ARTÍCULO CIENTÍFICO

 

Experimental study in rats of mandibular bone regeneration with different biomaterials

Estudio experimental sobre la regeneración ósea mandibular de la rata con diferentes biomateriales

 

 

B. Peral Cagigal¹, L.M. Redondo González¹, A. Verrier Hernández², A. Serrat Soto¹, M.Á. Torres Nieto³, C. Vaquero Puerta⁴

¹ Médico Adjunto. Servicio de Cirugía Oral y Maxilofacial. Hospital del Río Hortega de Valladolid. España
² Jefe de Servicio. Cirugía Oral y Maxilofacial. Hospital del Río Hortega de Valladolid. España
³ Médico Adjunto. Servicio de Anatomía Patológica. Hospital del Río Hortega de Valladolid. España
⁴ Jefe de Servicio. Angiología y Cirugía Vascular. Hospital Clínico de Valladolid. Catedrático del Departamento de Cirugía. Director del Laboratorio de Investigación Quirúrgica y Técnicas Experimentales. Facultad de Medicina de Valladolid. España

Correspondence

 

 

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ABSTRACT

Objective. Mandibular bone defects can occur as a result of trauma, neoplasm, or infectious conditions. Such conditions often are associated with severe funtional and esthetic problems. Corrective treatment often is complicated by limitations in tissue adaptation. The aim of this study was to compare new bone formation following application of a bioabsorbable membrane and two types of bone substitutes.
Material and method. In the present study, 24 fourmonth- old male Wistar rats were used. Standardized round throughand- through bone defects (4 mm in diameter) were made in both mandibles and the rats were divided into four groups: one control group and 3 experimental groups. Animals were killed 3 and 6 weeks after surgery. Bone defect healing was assessed by radiologic and histologic analysis.
Results. The control defects showed no bone formation; holes were filled with fibrous connective tissue. Bone membrane alone was an efficient barrier, excluding nonosteogenic tissue. However, new bone formation underneath the membrane was incomplete. The Colloss^® + membrane group showed complete healing after 6 weeks. The NovaBone^® + membrane group showed no bone formation and particles appeared in the defect.
Conclusions. The percentage bone regeneration was significantly better in the defects filled with Colloss^® and covered with Lambone^® than the other experimental groups.

Key words: Guided bone regeneration; Bone substitutes; Demineralized laminar bone membrane; Bovine bone collagenprotein extracts; Bioactive glass.

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RESUMEN

Objetivo. Los defectos óseos mandibulares resultantes de infecciones, traumatismos o resecciones oncológicas, van a producir severos problemas funcionales y/o estéticos, que van a precisar de un tratamiento complejo. Durante los últimos años, las aportaciones al terreno de la reconstrucción ósea se han debatido entre métodos tan dispares como la distracción ósea o la utilización de colgajos libres microvascularizados, pasando por un sin fin de biomateriales. El objetivo de este estudio fue comparar la formación de hueso nuevo tras la aplicación de una membrana reabsorbible y dos tipos de sustitutivos óseos.
Material y método. Se utilizaron 24 ratas adultas macho tipo Wistar, en las que se crearon defectos circulares de 4 mm de diámetro en ambos lados de la mandíbula. Se formaron 4 grupos, un grupo control y 3 grupos experimentales. Los animales fueron sacrificados a las 3 y 6 semanas de la cirugía, realizándose un análisis radiológico e histológico.
Resultados. Los defectos control no mostraron formación ósea, apareciendo una reparación por tejido fibroso. La membrana de hueso utilizada de forma aislada, actuó como una barrera eficaz excluyendo los tejidos no osteogénicos, pero no se produjo reparación total del defecto en ningún caso. El grupo de Colloss^® y membrana, mostró una regeneración ósea completa del defecto a las 6 semanas. El grupo de NovaBone^® y membrana, no mostró formación ósea, apareciendo las partículas del biomaterial ocupando el defecto.
Conclusiones. La regeneración ósea fue significativamente mayor en los defectos rellenos con Colloss^® y cubiertos con la membrana de Lambone^®, comparado con los otros grupos experimentales.

