OBJECTIVE The present work aimed to evaluate the systemic effect of H.
speciosa latex on bone neoformation.
METHODS For this, the latex was collected and diluted to 3% and 50%. A total of 28 Wistar rats were submitted to surgery to create a 5 mm diameter defect in the parietal bone. This experiment was conducted in 2 different periods: 1 and 2. For each period, the rats were divided into 3 groups: Control Group, Latex3 Group, and Latex50 Group, which received, respectively, daily administrations of 0.5 mL of distilled water, latex to 3% and latex to 50% by gavage, orally. The rats of periods 1 and 2 were euthanized, respectively, 15 and 30 days after the surgery, and the calvaria was collected. The results were analyzed using the ANOVA and Tukey tests; the significance level was 0.05.
RESULTS We show that, in each analyzed period, the experimental groups had the same amount of newly formed bone in the calvaria defect.
CONCLUSION We conclude that daily and oral administrations of H. speciosa latex to 3% and to 50% over a period of 15 and 30 days does not contribute to the increase of the area of the newly formed bone in the calvaria defect.
Keywords: apocynaceae; hematoxylin; histology; latex; bone and bones; complementary therapies.
OBJETIVO Este trabalho objetivou avaliar o efeito sistêmico do látex de H.
speciosas obre a neoformação óssea.
MÉTODOS Para isso, o látex foi coletado e diluído a 3% e a 50%. Um total de 28 ratos Wistar foi submetido a cirurgia para a criação de um defeito de 5 mm de diâmetro no osso parietal. Esse experimento foi conduzido em dois períodos distintos: 1 e 2. Para cada período, os ratos foram divididos em 3 grupos: Grupo Controle, Grupo Látex3 e Grupo Látex50 que receberam, respectivamente, administrações diárias de 0,5 mL de água destilada, látex a 3% e látex a 50% por gavagem, via oral. Os ratos dos períodos 1 e 2 foram eutanasiados, respectivamente, 15 e 30 dias após a cirurgia e a calvária foi coletada. Os resultados foram analisados utilizando os testes ANOVA e Tukey; o nível de significância estabelecido foi 0,05.
RESULTADOS Mostramos que, em cada período analisado, os grupos experimentais tiveram a mesma quantidade de osso neoformado no defeito da calvária.
CONCLUSÃO Portanto, concluímos que administrações diárias e orais do látex de H. speciosa a 3% e a 50% durante um período de 15 e 30 dias não contribui para o aumento da área do osso neoformado no defeito da calvária.
Palavras-chave: apocynaceae; hematoxilina; histologia; látex; osso e ossos; terapias complementares.
|Citation: Felipetti FA, Bereta RM, Piedade SMS, Novaes PD. Administrações Orais do Látex da Hancornia speciosa Gomes não aumentam a neoformação óssea*. 54(6):692. doi:10.1055/s-0039-1697019|
|Note: * Study developed at the Department of Morphology, Faculdade de Odontologia de Piracicaba, Universidade Estadual de Campinas, Piracicaba, SP, Brazil.|
|Received: June 04 2018; Accepted: August 08 2018|
Bone is a highly dynamic mineralized connective tissue. Major bone cells, such as osteoblasts, osteoclasts and osteocytes, are essential for their remodeling.1,2 In the early stages of bone production, osteoblasts deposit the organic matrix, release calcium (Ca)-containing vesicles and induce pyrophosphate degradation, releasing phosphate ions.2-4 Phosphate and Ca form hydroxyapatite crystals and mineralize the bone matrix, forming new bone.1 Through this process of bone remodeling, bone can regenerate spontaneously after the incidence of fractures, injuries or defects.5 However, physiological regeneration is unable to restore bone integrity in extensive bone defects, where the repair process must be supported by some complementary therapies.6,7
Latex obtained from the trunk of the Hevea brasiliensis (a native Brazilian tree known as “Seringueira”) can be an innovative biomaterial for bone repair. Some experiments that have been performed with this product include bone repair of dental cavities in rats,8 guided regeneration of rabbit calvaria,6 osseointegration of dental implants in dogs,9 and bone repair of a critical defect in rat calvaria.10,11
Latex extracted from the trunk of Hancornia speciosa Gomes (another Brazilian native tree known as “Mangabeira”, belonging to the Apocynaceae family) also demonstrated bone repair potential. The research group of the Faculdade de Odontologia de Piracicaba (FOP, in the Portuguese acronym) of the Universidade Estadual de Campinas (UNICAMP, in the Portuguese acronym), demonstrated that topical application of H. speciosa latex on the defects on the skullcap of Wistar rats increased the area of the newly formed bone at the edges of the defect.12
Some communities located in northeastern Brazil produced a milky juice called “leite de mangaba” (mangaba milk) from the trunk latex of H. speciosa. Ethnobotanical surveys have confirmed the use of this milky juice for therapeutic treatment of bone fractures.13 However, to date, no studies have been conducted to confirm this effect.
