implantation of sepiolite-collagen complexes in surgically created rat calvaria defects

Biomateriols 16 (1995) 625-631

0 1995 Else&r Science Limited

Printed in Great Britain. All rights reserved

0142.9612/95/$10,00

Implantation of sepiolite-collagen complexes in surgically created rat calvaria defects

Jo& I. Herrera, Nieves Olmo*, Javier Turnay*, Albert0 Sicilia+, Antonio Bascones, Jose G. Gavilanes* and Ma Antonia Liza.rbe* Departamento de Medicina y Cirugia Bucofacial (Periodoncia), Facultad de Odontologia, Universidad Complutense, 28040-Madrid, Spain; ‘Departamento de Bioquimica y Biologia Molecular, Faculfad de Ciencias Quimicas, Univer- sidad Complutense, 28040-Madrid, Spain; +Departamento de Cirugia y Especialidades MBdico-Quirtirgicas, Escuela de Estomatologia, Universidad de Oviedo, Oviedo, Spain

The response of osseous tissue to the implantation of sepiolite-collagen complexes has been studied.

Sepiolite, sepiolite-collagen complex and 0.5% glutaraldehyde-treated sepiolite-collagen complex

were implanted in created circular defects in rat calvaria. The tissue reactions were analysed using

light, transmission and scanning electron microscopies. The patterns of bone growth were radiogra-

phically analysed and the bone activity was indirectly quantified by using a point-count method. The

reaction against the three implanted materials is characteristic of a foreign body reaction with

abundant macrophages and giant cells. Implanted products have been detected in macrophages,

which suggest the involvement of phagocytosis in the resorptive process. Bone grew at the implanta-

tion sites originating excrescences or sometimes a thin bridge at the defect margins. The studied

materials, after implantation in contact with bone tissue, did not produce any toxic effect or necrosis,

allowing bone activity.

Keywords: Sepiolite-collagen complexes, collagen-based biomaterials, composites, in vivo implanta-

tion

Received 10 August 1994; accepted 19 October 1994

Sepiolite-collagen (SC] complex has been described previously as a composite material obtained from the interaction of a mineral compound, sepiolite (hydrated magnesium silicate), and a protein, type I collagen- the main component of the extracellular matrixle3. Treatment of this complex with glutaraldehyde results in collagen cross-linking originating the so-called sepiolite-collagen-glutaraldehyde treated (SCG) complex4. Several in vitro studies using human skin fibroblasts have shown that these two types of complexes do not exhibit any cytotoxic effects, since they allow normal cell adhesion and spreading on their surfaces4z5. The growth, morphology and collagen biosynthesis of human fibroblasts cultured on the above-mentioned complexes are normal compared with cells cultured on standard substrata”37. These results suggested the potential usefulness of sudh complexes as biomaterials.

In vivo implantation studies of SC complexes in rat soft tissues (subcutaneous and intramuscular) showed that these materials elicit a foreign body reaction. Abundant macrophages and multinucleated giant cells appeared initially, being progressively replaced by

Correspondence to Dr M” Antonia Lizarbe.

granulation tissue and further by dense connective tissue’. While SC complex is invaded and divided by inflammatory cells becoming resorbed after 3 months, SCG complex remains nearly unaltered for the entire period of study (4 months), being finally encapsulated by fibrous connective tissue. This suggested that both types of complexes exhibit different behaviour. On the other hand, the measurements of collagen antibody levels in sera showed that SC complex induces a low immunological response which is negligible for the SCG complex’.

The peculiar composition of the SC complexes, an inorganic component and a protein matrix, a priori suggests that they may be interesting biomaterials for implantation in osseous tissue. The aim of this study is the evaluation of the generic response of the osseous tissue to the implantation of SC complexes.

MATERIALS AND METHODS

Sepiolite, SC complex and 0.5% glutaraldehyde-treated SC complex were implanted in rat calvaria according to the protocol further described in order to study the bony tissue response. Animals were killed at 2, 4, a,

625 Biomaterials 1995, Vol. 16 No. 8

626 Implantation of sepiolite-collagen complexes: J.I. Herrera et al.

12, 24, 36 and 52 wk. Three specimens for each implantation time were histologically evaluated and additional ones used for electron microscopic evaluation. Three to six specimens were radiographically examined for each time period.

Material preparation

SC complexes were routinely prepared at a 0.6 protein/ sepiolite weight ratio’. Sepiolite was kindly supplied by Tolsa S.A. (Madrid, Spain) and type I fetal calf skin collagen was purified and characterized as previously described’,‘. Treatment of the SC complex with glutaraldehyde was performed for 20min at room temperature in 0.1~ sodium phosphate buffer, pH 7.4, containing 0.5% glutaraldehyde4. The materials obtained were dried and sterilized under ultraviolet light before use.