Palabras clave: Regeneración ósea guiada; Sustitutivos óseos; Membrana de hueso desmineralizado; Colágeno liofilizado bovino; Vidrio bioactivo.

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Introduction

In oral and maxillofacial surgery, we often have to deal with bone defects of different origins (trauma, tumors, and other) that constitute a therapeutic challenge because reconstruction must guarantee acceptable aesthetic and functional results.

Autologous cortical-cancellous bone is the ideal reconstructive material because it is completely biocompatible, has good osteogenic potential, mechanical resistance and resistance to infection, and ensures rapid consolidation.¹ The objective is to find a biological or synthetic substitute for autologous bone that allows bone defects to be repaired without the disadvantages of donor-site morbidity, limitations in the amount of bone that can be obtained, and the anatomic morphology of the bone. In the present experimental study, we hypothesized that the potential of membranes to induce bone regeneration can be increased by using filler in the defect, as the filler impedes membrane collapse, increases the concentration of osteogenic factors in the defect, and defines the anatomic contours of the neoformed bone.

Guided bone regeneration (GBR) uses membrane barrier systems to isolate the bone defect, in order to impede the growth of tissues with rapid repair capacity, such as connective tissue, which interfere with the osteogenic potential of the defect.

The membrane used in our study was sheets of demineralized cadaveric cortical bone (Lambone^®, Pacific Coast Tissue Bank, Los Angeles, CA, USA), which is radiotransparent, biocompatible, and resorbed in 6-8 months. This membrane has osteoinductive and osteoconductive capacity.

NovaBone-C/M^® (Porex Surgical, Inc., Newnan, GA, USA) is a synthetic, bioactive, osteoconductive, biocompatible, antimicrobial, radiotransparent glass that is resorbed by dissolution in 6-9 months. This ceramic binds to bone by bioactive fixation, forming a hydroxycarbonate apatite layer that is chemically and structurally equivalent to the mineral phase of bone.

Colloss^® (Ossacur^® Medical Products, Germany) is a preparation based on bovine diaphyseal bone matrix extract, which contains mainly collagen type I and insoluble proteins. This freeze-dried collagen has osteoinductive activity, is biocompatible and radiotransparent, and resorbs in 6 to 8 weeks.

 

Material and Method

Experimental model and study design

Twenty-four white male Wistar rats were used, age 3- 4 months (adult). The animals were distributed into 4 groups according to the type of biomaterial filler and membrane used (Table 1).

The animals were anesthetized using an intraperitoneal injection of ketamine (80 mg/Kg weight), followed by local infiltration of articaine (Ultracaine^® 0.5 ml without vasoconstrictor). A submandibular approach was used to expose the mandibular angle and ramus, where a circular bone defect 4 mm in diameter was made (a critically sized defect) (Fig. 1). The ostectomy was performed with an electrical motor and a 4-mm carbon burr under continuous irrigation with physiologic serum. The experiment was bilateral.

The defect was left open and uncovered in the control group (group I). In group II, the defects were covered on the vestibular and lingual side with demineralized bone membrane (Lambone^®) measuring 5 mm on the side. In group III, the defects were filled with freeze-dried collagen (Colloss^®) and covered with Lambone^®. In group IV, the defects were filled with bioactive glass (NovaBone^®) and covered with Lambone^®.

A series of parameters were evaluated in the postoperative clinical follow-up: general condition of animal, appearance of the wound and intervention zone, bleeding, exudates or collections, extrusion of biomaterials or membranes, and degenerative changes caused by dental injuries. Once the experimental period concluded, the animals were killed by injecting an intraperitoneal anesthetic overdose of ketamine (Ketolar^®).

Specimens were obtained by block excision of each of mandible. A total of 48 specimens was obtained. A macroscopic evaluation was made and then the piece was immersed in buffered formolin 10% for radiologic and histologic processing.

