Macroscopic and histological study of biologically acceptable osteochondral repairs

Abstracts

Este estudo teve como finalidade avaliar macroscopicamente e histológicamente defeitos osteocondrais já cicatrizados, também conhecidos como reparos. Foram utilizados seis coelhos machos, adultos, albinos da raça Nova Zelândia. Defeitos cilíndricos osteocondrais de 3.2 mm de diâmetro por 4.0 mm de profundidade foram criados cirurgicamente em ambos côndilos femorais mediais. O cilindro osteocondral retirado do joelho esquerdo (defeito não tratado) foi implantado no joelho direito (joelho tratado). Comparou-se macroscopicamente e histológicamente ambos tipos de defeitos após doze semanas de evolução. A avaliação macroscópica de todos os defeitos mostrou evolução para reparos denominados biologicamente aceitáveis. O termo "biologicamente aceitável" foi utilizado para definir reparos, que à observação macroscópica, se apresentaram como tecido neo-formado semelhante à fibrocartilagem, brilhante, liso, firme, em continuidade com a cartilagem adjacente. Como todos os defeitos, tratados e não tratados, eram macroscopicamente semelhantes, realizou-se um estudo histológico comparativo para averiguar qual tipo de tecido de reparação se formava em ambos defeitos. Pela análise histológica dos reparos biologicamente aceitáveis, concluiu-se que houve formação de tecido cartilaginoso hialino nos defeitos tratados com enxerto autólogo e de tecido fibrocartilaginoso nos defeitos não tratados.

Joelho; Transplante autólogo; Cartilagem articular; Defeito osteocondral; Coelho

ORIGINAL ARTICLE

Macroscopic and histological study of biologically acceptable osteochondral repairs

José Leão Ribeiro^I; Gilberto Luis Camanho^II; Luiz Carlos Takita^III

^IAdjuct Professor of Orthopedics of the Universidade Federal de Mato Grosso

^IIAssociate Professor of the Universidade de São Paulo - FMUSP

^IIIAssistant Professor of Special Pathological Anatomy of the Universidade Federal de Mato Grosso do Sul

^{Correspondence}

SUMMARY

The aim of this study was to evaluate macroscopically and histologically healed osteochondral defects, also known as repairs. Six adult, male, New Zealander White rabbits were used. Cylindrical osteochondral defects of 3.2 mm in diameter by 4.0 mm in depth were artificially created in the load-bearing surfaces of both medial femoral condyles. The osteochondral graft collected from the left knee (untreated defect) was implanted in the right knee (treated defect). Both defects were compared macroscopically ad histologically after twelve weeks. The macroscopic evaluation of all defects demostrated evolution in repairs called "biologically acceptable". The "biologically acceptable" term was used to define repairs, that at macrocospic observation, presented as neo-formed tissue similar to fibrocartilage, brilliant, smooth, firm, in continuity with the adjacent cartilage. As all the defects, treated and untreated, were macroscopically similar, a histological comparative study was made to verify which type of repair tissue was formed in the surface of both defects. By histologic analysis of the biologically acceptable repairs, the authors conclude that: treated defects with autologous grafts were filled with hyaline cartilaginous tissue and untreated defects were filled with fibrocartilaginous tissue.

Key words: Knee; Autologous transplantation; Articular cartilage; Osteochondral defect; Rabbit

INTRODUCTION

The surgical treatment of the chondral and osteochondral injuries that affect articulations that support load, mainly the knee, still represents a challenge for the orthopedist. This is due to the characteristics of the articular hyaline cartilage, that is unprovided of vascularization and so has limited potential of cicatrization. According to Mankim⁽¹⁵⁾, the cicatrization of an injury restricted to the articular hyaline cartilage (chondral defect) doesn't follow the three natural phases - the necrosis, the inflammation and the repair - exactly because of its avascular condition. As in any other injured body tissue, the same phase of initial necrosis occurs and consequently there is loss of cells and matrix. As the chondrocytes are relatively insensitive to the condition of installed hypoxia; there is a smaller number of dead cells than in any other organical tissue. A second phase of inflammation, as is entirely mediated for the vascular system, is absent. Consequently, the hematoma formation doesn't occur, and so it does not produce fibrin or coagulum of fibrin that would work as a frame for the new repair tissue growth. This way there isn't continuity in this process. Considering the third phase that is the repair, the absence of a vascular or inflammatory phase limits the number of available cells to respond to the trauma and the repair capacity fails in the remaining chondrocytes, blocking the cicatrization process definitively.