In summary, these findings motivated us to investigate the therapeutic potential of H. speciosa latex. Therefore, the present study reports the systemic effect of H. speciosa latex in bone neoformation in Wistar rats.
MATERIALS AND METHODS
Latex Collection and Analysis
Hancornia speciosa latex was collected at the city of Mata de São João, Bahia, Brazil (12º27'42'S 37º56'38'O 69NE) in summer. A sample of this was deposited in the Escola Superior de Agricultura Luiz de Queiroz (ESALQ, in the Portuguese acronym) da Universidade de São Paulo (USP, in the Portuguese acronym) and received the number ESA 121.402. After drilling ∼ 10 tree trunks, 250 ml of the dripping latex was collected in a sterile container and mixed with distilled water to obtain a 3% latex solution and a 50% latex solution. The solutions were stored in syringes, protected from light and stored at 4ºC.
A total of 28 Wistar rats (mean body weight: 390 g; age: 10 weeks) were used. The rats were housed in individual cages under controlled temperature and light conditions (12/12 h light/dark cycle). All of the animals received food and water ad libitum. Prior to the surgery, the rats were anesthetized with intraperitoneal injection of 80 mg/Kg of ketamine (Dopalen, Sespo Indústria e Comércio Ltda., Paulínia, SP) and 8 mg/Kg of Xylazine (Rompun, Bayer SA, São Paulo, SP, Brazil). The surgery consisted in the creation of a critical defect in the left parietal calvarial bone (5 mm diameter) using a dental trephine drill mounted on a low-speed dental motor. After the surgery, 2 mg/ml tramadol hydrochloride (Tramal, Grunenthal do Brasil Pharmaceutical Ltda, São Paulo, SP, Brazil) were administered intramuscularly to the rats and they rested for 24 hours. The present experiment was conducted in 2 distinct periods: 1 and 2. For period 1, the rats were divided into 3 groups: Control 1 Group (C1, n = 4), Latex 31 Group (L31, n = 4) and Latex 501 Group (L501, n = 5) who received, respectively, daily administrations of 0.5 mL distilled water, 3% latex, and 50% latex by gavage, orally, for 15 days. For period 2, the rats were divided into 3 groups: Control 2 Group (C2, n = 5), Latex 32 Group (L32, n = 5) and Latex 502 Group (L502, n = 5) who received, respectively, daily administrations of 0.5 ml distilled water, 3% latex, and 50% latex by gavage orally for 30 days. The rats from periods 1 and 2 were euthanized, respectively, 15 days and 30 days after the surgery. After euthanasia, the calvaria was collected for analysis. All of the procedures were approved by the Animal Use Ethics Committee (CEUA, in the Portuguese acronym) with the protocol number 3427-1.
Samples Cleavage and Histological Processing
The calvaria was fixed in formalin solution and submitted to conventional histological processing.14 First, the calvaria was sectioned transversely and only the bone portion was obtained. Subsequently, the calvaria was decalcified with PLANK solution for 1 week with 2 daily changes. After decalcification, the calvaria was cleaved and only the bone defect region was preserved for further processing. Then, the calvaria bone defect was dehydrated, diaphanized and embedded in paraffin to obtain the blocks.14
Preparation of the Histological Slides
Once the paraffin blocks were obtained, coronal sections of the calvaria were collected. All of the sections had 6 µm of thickness. By default, eight consecutive sections of the calvaria blocks were first collected; 2 histological slides were prepared, and 15 sections were, finally, discarded; these steps were performed repeatedly until 20 slides were obtained. All of the slides were stained with hematoxylin and eosin and observed under a light microscope.
Descriptive and Histomorphometric Analysis
The slides were photographed using the software Optica View 7, and the images were recorded at 100x magnification for the entire calvaria bone defect. These images were analyzed by a single examiner using the software Image J (National Institutes of Health, Bethesda, MD, USA). Calvaria repair processes were demonstrated by descriptive analysis. The newly formed bone area throughout the calvaria bone defect was examined by histomorphometric analysis. For this, a millimeter ruler was first photographed at 100x magnification and then measured from end to end; the distance in pixels was recorded; the known distance was established according to the size of the ruler (1 mm); and the length unit was adjusted in millimeters. This information was recorded on each calvaria defect photo to define scale. Finally, a line was drawn marking all the newly formed bone within the calvaria defect, and then the area of the new bone was calculated. Quantified values were compared between groups.
