheses caused an early healing of this bone defect, and we also based our trial on these stages.
In a literature review, Croci (3) described BMP (the bone morphogenetic protein) that would act in four specifically differentiated stages, with the formation of lamellar primitive bone, although experimentally only, with no details about the specific stage when the BMP should be introduced; this protein might be present even in the blood elements that might be used to stimulate bone callus formation.
Besides the above-mentioned fact, we used plasma concentrate to stimulate bone healing also because this plasma can be easily obtained from patients themselves, being thus a biological alternative that does not bring any relevant complications to the patient.
In order to better obtain the plasma concentrate, we used an isogenic mouse, which is genetically equal to the animals of the trial; this prevented the presence of a potential immunogenic reaction capable of interfering with the study.The objective of the study was to test the crossed use of plasma in the test of bone callus formation.
The method was homogeneized for bone defect and the right and left femurs were alternated to prevent the predominance of either member; also, during the postoperative period, the same biologist monitored the feeding and care of the mice while they were confined.
The anesthesia protocol consisted of an intraperitoneally administered combination of ketamine plus xylazine hydrochloride, because it is easy to use and the animals can be controlled; the surgical technique was carefully applied always with the same pattern and by the same team if order to prevent discrepancies; the rats were killed using the same anesthetic combination followed by ether inhalation until the final respiratory and cardiac arrest.
The pattern used for the anatomopathological evaluation was the one used by most pathologists, being followed by the analysis of all sections prepared during the trial.
Because the fracture area was small, the number of miroscope fields did not exceed five HPFs in a 0.5-cm long area of each section analyzed.
During the bone repairment processes, established anatomopathological criteria were used and the alterations below were observed.
A -Hemorrhage: After the bone injury, cells become damaged, mainly the blood vessels, when blood overflows into the medullar canal in the presence of local hemosiderin.Where no differences were noticed between Groups I and II analyzed, hemorrhage was evaluated as absent or focal (up to one field) and present (more than two fields).B -Polymorphonuclears: Inflammatory mediators released by the dead and injured cells cause blood vessels to dilate and exsudate plasma, with the formation of inflammatory cells such as polymorphonuclear cells, or neutrophils.The evaluation parameters used were absent (0) and present (1 to 4 neutrophils per 10 HPFs).No differences were noticed between Groups I and II analyzed.área de 0,5 cm de extensão na lâmina analisada.
Os achados em relação à matriz óssea mostraram uma possível aceleração do processo de osteogênese, principalmente C -Granulation tissue: During the tissue repair stage the granulation tissue characterizes by the appearance of fibroblasts and neovascularization as a result of the presence of chemical mediators.The evaluation criterion used was absent or scarce (up to 2 HPFs) and present (more than 3 HPFs).No differences were noticed between groups.D -Osteoblasts: Originating from the medullar stroma and responsible for the synthesis of the matrix for repair and growth, morphologically osteoblasts include Golgi's apparatus, the endoplasmic reticulum and large amounts of canaliculi, their main function being osteogenesis.The criteria used to analyze them were absent or mild (up to 2 HPFs) and present (3 or more HPFs).No differences were noticed between the groups analyzed.E -Bone matrix: Includes about one third of the bone mass and is responsible for bone viscoelasticity and tensile forces.Type 1 collagen includes 90% of the matrix, and degrades into hydroxyproline, giving origin to mature bone tissue.For the analysis we used the absent or focal parameters (up to 4 HPFs) and present (more than 5 HPFs).No significant differences were noticed according to the specific 5% parameter, but very close to it (p= 0.057); therefore, a tendency exists to use plasma concentrate to produce more bone matrix, and probably a trial would be required with a higher number of individuals; or maybe the amount of stimulating elements present in the plasma is too small to achieve complete stimulation.Also, in order to reach further conclusions, more trials would be required, perhaps differentiating morphogenetic protein subtypes.F -Osteoclasts: These are larger cells that derive from macrophages or from the hematopoetic system that includes lysosome enzymes such as acid phosphatase, collagenase and catepsin.Osteoclasts are the mains cells in the bone remodeling process.The parameters used to evaluate osteoclasts were absent or minimal (up to 5 HPFs) and present (more than 5 HPFs).No differences were noticed between the groups analyzed.
The 5% significance value (p=0.05) was used in all analyses, since this is the parameter adopted in most scientific papers.
The bone matrix results showed a possible acceleration in the osteogenesis process, mainly as regards osteoid bone formation in animals exposed to plasma concentrate stimulation.This may correspond to a higher speed in the process of bone callus formation.
For the rest of the elements studied, no different responses were found between the control group and the plasma group.Various factors may have contributed for these results.We believe that a higher number of animals might provide significance to some of the studied elements or, perhaps, a higher plasma depuration, in order to increase the plasma concentration of factors stimulating bone callus formation.This is a rather expensive resource that still does not exist in our area, making it impossible to perform many trials.
Based on this, it seems more appropriate to use a methodology including osteoclast and osteoblast counts and possibly immunohistochemical methods to assess cell proliferation, for a more ample approach.
We believe that in the future, with improved techniques and better understanding of the healing steps, it will be possible to have trials based on the use of various substances, among them plasma itself, that will have practical application in Orthopedics no que se refere na formação de tecido osteóide nos animais expostos à estimulação do concentrado de plasma.Isto pode corresponder a uma maior velocidade no processo de formação do calo ósseo.
3. Há uma tendência em formar mais matriz óssea com o uso de concentrado de plasma.and Surgery, to heal and repair many types of injuries such as fractures, healing delay and even pseudoarthroses.

1.
The use of plasma concentrate in bone defects of mice femurs does not lead to stimulation of bone callus formation.

2.
The use of plasma concentrate does not increase the inflammatory process at bone defect sites.

3.
The use of plasma concentrate promotes a tendency to form more bone matrix.