Each specimen was radiographed using Trophy CCX digital dental radiologic equipment. All radiographs were made under the same exposure conditions. Each specimen was laid on its lingual surface and placed on the dental radiographic plate (Fig. 2). An aluminum (Al) plate with a total of 6 grades, each of which corresponded to an increase in thickness of 1/3 mm Al, was placed next to the mandible. The range of the scale was 0.33 mm to 2 mm Al.

A square block of bone was excised from each mandible, which included the bone defect to be studied and a margin of mandibular bone (cube with a 7 mm side). The bone blocks were submerged in a decalcifying solution of formic acid and processed, embedded in paraffin, sliced with a rotation microtome, and finally stained with hematoxylin-eosin.

Study variables

Macroscopic evaluation: At the time of death of the rat and dissection of the specimens, a descriptive evaluation was made of the following parameters: a) anatomic and tissue organization; b) infections, exudates or tissue collections; c) biomaterial displacement; d) presence of fractures; e) dental degenerative changes; f) bone sequestration; and g) consistency and superficial form of the defect.

Radiologic evaluation: A densitometric study was made of the radiologic repair of the mandibular defect. The radiologic images were transferred to a computer and digitalized according to a gray scale (256 levels), using the Sigma Scan Pro Image Analysis 5.0 for Windows computer program. The study area was defined as a circular cylinder 4 mm in diameter, similar to the original surgical defect. The optical density of the study area was expressed in relation to the Al grades described above. The density of the mandibular defect was expressed as an equivalent to one-third millimeter of Al, resulting in a value from 0 to 6.

Histologic evaluation: The histologic reading of the preparations was made with an Olympus BX41 optical microscope adapted to an Olympus DP70 digital camera, which was connected to a computer for recording the images. The histologic data in the study groups were analyzed using four parameters according to a scale of numerical scores assigned to each parameter, according to the model proposed by Heiple (Table 2).³

Statistical analysis: SPSS for Windows was the statistical program used. Each parameter was studied according to the basic statistical descriptors and using central tendency and dispersion measures. In the statistical test used to compare hypotheses, the level of statistical significance was 5%.

 

Results

Postoperative clinical evaluation: The general condition of the animals was satisfactory in all the groups. Only soft-tissue swelling due to ostectomy was observed, which disappeared in 24-48 hours without treatment.

Macroscopic results: Two study specimens were discarded (due to abscess and mandibular fracture). The results refer to the 46 remaining mandibles.

In the control group, softtissue occupation of the defect occurred. In the experimental groups, no displacement of the bone substitutes or membranes was observed, which were examined at 3 weeks and 6 weeks. Mild peripheral resorption occurred, but no displacement in relation to the defect. Adherence and integration into the mandibular bone were good.

Descriptive radiologic results

Group I (control group): At both week 3 and week 6, minimum signs of radiologic repair were found; generally, mandibular radiotransparent circular defects were observed (Fig. 3A).

Group II (Lambone^® membrane): At 3 weeks of evolution, low levels of radiopacity were observed in the defect, although the circular form of the defect was conserved. At 6 weeks, the radiopacity increased and signs of bone repair with centripetal growth from the edges of the defect was evident (Fig. 4A).

Group III (Lambone^® membrane and Colloss^®): At 3 weeks, homogeneous levels of radiopacity were evident in the center of the defect, although the continuity around the edge still was not very marked. At 6 weeks, the radiologic repair of the defects was practically complete in every case (80- 100%), showing strong continuity with the surrounding bone and a highly homogeneous distribution of the radiologic density (Fig. 5A).

Group III (Lambone^® membrane and NovaBone-C/M^®): At 3 weeks, the radiopacity of the lesion was very heterogeneous, with a «granulated» appearance at the center of the defect. At 6 weeks, the radiopacity was more intense and more homogeneous, although the granular aspect of the radiologic repair and lack of continuity with the bone surrounding the defect persisted (Fig. 6A).