However it is known that if the injury extends until the subchondral bone (osteochondral defect), that is well vascularized, all the three phases occur naturally^(3,5,11,15). The hematoma is quickly organized with coagulum of fibrin, white globules and elements of the bone marrow. Undifferentiated cells of the marrow and vascular endothelium are transformed into primitive fibroblasts and become fibroblastic vascularized tissues of repair by receiving capillaries and coagulum of fibrin⁽¹⁵⁾. The study of the cicatrization of the cartilaginous injuries has been performed in diverse animal experimental models. Probably, no other experiment has been so frequently performed, concerning the study of cartilage, experimental surgical injury and the observation of the cartilage behavior by macroscopical, radiological, biochemical, biomechanic, biomolecular and ultrastructural view^(3,4,7,15). The focal injuries of the articular cartilage require surgical treatment that includes the classic methods of stimulation of the bone marrow such as debridement, multiple perforations, abrasions, microfractures^{(12,19,22,23)}, and modern biological methods such as periosteal and perichondral transplants, implantation of cultivated autologous chondrocytes, and autologous osteochondral grafts^{(1,9,17,18,21)}. Because of the high cost and technical restrictions imposed to some biological methods of treatment of the osteochondral injuries, some authors have also searched efficient methods and easier to perform.

One of these simple and direct methods is the transplant or autologous osteochondral graft that represents a good alternative for biological repair of the focal injury of the articular surface. It consists of filling the defect with a unique osteochondral cylinder, or according to the size of the injury, with a series of small osteochondral grafts. This technique is known as mosaicplasty. It has been used in dogs, horses and human corpses by Hangody⁽⁹⁾ since 1991 and the donor and receiver portions have been studied in order to verify the injuries behavior and supervene of the transplanted hyaline cartilage.

The method of treatment of the osteochondral injuries described and developed by Hangody⁽⁹⁾ was the base for this experimental model. Osteochondral defects in both femoral condyles of rabbits were surgically produced. The defect of the right knee (D) was filled by autologous osteochondral graft (treated) and the defect of the left knee (E) remained empty (untreated). The aim of this study was to compare macroscopically and histologically the osteochondral defects healed after twelve weeks of evolution.

MATERIAL AND METHOD

1 - Animals of experimentation

Six male, albinic, adult rabbits of the New Zealander race, weight between 3.4 kg and 3,6 kg were used in this study. The animals were lodged in appropriate room and confined in metallic cages measuring 70X70X70. They were fed with standard pelleted ration and water ad libitum.

2 - Surgical technique

As a pre-anesthetical drug, each animal received 0,2 mg/kg of weight of acepromazine 1% by intramuscular. The anesthesia consisted of the intramuscular application of hydrochloride of ketamine 5% in the dose of 50 mg/kg of weight associated with xylazine 2% with the dose of 3mg/kg of weight.

After the trichotomy of the surgical area it was performed a parapatellar medial incision followed by arthrotomy and lateral dislocation of patella.

As the knee was flexed, the femoral medial condyle was exposed and with the help of a metallic trephine an osteochondral cylinder of 3.2 mm/4.0 mm was removed from the knee E (Figure 1). Through sequential perforations with up to 3.2-mm drills, the osteochondral defect in knee D was created.

Osteochondral grafts removed from knee E (untreated defect) were then transferred to knee D (treated defect) (Figure 2). The synthesis of the surgical wounds was performed and the animals returned to their respective cages for postoperative recovery.

3 - Euthanasia

It was performed in the animals after 90 days of evolution by a similar anesthesia used in the preoperative, followed by endovenous injection of 60 mg/kg of potassium chloride.

Both knees were removed in order to be carefully examined. All the data of the macroscopic evaluation of the obtained repairs were noted. The parts then were led to histologic study.

4 - Criteria of evaluation

Specifically for the proposed study, the analysis of two criteria of evaluation was considered: macroscopic and histologic.

4.1 - Macroscopic evaluation

Five parameters were analyzed through the macroscopic inspection: the repair concerns the defect cicatrization, the continuity is the levelling and integration of the repair with the adjacent cartilage.

The other three parameters show the external characteristics of the repair and they are the surface, the brightness and the consistency of the tissue formed. A considered good result (biologically acceptable) should present the five satisfactory parameters. On the other hand, a considered not good result (biologically not acceptable) would present one or more of these parameters not satisfactory.

4.2 - Histologic evaluation

After the euthanasia the distal portion of femur was removed in block, settled in formol 10%, decalcificated in solution of nitric acid and formalin and studied histologically. The blades were tinged with Hematoxylin-Eosine. in cuts in the sagittal plan, including the long axle of distal femur for a better view of the extension of the defect.

In the histologic examination of the cuts of distal femur, the attention was particularly directed to the following elements: 1- the type of regenerated tissue in the surface of the defect: cartilage, fibrocartilage or fibrous tissue; 2- the appearance of the cartilaginous surface: smooth, depressed or irregular; 3- presence or absence of the regenerated subchondral bone.

