The use of inhaled bone marrow of ileum in bone failures of femurs of rats: experimental study

Abstracts

Os autores estudam a utilização de medula óssea em camundongos como estimulação da formação de calo ósseo. Foram utilizados dez camundongos adultos machos de linhagem isogênica gioto com peso de aproximadamente 250 gramas, e realizadas falhas ósseas na região distal do fêmur com alternância do lado direito e esquerdo, divididos em grupos A e B, sendo como controle camundongos com falha óssea isolado e com falhas ósseas com medula óssea colhida previamente de cada camundongo. Após análise qualitativa e quantitativa foi observado que o uso do aspirado de medula óssea não leva à estimulação da formação do calo ósseo e não há o aumento de processo inflamatório local.

Camundongos de cepas endogâmicas; Medula óssea; Calo ósseo

ORIGINAL ARTICLE

The use of inhaled bone marrow of ileum in bone failures of femurs of rats – Experimental study

Alberto Tesconi Croci^I; Olavo Pires de Camargo^I; George Bitar^II; Silvio Luiz Borges Pereira^III; Talmo de Melo Freire^IV; Sérgio Cristiano Inácio Cardoso^IV

^IAssociate Professor of the Orthopedics and Traumatology Department at the São Paulo University Medical School and of the Orthopedics and Traumatology Institute at the General Hospital of SPUMS

^IIHead of the Medical Residence Service of Orthopedics and Traumatology in the Santa Casa de São Vicente and Santos Medical School – UNILUS

^IIIPreceptor of the Medical Residence Service of Orthopedics and Traumatology in the Santa Casa de São Vicente e Santos Medical School – UNILUS

^IVEx-resident in Orthopedics and Traumatology of the Santa Casa de São Vicente and Santos Medical School – UNILUS

^{Correspondence}

SUMMARY

The aim of this study is to analyze the bone marrow employment in rats to stimulate the bone callus formation. Ten adult rats were used, male, isogenic, gioto lineage, approximate weight of 250 grams. Bone failures were produced at femur distal portion, alternating the right and left sides, and they were divided in group A and B. The control was held in rats presenting an isolated bone failure or having their bone marrow previously collected

After quantitative and qualitative analysis, it was observed that the bone marrow utilization does not lead to the bone callus formation and there isn't an increase in the local inflammation process.

Key words: Rats, endogamous strains; Bone marrow; bone callus.

INTRODUCTION

The bone is an specialized connective tissue with a mineralized collagen structure for the skeletal support of the body. Its structure can be spongy ( trabecular) or compact (cortical).

The spongy bone consists of interlaced bone and linkage (trabecula) of different forms and thickness and the bone marrow places among them. The compact bone is a continuous osseous mass presenting linked vascular canals of microscopical dimensions.

The bone appears for the first time after the seventh embryonal week. This bone can be of two types: the membranous that is which directly forms the membranous folium and the cartilaginous that is the one where the structure is formed at first as cartilage and after that substituted by bone.

Although the histogeny of the bone is identical in each case, in the last type the cartilage must first be removed before the bone can be deposited^(6,8). The membrane of the mesenchymal or connective tissue first forms the original model of the face and cranial bones. The intramembranous ossification starts in one or more central points of the membrane

These ossification centers characterize for the appearance of osteoblasts that deposit a net of radially spread osseous trabeculas in some directions⁽²⁾. The peripheral mesenchyma differs in a fibrous sheath (periosteum), whose inferior surface differs in osteoblasts that deposit parallel plates of compact bone (coverglass).

The trabeculas are distributed mainly throughout lines of higher tension.

A cartilaginous model of the structure precedes the destruction of the cartilage and its substitution by bone. Two processes are involved: the central ossification of the cartilage or endochondral ossification, and the peripheral ossification below the perichondrium or periosteum or perichondral ossification (periosteal).

In the cartilaginous precursor center, the cells become hypertrophied and are distributed radially. Calcium sal is deposited in the matrix. This calcificated cartilage disintegrates and is destroyed by the neoformed vascular tissue from the perichondrium. At the same time the neoformed germiform mass also originates the osteoblasts that deposit the new bone in many points and in the calcificated cartilage.