Quantitative radiologic results: Bone densitometry showed statistically significant differences (p < 0.001) at 6 weeks of evolution in all the experimental groups (II, III and IV) with respect to the control group (I). Group III (membrane + Colloss ^®) achieved the highest mean value (4.89 ± 0.66).

Descriptive histologic results: Group I (control group): The defect healed at the expense of soft tissue (fundamentally connective, muscular, and adipose tissue). No ossification nuclei or bone tissue were present (Fig. 3B).

Group II (Lambone^® membrane): Two fronts of bone growth were observed that started from the ends. Bone growth was guided by the membranes, which remained stable, delimiting the area of the defect, and intimately adhered to the bone at each end and no collapse. At 3 weeks, bone regeneration occupied approximately one third of the defect, whereas at 6 weeks it extended to the middle of the defect (Fig. 4B). However, complete bone substitution was not observed in any animal.

Group III (Lambone^® membrane and Colloss^®): At 3 weeks, a mandibular defect was observed that was perfectly delimited by membranes, with extensive repair by immature cancellous bone tissue that occupied about two thirds of the defect, but with a certain lack of continuity at the central level. At 6 weeks, the bone was more compact, voluminous and mature, showing continuity at the mandibular ends. It filled the total volume of the defect and the space delimited by the membranes (Fig. 5B). In no case was bovine collagen detected.

Group IV (Lambone^® membrane and NovaBone^®): At 3 weeks and at 6 weeks, a membrane-delimited defect was observed in which bone formation was absent and connective tissue formation, intense foreign-body inflammatory cellularity, and abundant polymorphonuclear cells around particles of biomaterial (NovaBone ^®); these particles appeared as transparent, birefringent granules (Fig. 6B).

Qualitative histologic results: In Table 3 are shown the mean values of the points obtained in different treatment groups, according to the scale of scores proposed by Heiple.³

Bone maturity grade: At 3 weeks and 6 weeks of evolution, significant differences (p < 0.01) appeared in groups II and III with respect to the control group (group I) and group IV, as well as group III with group II.

Bone marrow presence and quality: At 3 weeks and at 6 weeks, significant differences (p < 0.01) were found in group III with respect to groups I, II and IV.

Bone junction: At 3 weeks, significant differences (p < 0.01) were observed in group III with respect to the control group (group I), group II, and group IV At 6 weeks of evolution, differences (p < 0.05) also were observed in group II with respect to the control group.

Peripheral formation: At 3 weeks and 6 weeks of evolution, significant differences (p < 0.05) were evident in group III with respect to the control group (group I).

 

Discussion and conclusions

Clinical and macroscopic

The animals tolerated the surgical procedure without complications. Bone substitutes and membranes were considered biocompatible and clinically well tolerated, because there were no problems in relation to their biological behavior. The implanted membranes, alone or associated with a bone filler, showed good surface adherence, adequate osteointegration, no displacement, and no tendency to collapse.

Radiologic and histologic

The absence of radiologic and histologic evidence of bone repair in all the mandibular defects of the control group indicates that this experimental model is valid for our study, as it does not have self-regenerating capacity.

Group II (Lambone^® membrane): The isolated use of demineralized bone membranes significantly improved the bone regeneration of the mandibular defect at 3 weeks and at 6 weeks, compared with the control group.

In the radiologic study, this difference was significant only at 6 weeks, possibly because the immature, hardly calcified bone tissue observed in the histologic study was not evident on radiologic analysis. In our study, we did not observe complete bone regeneration of the defect in any of the animals at 6 weeks, only 50% bone regeneration, which coincides with the results of other authors who have used barrier membranes.⁴

This type of membranes, according to the studies of Hämmer 5 on histologic repair with GBR techniques, originates more bone formation in peripheral areas than the central areas. This coincides with the radiologic results, which showed that radiopacity increased centripetally from the edges of the defect. Our results coincided with the classic histologic descriptions by Urist⁶ of ectopic osteoinduction in relation to the process of ossification of demineralized grafts by intermediate cartilage formation.⁷ In our study, in addition to macroscopic and radiologic results, we were able to confirm histologically that the membranes acted as a barrier, impeding soft-tissue invasion and proliferation in the defect and improving the amount of bone regenerated with respect to controls.