RESULTS

1 - Macroscopic aspect

The typical macroscopic aspect in the treated and untreated defects can be seen in the Figures 3-A and 4-A; the results were considered as good or biologically acceptable. "The biologically acceptable" term, according to Amiel⁽¹⁾, is used to define repairs, that in macroscopical observation, present as neo-formed tissues similar to the fibrocartilage. They are smooth, brilliant, firm, and in continuity with the adjacent cartilage. As this is a macroscopic criterion of evaluation, it is not possible to settle an accurate correlation among the histological, biochemical or biomechanic characteristics of the repair. The relative data to the result of the macroscopic evaluation, that include the five parameters already mentioned, are summarized in Tables 1 and 2. It was observed the predominance of the formation of a firm repair tissue, with total fulfilling of the injury, presenting a plain and regular surface in continuity with the adjacent cartilage in the treated defects.

In spite of presenting minimum alterations of the surface these characteristics were similar in the treated defects. However, all the repairs of untreated defects or not were considered satisfactory, therefore biologically acceptable after twelve weeks of evolution.

2 - Histologic aspect

The histologic repair would only be considered satisfactory if the surface of the tissue regenerated smoothly, were covered by cartilage were in the same level of the adjacent cartilage. The differences in the types of cicatrization tissue of the repairs were clear. For the treated defects the surface of the repairs presented the preservation of the smooth, regular original hyaline cartilage and in the same level of the adjacent hostess cartilage. The deep part of the repair became bone tissue with trabecular standard close to normality (Figure 3-B). The surface of the repairs proceeding from treated defects was not regularly smooth. It presented small depression and was predominantly re-covered by fibrous tissue. The deep part presented rudimentarily neoformed trabecular bone. It practically did not present formation of subchondral bone (Figure 4-B).

DISCUSSION

Not all the cartilaginous injuries are gradual and clinically symptomatic. The articulation can return to its normal state^(4,14). The uncertainty about which injury should require surgical repair still remains and it is not sure that the prophylactic treatment would modify for better the particular evolution of each injury.

The rabbit was chosen, in this study, because it is an animal widely used in repairing models of the articular surface of the defect and the cylindrical form can also be unanimously chosen if the case concerns the study of cicatrization of osteochondral focal injuries^{(9,12,15,16,21,22)}.

Regarding the size of the injury we decided to develop defects of 3.2 mm/4.0 mm in animals over 3.5 kg of weight in order to maintain an adequate proportionality.

The defects larger than 4 mm of diameter can practically take all medial femoral condyle. The ones 5-mm height can cause destruction to the bone marrow and such factors modify the sensitivity of the experimental model and the expected result⁽²⁴⁾.

The amount of involved animals in this research was based on bioethic principles. We used a minimum number for validation of hypothesis^(8,13). The six animals of the present series were selected from a bigger group (n=20) of a wider study, exactly because all the repairs were biologically acceptable independently if they were from treated or untreated defects. All the knees of these 06 animals underwent magnetic resonance, in a previous study of a graduation thesis of the main author⁽²⁰⁾. The examinations were performed at the end of the1st, 4th, 8th and 12th weeks of evolution with homogeneously good results in what concerns the cicatrization, but not to the type of tissue formed on the surface of the repairs. The aim of this present animal model was to perform a basic histologic study, in a minimum sample that was homogeneous and known, in order to verify the type of tissue formed in both types of repairs.

The result of 100% of biologically acceptable repairs according to the settled macroscopic criteria, the absence of macroscopic alterations in the articular tibia cartilage in contact with the femoral condyles, the absence of infection and the maintenance of the articular stability during the twelve weeks of evolution demonstrated the validity of the experimental method applied. Biologically acceptable repairs can be formed from treated defects or not and can be indistinguishable by the macroscopic appearance. The histologic study allowed us to differentiate both types of repairs. In the circumstances of the histologic study it was well evidenced the type of neoformed tissue for each type of repair. The inferiority of the repairs of treated defects was due to the delay in the formation of the subchondral bone, what shows the basic importance of the subchondral bone in the tissular reorganization. The previsibility of the cartilaginous tissue formation in the treated defects and the fibrous tissue in the untreated defects is in accordance with the literature data^(10,17,22).

CONCLUSIONS

After twelve weeks under the described experimental conditions in the present study, we can conclude:

1 - All the treated and untreated osteochondral defects became biologically acceptable repairs.

2 - histologic analysis of the biologically acceptable repairs demonstrated cartilaginous tissue and fibrocartilaginous tissue formation in the treated or untreated defects respectively.

REFERÊNCIAS BIBLIOGRÁFICAS

Work performed at the Orthopedics Service of the Surgical Clinical Department of the Universidade Federal de Mato Grosso

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Publication Dates

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