This spongy osseous formation continues to substitute the cartilage, extending itself in the proximal and distal direction⁽²⁾.

The formation of the spongy central bone occurs simultaneously to the internal layer of perichondrium (now called more adequately of periosteum) deposit in parallel layers of the compact bone. The process of endochondral ossification continues during the growth period for the existence of a cartilage layer next to the epiphyses, and it is responsible for the growth in length of the structure. The periosteal ossification contributes to the growth in thickness of the structure.

The consolidation of the fractures occurs by a similar physiological process to the cicatrization of the wounds of soft tissues and it only occurs in a longer time, therefore it is a tissue with a great amount of mineral osseous structure. But it has a basic advantage: after the maturation of the bone callus, its histological properties and previous structures are all settled, and this does not occur with any other tissue

The osseous consolidation depends on some factors, such as the vascularization, the stability, and the contact of the fragment extremities and the time so that the neoformed bone restores a strong link between the osseous fragments.

Sometimes the consolidation process deviates from its normal course producing three types of delayed complications:

1. delay of consolidation, 2. pseudo-arthrosis and 3. vicious consolidation⁽²⁾.

The habitual model used to explain the consolidation is based on a closed fracture of diaphysis of long bone, therefore the repair principles can be applied in any fracture.

It is supposed that the process is divided in three phases based on the morphologic changes: the inflammatory initial phase, the repair phase and the phase of remodeling.

The duration of the cicatrization process, as well as the one of each phase varies in accordance to innumerable factors, including the species of the studied animal.

In the inflammatory phase the hematoma is formed from the rupture of the injured blood vessels of the medulla and the osseous cortical.

Some of the vessels thrombose, determining necrosis of some osseous tissue to the level of the fracture. The inflammatory cells then invade the hematoma.

In the repair phase there are mesenchymal pluripotential cells and new capillaries invade the organized hematoma, differentiating in fibroblasts, chondroblasts or osteoblasts, forming the fracture callus. In the remodeling phase the new bone is formed by the predominant osteoblasts in relation to the other elements of the callus and more and more it assumes its initial characteristics.

In the formation of the bone callus, injury of some vessels occurs, and this determines the development of an hematoma that fills the space among the osseous fragments, and the soft tissues around.

As in the repair of all the wounds, the cicatrization starts with the coagulation of the overflown blood. The organization of the hematoma starts in the first 24 hours, and consists in the invasion of coagulum by the granulation tissue (capillaries and new fibroblasts). The periosteum, the endosteum and the bone marrow next to the fracture focus provide cells that proliferate and differentiate in fibrous tissue, fibrocartiloginous and hyaline cartilage, all of them part of the formation of new bone^(4,7).

The undifferentiated cells of the bone marrow also contribute to the osseous neoformation. This starts in young individuals in the internal and external surfaces of the injured bone, in the first 48 hours. At the moment of the trauma, a certain rise of the periosteum in the bone is produced above and below the fracture. This displacement of the periosteum is a great stimulus for the cellular proliferation of the deep layers. Microscopically one can see an osseous neoformation two days after the trauma.

The osseous neoformation initiates in a certain distance from the fragment extremities and the closer cells produce a cartilaginous or fibrocartilaginous tissue. The amount of bone callus depends on the type, localization, duration, patient's age and treatment of the fracture.

The osseous consolidation means the reestablishment of the osseous structure resistance that occurs in few weeks, but the microscopical establishment of the original structure can delay until the internal remodeling.

Two prerequisite are essential for the adequate osseous consolidation: the biological capacity (vascularization) and the biomechanic condition that control the activity of the cells.

Croci⁽²⁾ describes, in a revision article, the mediating factors involved in the osseous consolidation, among them: prostaglandins PGE 1 and 2, the somatomedin, the angiogenic factors, the epidermal growth factors, the fibronectin, interleukin 1 and 2, the factor of platelet growth, the activator factor of osteoclasts, the bradykinin and the factor of tumorous necrosis.

Regarding the intercellular communication, the studies suggest the presence of a factor, that would be important in the acceleration of the consolidation phenomena , as RAP (rapid accelerator phenomenon), that could be considered as a new phase in the consolidation. The trauma would initiate and speed up this process, that would have started some days after the fracture, with a peak of action from one to two months, lasting from six months to two years. This phenomenon, according to the author, would not only occur in about 3% of the fractures⁽⁵⁾.