In previous studies, it has been demonstrated that bone regeneration in critically sized mandibular defects in rats is possible after 6 weeks using nonresorbable Gore-Tex^® membranes.^8,9 We believe that in our study complete repair did not occur (40-50%), not as a result of membrane collapse, but because the regenerative process was slower, which lead to failure of the technique at this level.

Group III (Lambone^® membrane and Colloss^®): The association of membrane and freeze-dried collagen improved bone regeneration with respect to the use of membrane alone. The differences in both the radiologic and histologic study were statistically significant differences at 3 weeks and at 6 weeks compared to the control group. These results, as suggested by other studies, demonstrated that the regenerative effects of biodegradable membranes can be potentiated by associating the membranes with a biomaterial defect filler.^10,11 Our results coincide with the results of other authors who have postulated that freeze-dried collagen accelerates bone regeneration in the early phase (first 2 weeks).¹² Radiographically, at 3 weeks the defects showed homogeneous levels of radiopacity, although with a certain lack of continuity with the adjacent bone. However, at 6 weeks we confirmed the repair of 80 to 100% of the defect, with high radiologic homogeneity and continuity.

Histologically, at 3 weeks bone formation was greater in peripheral areas, but at 6 weeks, there were no differences between the areas of the defect, indicating greater uniformity in the bone repair. There were even cases of total and homogeneous repair of the defect. When freeze-dried collagen is used alone, bone formation frequently advances away from the area of the defect, which means that bone regeneration does not conform to the defect.¹³ The use of membranes prevents the dispersion of the biomaterial, increasing its concentration in the defect and enhancing the osteoinductive and osteoconductive stimulus. The membranes have the advantage of being biodegradable and not requiring a second surgical intervention for removal.

Histologic analysis of the specimens confirmed the radiographic and macroscopic results, yielding new data that suggested that combined treatment with freeze-dried collagen and resorbable demineralized bone membranes has advantages in the regeneration of critical mandibular defects.

Group IV (Lambone^® membrane and NovaBone-C/M^®): The bone regeneration achieved with membranes and bioactive glass did not differ significantly at any period in the evolution from the control group from a histologic vantage point. Interestingly, the radiologic study at 6 weeks showed statistically significant differences compared to the control group, which may contradict the histologic results. However, these results also have been found in other studies, such as the one by Dorea,¹⁴ in which femoral defects in cats repaired with bioactive glass filler regenerated radiologically at 6 weeks without concordant histologic findings. For that reason, radiopacity per se should not be used as the only criterion for evaluating bone regeneration and should be correlated, as has been proposed, with the histologic findings.¹⁴ In fact, the apparent decrease in the size of the defect may be due to the precipitation of silica gel and the formation of calcium phosphate on the periphery of the defect, which is difficult to differentiate radiologically from host tissue.¹⁴ Histologically, the defects had not regenerated by 6 weeks; they were occupied by fibrous tissue and exhibited a foreign-body inflammatory reaction. There also were empty spaces between the particles of biomaterial, possibly due to the dissolution of granules and release of silica gel.¹⁶ The association of bioactive glass with demineralized bone membrane appeared to interfere with the process of bone regeneration, since there was less bone formation in the defect than when the membrane was used alone. Therefore, this experimental model did not demonstrate the osteoconductive properties of bioactive glass.

 

 

Correspondence:
Dra. Beatriz Peral Cagigal
Servicio Regional de Cirugía Oral y Maxilofacial
Hospital Universitario del Río Hortega
C/ Cardenal Torquemada s/n
47010 Valladolid, España
E-mail: beaperal77@yahoo.es

Recibido: 11.04.2008
Aceptado: 15.10.2008

 

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