Effect of the ossein-hydroxyapatite complex on fracture consolidation under protein malnutrition: experimental study using rats

Abstracts

Foi realizado trabalho experimental com 40 ratos da raça Wistar, machos, para avaliar o efeito do complexo osseína-hidroxiapatita (COH) , na consolidação das fraturas, em animais submetidos a dieta protéica e dieta aprotéica, divididos em quatro grupos distribuídos aleatoriamente, que receberam ou não a medicação. Cada grupo com 10 animais foi assim constituído: grupo I, protéico sem COH; grupo II, protéico com COH; grupo III, aprotéico sem COH; grupo IV, aprotéico com COH. Procurou-se fazer fraturas semelhantes quanto ao traço e a localização em todos os grupos no 15º dia, passando os grupos II e IV a receber a dose diária de 20mg do COH, por gavagem. Todos os grupos foram submetidos à coleta de sangue no 1º, 15º e 43º dia, que foi o dia da eutanásia dos animais. Além do controle de peso fez-se a dosagem de cálcio, fósforo, fosfatase alcalina, proteínas totais, albumina e osteocalcina. O membro fraturado foi desarticulado na articulação coxofemural, no 43º dia, sendo submetido o calo ósseo à avaliação histológica, histomorfométrica, radiográfica e densitométrica e planimétrica. Concluiu-se, após análise estatística, que o COH não interferiu, de maneira significativa, na avaliação geral, na formação do calo ósseo dos animais nutridos e desnutridos. Entretanto mostrou interferência em alguns resultados como na dosagem de albumina e de fosfatase alcalina, na planimetria e no peso dos animais.

Estudo Experimental; Consolidação Óssea; Complexo Osseína-Hidroxiapatita

ARTIGO ORIGINAL

Effect of the ossein-hydroxyapatite complex on fracture consolidation under protein malnutrition: experimental study using rats

Wilson Galego Campos

Professor Adjunto da Universidade Estadual de Londrina

^{Correspondence}

SUMMARY

Assessement of the effect of ossein-hidroxiapatite compound in the fracture healing under protein malnutrition: experimental essay in rats.

An experimental essay was carried out with 40 Wistar male rats to assess the effect of the ossein-hidroxiapatite compound (OHC) in the fracture healing in animal under protein diet and no protein diet, which were divided into four groups, at random, which received or did not receive the compound. Each group with 10 animals, was divided into: group I, protein diet, without OHC; group II, protein diet, with OHC; group III, no protein diet, without OHC; group IV, no protein diet, with OHC. The fractures were made similarly as to the trace and site in all groups in the 15^th day, when groups II and IV started receiving a daily dose of 20mg of the OHC, orally. Blood samples were drawn from all groups on the 1^st , 15^th and 43^rd days. The animals underwent euthanasia on the 43^rd day. Weight control as well as calcium, phosphorus, alkaline phosphatase total proteins, albumin, and osteocalcine were determined. The fractured limb was desarticulated in the hip joint on the 43^rd day, and the bone callus was submitted to histological, histomorphometric, radiographic, planimetric and densitometric examination. It was concluded, after statistical analysis, that the OHC did not interfere significantly, in general assessment in bone callus formation in malnutrition and nourished animals. However it showed significant interference in some results such as albumin and alkaline phosphatase blood samples, in the planimetry and weight of the animals.

Key words: Experimental Study, Osseous Consolidation, Ossein-Hidroxiapatite Compound

INTRODUCTION

Traditional drugs have not been focused by experimental, comparative and recent studies to establish their actual validity and improve the achievement of the wanted effects.

The high prevalence of proteic malnutrition in our environment causes harm to the bone tissue and deserves more study; malnutrition must be considered when drugs are administered to reduce the consolidation period of a fracture. The aim of this study was to experimentally evaluate the effect of the ossein-hydroxyapatite complex (OHC) on male, adult, nourished and undernourished rats with fractures provoked in their tibias.

OHC has been clinically experimented as concerns mineral deficiency and osteoporosis in humans, besides being considered an adjuvant to promote faster fracture consolidation.

The biochemical behavior of calcium, phosphorus and alkaline phosphatase as well as total protein and albumine was observed. The bone formation biochemical marker, osteocalcin, was determined in order to establish correlations concerning OHC, malnutrition and bone consolidation. After ponderal, radiographic, densitometric, microscopic, histological and hystomorphometric determinations the data from the statistical analysis were analyzed.

MATERIAL AND METHOD

Forty male, albinus, Wistar rats weighing initially 250-400 g were utilized in this study.

The forty animals were randomly allocated into four groups: Group I - proteic diet without OHC (10 rats); Group II - proteic diet with OHC (10 rats); Group III - non-proteic diet without OHC (10 rats); Group IV - non-proteic diet with OHC (10 rats).

After two weeks, 20 mg/day OHC were orally administered to groups II and IV.