CASES AND METHODS

Ten rats are used in this experiment of the isogenic ancestry gioto, proceeding from the Biotério of the College of Medical Sciences Lusíadas de Santos.

These animals are placed in cages separated and numbered from 01 to 10. They are submitted to surgical procedures in femurs, alternating the right and left side. There are 10 femurs (five right and five left femurs as control group and five right and five left femurs as the group where the bone marrow is used).

OBJECTIVE

The aim of this paper is to evaluate the osseous consolidation in an experimental study in rats, with the inhaled of bone marrow used in the stimulation of the bone callus formation, in osseous failures produced in animals femurs.

MATERIAL

Ten male adult rats of isogenic ancestry gioto are used, proceeding from the Biotério of the College of Medical Sciences Lusíadas de Santos of the State of São Paulo, with weight around 250 grams. After the objective examination, the physically apt animals are submitted to the procedures. They are divided in two Groups and their right and left femurs are alternated for the experiments (Group A and B).

In the Group A osseous failures are performed, with segment perforations of the femur of two millimeters, in right and left sides, in such way that we have five femurs in the right side and five in the left side. These failures are not substituted by any element (Control Group).

In Group B osseous failures are performed, similar to the ones of the Group A, followed by placement, in this failure of medullary inhaled, collected previously from the same rat, in its iliac crest, by puncture with needle and syringe. This way, we have five operated right femurs and five in the left side, all with placement in the osseous failure of medullary inhaled.

Summarizing, we have 10 rats, and 20 operated femurs, 10 in the right side and 10 in the left side. Group A is the Control Group where the osseous failures were induced, without any substitution and the Group B is where it is used the medullary inhaled removed from the iliac crest of the same rat.

METHOD

Protocol anaesthetic: The animals are anesthetized by the intraperitoneal way, with Virboxil® 2% (xylazine hydrochloride) in the dose of 0,01 ml/kg, associated to Francotar® (Quetamina) in the dose of 0,50mg/100 gram.

Surgical technique: After bilateral trichotomy of the thighs, it is performed the antisepsis with topical polivinil pirolidona 1% and the aseptic fields are placed. The Incision in the skin is of approximately one centimeter from the distal portion of femur, with control of the hemostasis, dieresis of soft tissues with Kelly's speculum and soft scissors of Metzembaum, and revision of the hemostasis.

The subperiosteal elevation of femurs is performed with the unsticky of Freer followed by perforations of right and left femurs one centimeter above of the articular interline of the knee, from lateral to medial, using a perforator under manual pressure, with titanium drill of two millimeters of diameter under irrigation with saline solution 0.9% (Figure 1A).

About 0,3 ml of the inhaled of bone marrow is collected, with an aseptic syringe of 10 ml and needle of 40X16 millimeters in the crest of ilium. It is and immediately injected in the bone failures (Figure 1B).

The suture of soft tissues to close the surgical wound is made with mononylon wire 4-0 in isolated points. The rats are placed in individual boxes, numbered from 01 to 10, and r in the first 12 hours postoperative, they receive analgesia with 0,1 ml of sodium dipyrone intraperitoneal. The animals are fed with standard normoprotean ration and water is supplied "ad libitum", in ambient temperature from 19 to 20 centigrade degrees, in the individual cages.

After two weeks of confinement, the animals underwent euthanasia. The parts (femurs) are removed and prepared for anatomicopathological examination, where they are qualitatively and quantitatively analyzed.

Protocol of euthanasia: The euthanasias are performed in the groups A and B, with anesthetized rats intraperitoneal with xylazine hydrochloride in the dose 0,01ml/kg, associated to quetamina in the dose of 0,50mg/100 gram. After the anesthesia, an inhalant mask with ether in lethal dose is used, until the cardiorespiratory stop.

Anatomicopathological protocol: The removed parts and side are identified according to the number of the rat and to the protocol of the experiment, respectively. They are fixed in formol 10% for twelve hours, and then decalcified in nitric acid, for twenty four hours. After that, they are parted longitudinally in their longer axis.