Data were analyzed using the software R (R Core Team). The Bartlett and Shapiro-Wilk tests were used to verify the deviation of the assumptions. Once the homogeneity of variance and normal distribution were confirmed, the analysis of variance (ANOVA) and Tukey tests were used to compare the period 1 and 2 groups separately. The significance level was set at 5% (p < 0.05).
Descriptive Histological Analysis of the Calvaria Bone Defect
Conventional histological technique using light microscopy is widely used for bone repair analysis. This technique allows a descriptive analysis of the tissue and the measurement of the area of the components of interest (histomorphometry).15 We used these analyses to evaluate the newly formed bone in the calvaria defect.
The descriptive analysis revealed a similar stage of bone repair between groups C1, L31 and L501 and between groups C2, L32 and L502 (Figure 1). Mineralized trabeculae of the newly formed bone extended from the edges of the basal bone toward the center of the defect.We observed several osteocytes trapped in the bone matrix and medullary spaces filled with blood cells along the trabeculae. Numerous active osteoblasts have been noted around the trabeculae. Abundant connective tissue, widely vascularized, filled the central portions of the defect.
Histomorphometric Histological Analysis of the Calvaria
The histomorphometric analysis of the calvaria is represented in Figure 2. We measured the area of newly formed bone in each group. Results showed that the amount of new bone in groups L31 and L501 was similar in group C1. Concurrently, the amount of new bone in groups L32 and L502 was similar in group C2. These data are consistent with those of the descriptive analysis.
The present work investigated the systemic effect of H. speciosa latex on bone neoformation in Wistar rats.
In the presence of a fracture, healing-related events result in the formation of a new bone.16 In the early stages of bone production, osteoblasts deposit the organic matrix, forming a tissue known as an osteoid. Then, osteoblasts release vesicles containing Ca reserves that are degraded by enzymes to release this ion into the newly deposited matrix. Osteoblasts also secrete alkaline phosphatase, an enzyme that degrades pyrophosphate, releasing phosphate ions.2,4,17 Phosphate and Ca form hydroxyapatite crystals and mineralize the bone matrix, forming new bone.1 In the present study, the histological analysis showed newly formed bone trabeculae along the calvaria defect. According to the literature, in the presence of a fracture, healing-related events result in the formation of a bone callus that progresses to interconnect the two ends.16 These concepts explain why the process of bone repair began at the edges of the basal bone toward the center of the calvaria defect in all groups.
The histomorphometric analysis revealed that oral administration of latex at 3% and 50% did not increase the area of newly formed bone in the calvaria defect even after 15 and 30 days of treatment. On the other hand, the literature confirms that there is an increase in the area of the newly formed bone both in the calvaria12 and in the mandible socket8 of rats treated with natural latex using local product applications. This effect can be attributed to the presence of some phytochemicals in the latex. Dos Santos Neves et al12 identified that H. speciosa latex contains chlorogenic acid and naringenin-7-O-glycoside. There is evidence that these compounds stimulate osteoblastic activities.18,19 Active osteoblasts secrete a large amount of alkaline phosphatase and osteocalcin, important molecules for Ca deposition and bone mineralization (Florence et al., 2017). Thus, these phytochemicals can increase bone mineral density and improve bone microarchitecture.18,19 Possibly, our results are different from those found in the literature because of the different routes of product administration. In the present study, we evaluated the effect of the orally-administered product, whereas studies described in the literature demonstrate product results when administered topically. To confirm the effect of oral latex administration on bone neoformation, further doses need to be evaluated in future studies. Also, the chemical composition of newly formed bone also needs to be analyzed to understand the popular belief about the benefit of consuming mangaba milk daily for the treatment of fractures.3
In the present article, we have found that daily and oral administrations of 3% and 50% H. speciosa latex over a 15- and 30-day period did not contribute to the increased area of newly formed bone in the calvaria defect. To confirm other effects of this latex on bone mass, different doses of the product need to be tested and the chemical composition of newly formed bone needs to be evaluated in future studies. In this way, we will be able to understand the popular belief about the benefit of consuming mangaba milk daily for fracture treatment.
The authors would like to acknowledge the National Council for Scientific and Technological Development (CNPq, in the Portuguese acronym), number 141226/2014-5, for the support given to the present study.