Blood was collected through intracardiac puncture under general anesthesia at days 1, 15 and 43 and submitted to biochemical analysis. In day 15, closed fracture in the left tibia middle third was produced, by manual flexure, under ethyl ether anesthesia. Four weeks after the fracture all the animals were sacrificed using an overdose of ethyl ether. The fractured limb was coxofemorally disarticulated and radiographic, planimetric, densitometric, histological and histomorphometric determinations were made. Data from the biochemical, ponderal, radiographic, densitometric, microscopic and histomorphometric analysis were submitted to statistical analysis, at the 5% significance level; the tests were considered significant when p < 0.05%.

DISCUSSION

OHC, administered to nourished and undernourished animals, has important organic components to consolidate fractures so the importance of these factors must be evaluated.

The Wistar rat is extremely useful in research for the following reasons: a) provides data similar to those observed in the human skeleton; b) is a manageable animal; c) its fracture show fast consolidation, dispensing immobilization, OTTO et al. (1995); d) bone alterations can be easily analyzed (LI et al., 1990; MAEDA et al., 1993); e) similar experimental fractures can be obtained; f) formation of bone callus with established diets (GUARNIERO, 1987) and hormonal substances, among them the calcitonin, according to GUARNIERO et al. (1995).

The similarity of the fractures, preconized by URIST and McLEAN (1950), was satisfactory, and it was possible to detect small differences in the fractures; however, under our point of view, the elaboration of special mechanical devices to make the fracture is not necessary. Osteosynthesis materials were not used in the fracture focus, since the presence of these materials could interfere in bone consolidation. The design of the study, with four groups with ten pacients each provided a consistent statistical analysis. The non-proteic diet caused alterations pointed out by the total protein and albumin values; the protein malnutrition thus produced exerted direct interference in the fracture consolidation, in agreement with PARK (1964) and GUARNIERO (1987) observations. Euthanasia after six weeks was convenient due to the animals suffering and because in this occasion the callus was very visible, (ARO, 1985).

Tests of the fracture focus mobility according to HEARD et al. (1982) were not effected because they are operator dependent. Mechanical resistance studies of the callus were not made, but this would certainly improve the quality of the study.

Anesthesia was efficient with ethyl ether, however other anesthetics should be considered taking into consideration possible interferences in the experiments.

The OHC effect on the diets evaluated after 15 and 43 days, and the mean weights of the groups with proteic and non-proteic diets were significantly different (p < 0.0001). In the third evaluation a significant effect of the CHO addition was observed only in the non-proteic diet group (p = 0.0104).

In the total protein analysis there was a significant effect of the diets only after 43 days (p < 0.0001). Diet influenced the mean total protein levels, however OHC addition did not.

The albumin determination indicated that its mean levels showed the significant effect of the diets after 43 days (p = 0.0003), and the OHC addition effect was also observed after 43 days (p = 0.0282), only in the non-proteic diet group (p = 0.0094).

Osteocalcin synthetized by osteoblasts is incorporated to the bone matrix liberating a small fraction in the circulation, and serves as an important marker of bone formation (DELMAS, 1992). No interference of OHC was observed at the osteocalcin level, when determined in nourished and undernourished animals. Osteocalcin has affinity with calcium and non-crystalized hydroxyapatite and serves, as the alkaline phosphatase, as a guide for the osteoblastic activity degree (BERNE and LEVI, 1995).

A profile X-ray was sufficient to determine the fracture site and the general aspects of bone consolidation in the four different groups of animals, according to ARO et al. (1985) criteria. The anterior-posterior study after disarticulation of the knee and ankle can provide more significant data of the planimetric study.

In the densitometric study, the nourished animals presented the highest mineral densities when compared with the undernourished groups showing the significant effect of the diet (p = 0.0191) and the non-significance of OHC (p = 0.9567). In the histomorphometric evaluation of fibrosis, cartilaginous and bone tissues, no statistically significant differences were observed in the nourished and undernourished animals after OHC. The significant effect of the diet was observed when fibrosis (p = 0.0024) and bone (p = 0.0211) percentages were compared.

Concerning the laboratory determination of mean levels of calcium there were not significant differences in relation to diet and medication. Calcium and phosphorus from OHC did not interfere during the experiment.

The OHC effect on nourished and undernourished animals, mainly on their most important organic part, ossein (SCHMIDT, 1988), could not be established with the bone metabolism markers due to the complexity of the phenomena concerning bone consolidation.

The best comprehension of the fracture consolidation mechanism, in addition to more adequate analyses of the interaction between different organic and inorganic components of the bone tissue, certainly will possibilitate the synthesis of drugs which have a more determinant action on the fracture consolidation process.

CONCLUSIONS

1) OHC, 20 mg/day, orally administered to Wistar adult rats with or without protein malnutrition, did not interfere significantly in the process of consolidating experimental fractures.

2) OHC interfered significantly in the planimetric determination of the bone callus in nourished animals and in weight and dose of albumin and alkaline phosphatase in undernourished animals.

REFERENCES

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Correspondence to

Av. Robert Koch, 60 - Bairro Cervejaria

CEP 86038-250 - Londrina - Paraná

Publication Dates

• Publication in this collection
  17 May 2006
• Date of issue
  June 2001