They are identified according to the processing tissular protocol, and after that submitted to a sequential bath of alcohol, xylol and paraffin, and placed in paraffin block; they are submitted to the histologic cuts of four micras of thickness and then tinged with hematoxylin and eosine.

Two or three blades for block are produced and analyzed in microscope Nykon type - alpha phot I number 2, with 400 times of increase (figure 2A and ^B). The parameters are the following finds that will be taken in consideration for the present study:

1, Hemorrhage, 2. Polymorphonuclears, 3. Granulation tissue, 4. Osteoblastic activity, 5. Bone matrix and 6. Osteoclasts.

EVALUATION CRITERIA

The anatomicopathological evaluation follows the habitual parameters adopted by our Service of pathological anatomy to analyze the inflammatory process and formation of the bone callus. They were marked, in agreement with the presence or not of the studied findings, as Table 1.

STATISTIC ANALYSIS

We observed the frequency (absolute and relative) of the incidence of the anatomicopathological findings in the blades of the Groups A and B.

We performed the descriptive statistics of the quantitative parameters in the Groups A and B of the hemorrhage, polymorphonuclears, granulation tissue, osteoblastic activity, bone matrix and osteoclasts finds.

By analyzing the parametric results we used the accurate test of Fisher, comparing the events for each find in the Group A vs. Group B.

In all the results we adopted the probability of significance of 5% (p=0,05), and the significant results are marked with an asterisk.

RESULTS

The analysis of the results, according to criteria adopted for the present study are shown in Table 2, for the group A (control) and table 3 for group B (bone marrow).

COMPARATIVE STUDY

The results of the hemorrhage findings, polymorphonuclears, granulation tissue, osteoblastic activity, bone matrix and osteoclasts finds in the blades of the Group A (control) and Group B (bone marrow) can be observed in Table 3.

DISCUSSION

The necessity of reducing the consolidation time and of a good formation of the bone callus, justifies the attempts in experimental studies since we want to understand this process of consolidation better, and expect that these results lead to the discovery of biological and simple solutions.

By observing the primitive undifferentiated cells that can change into osteoblasts under the influence of osteoinductor substance⁽⁰⁸⁾ we could understand the process of formation of the bone callus. Carvalho et al. ⁽¹⁾ verified that the sciatic denervation does not result in alterations of the bone callus formation, and the nervous physiology does not interfere with the osseous consolidation.

The accelerator factor described for several authors⁽²⁾ is important and can be considered a new phase in the consolidation. Its function or presence is unchained by the trauma.

The use of the bone marrow to speed up the bone consolidation - as a simple method that can be obtained from the patient, in place of the autogenous graft that would compel to a surgical intervention for its withdrawal - makes us follow this line of research in order to obtain the result related to its use and degree of obtained result.

Therefore, we used isogenic rats that are genetically equal, and do not present interference in the immunity of the receiver or alteration in the bone formation. Moreover, the alternation of the side (right and left) was used to prevent eventual influences of a predominant member.

In the experiment we adopted the use of bone perforations, that was standardized with the same drill for all the animals. Besides that all the procedures were uniform regarding the team responsible for the surgery and the biologist responsible for feeding and diverse cares.

The intraperitoneal way was used as the anesthetical technique, that is an adequate procedure for the experiment. Drugs such as the quetamina and the xylazine hydrochloride were associated and regarding euthanasia, we used the standard protocol of the biotério, in which after the anesthesia, ether was used by inhalant way until the lethal dose followed by the cardiopulmonary stop.

Due to the area of the fracture is small, the microscopical fields number adopted did not exceed five fields HPF in an area of 0,5 cm of extension from the analyzed blade.

The anatomicopathological criteria were settled according to the rules established for the Pathological Service of Anatomy, analyzing the alterations present in the process of repairing the bone fractures taking into account:

1 - Hemorrhage: Defined as the presence of red blood cells in the interstice and presence of hemosiderin pendants, characterizing an alteration of recent nature after the trauma. The hemorrhage in organization was evaluated as absent or focal (0) or present (more than 2 fields) and didn't present alterations between the groups A and B.

2 - Polymorphonuclears: The inflammatory cells (neutrophils), present after the trauma, are responsible for the production of chemotaxis mediators and were evaluated as absent (0) and present (1 to 4 neutrophils per 10 HPF). Differences between the groups A and B weren't observed; the find can be due to the tissues have been studied two weeks after the initial trauma.

3 -Granulation Tissue: This corresponds to the phase of tissular result characterized by the fibroblastic and neovascular proliferation, resulting in the action of chemical mediators such as the fibroblastic growth factor and the angiogenic factors evaluated as absent (up to 2 fields HPF) scarce and present (with more than 3 fields HPF) It didn't occur any difference between the groups A and B.

4 -Osteoblasts: They are the cells that produce the bone tissue, the main ones in the process of osteogenesis. Morphologically, these cells are placed in the periphery of the osseous trabeculas of the mature bone or interspersing the new osseous matrix. They were evaluated as absent or focal when the osteoblastic activity is up to 4 HPF or present when over 4 HPF.

5 - Bone Matrix: Characterized by the deposit of osseous extracellular matrix, with hyaline aspect, observed through the coloration of hematoxylin and eosine. This new matrix will undergo mineralization originating mature bone tissue. It was evaluated as quantitatively absent or focal, in the observation of up to 4 fields HPF or present over 5 fields HPF. It was not observed any statistics difference between the groups A and B.

6 - Osteoclasts: They constitute the main cells in the bone modeling and are responsible of the reabsorption of the bone matrix. Morphologically, they are characterized by large cells, multinucleate and wide cytoplasm, situated preferentially in the mineralized osseous periphery of the trabeculas.

They were evaluated as absent or minimum during the observation of up to 5 HPF and present when over 5 HPF.

Between the groups A and B, there wasn't any difference in the osseous consolidation process histologically evaluated two weeks after the trauma.

For statistics analysis, we adopted the probability of significance of 5% (p = 0,05) as standard for all results.

The histological parameters of the hemorrhage, polymorphonuclears and tissue granulation can have been sub-evaluated due to the delayed period of the analysis.

The results of this paper show the necessity of posterior studies, focusing alterations in earlier periods after the trauma and a detailed morphologic analysis. It should include morphogenetic studies for evaluation of eventual differences in the process of consolidation and osseous remodeling by biological material (bone marrow) stimulating the formation of the bone callus.

CONCLUSIONS

1 - The use of the inhaled bone marrow of ileum, placed in the bone failures of femurs of rats, does not lead to the formation of the bone callus.

2 - There isn't an increase in the inflammatory process in the bone failures by using inhaled bone marrow of ileum.

REFERÊNCIAS BIBLIOGRÁFICAS

Work performed in the Medical Residence service in Orthopedics and Traumatology of Santa Casa de São Vicente e Faculdade Ciências Médicas de Santos – UNILUS

• 1. Carvalho EM, Cavalcante MA, Oliveira LP, Elias N, Mesquita KC. Efeitos da desnervação periférica sobre a formação do calo ósseo: estudo experimental em ratos. Rev Bras Ortop 28: 683-686, 1993.
• 2. Croci AT. Retarde de consolidação e pseudoartrose. Acta Ortop Bras 5: 26-34, 1997.
• 3. Kawai T, Mieki A, Ohno Y et al. Osteoinductive activity of composites of bone morphogenetic protein and pure titanium. Clin Orthop 290: 296-305, 1993.
• 4. Kirker-Head CA, Gerhart TN, Armstrong R. Healing bone using recombinant human bone morphogenetic protein 2 and copolymer. Clin Orthop 349: 205-217, 1998
• 5. Leung KS, Fung KP, Liu PP, Lee KM. Bone-specific alkaline phosphatase activities in plasma and callus during callotasis in rabbits. Life Sci 57: 637-643, 1995.
• 6. Santos Neto FL, Volpon JB. Experimental nonunion in dogs. Clin Orthop 187: 260- 271, 1984.
• 7. Street J, Winter D, Wang JH et al. Is human fracture hematoma inherently angiogenic? Clin Orthop 378: 224237, 2000.
• 8. Urist MR. Bone: formation by autoinduction. Science 150: 893-899, 1965.

Publication Dates

• Publication in this collection
  24 June 2004
• Date of issue
  Mar 2004

History

• Accepted
  18 Feb 2004
• Received
  10 June 2003