Implantation and resection

Male Wistar rats (ZOO-250g) were used in this study. After being anaesthetized, the hair overlaying the cranium was shaved and a midsagital incision was made with a scalpel. The cranial skin, the fascia and the periosteum were raised to expose the cranial vault. Then four holes (3.5mm in diameter) were drilled under copious saline coolant, two in the frontal bone and one at each parietal (Figure la), by using a trephine and motorized equipment. The two materials tested were hydrated for 10min using 0.1 M phosphate buffer and then placed into the holes (Figure lb).

Sepiolite was implanted in powder form. One of the holes was left empty as a control. Finally, the periosteum and the skin were replaced and sutured by layers. At the selected time points, the animals were killed by anaesthetic overdose. After exposing the area, the calvarium was resected by using a high speed handpiece; a thin layer of cerebral tissue was excised with a scalpel, remaining attached to the inner face of the cranium in order to prevent damage of the implantation areas. The entire piece was then removed and submerged in the fixative solution.

Radiographical analysis

After fixation, the cerebral tissue was removed, the calvaria radiographed and the radiograph mounted in slide frames. In order to quantitatively evaluate new bone ingrowth, a point-count method was used. The radiographs were projected at a standard magnification onto a grid with points marked at regular 4mm intervals; the number of points on the implantation areas exhibiting a radiodensity lower than the adjacent normal bone were counted. The percentage of such an area filled with radiographically detectable bone was calculated from the previously known standard defect at each experimental and control site and for each implantation period. Sites exhibiting any damage resulting from the calvaria extraction procedure were discarded.

Light microscopy studies

The specimens were fixed in 10% neutral buffered formalin and demineralized in 10% ethylenediamine tetraacetic acid. The skulls were then cut in such a way that every experimental and control area was

Biomaterials 1995, Vol. 16 No. 8

Figure 1 Rat calvarium showing the four surgically created defects before (a) and after (b) implantation.

separated and all of them were embedded in paraffin. Sections of 6pm were cut, stained with haematoxylin- eosin, and von Kossa stain, and three middle sections examined with a light microscope.

Transmission electron microscopy

Six animals were used for this study; three of them were killed at 2 wk implantation time and the others at 4 wk. The skulls were fixed in 0.1 M phosphate buffer, pH 7.4, containing 2.5% glutaraldehyde, and washed with phosphate buffer. Dehydration was performed in a graded series of acetone solutions. The specimens were treated with osmium tetroxide and embedded in AralditeH’. Sections (1 pm) were cut, stained with toluidine blue and observed by light microscopy in order to select the areas for further examination. Then ultrathin sections were cut, placed on a grid, contrasted with lead acetate and examined with an electron microscope (Hitachi, HU-12, Tokyo, Japan).

Implantation of sepiolite-collagen complexes: J.I. Herrera et al. 627

Scanning electron microscopy

Unstained histological sections previously processed for light microscopy were deparaffined by xylol treatment and newly hydrated by immersion in a graded series of ethanol. The specimens were then postfixed by treatment with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 45min at room temperature, dehydrated by a series of acetone solutions, and vacuum dried at room temperature. The coverslips were mounted on stubs and a coating of gold-palladium was applied under vacuum by using a sputter coater. The specimens were observed on a scanning electron microscope (Phillips SEM 500, Eindhoven, The Netherlands) operating at 10-20 mV with various tilting angles.

Statistical analysis

Quantitative data from the radiographic study were analysed by the Mann-Whitney U test looking for differences between the tested groups.

RESULTS

Histological analysis

A granulomatous infiltrate with abundant macrophages and multinucleated giant cells is observed at the implantation sites of the materials tested. Such an infiltrate is progressively replaced by granulation tissue and further by dense connective tissue. Since these findings are similar to the ones previously described for soft tissue implants*, in the present work attention is mainly focussed on the bone reaction.

Sepiolite and SC complex appear very fragmented and scattered over the above-mentioned granulomatous infiltrate (Figure Za, c). Foci of acute inflammatory infiltrate can be found in some specimens at 2 wk implantation. Osseous activity is observed at the defect margins from the early stages. Sometimes the bone growth originates excrescences. In other specimens, partial or complete bony bridges composed of compact and cancellous bone containing haematopoietic marrow can be observed after 2 wk implantation of SC complex (Figure 2~). The amount of implanted material decreases through the implantation period becoming completely resorbed in some areas. A complete bridge and areas where the material is still present are observed during the 9- to 12-postimplanta- tion period (Figure 2b, d).

Figure 2 Observation by light microscopy of haematoxylin- and eosin-stained section of sepiolite implants at 2 (a) and 12 (b) months and sepiolite-collagen (SC) complex implants at 2 weeks (c) and 9 months (d). (Original magnification: x40; b, bone margins; g, granulomatous infiltrate; bm, bone marrow; ob, osseous bridge; bb, bony bridge.)

Figure 3a. Abundant osseous regeneration can be observed in the majority of empty defects.

Histological analysis of the SCG complex implantation renders similar observations, also exhibiting granulomatous infiltrate. During the first 2 months of implantation, the SCG complex appears in the form of compact blocks surrounded by an acute inflammatory infiltrate consist- ing of polymorphonuclear leucocytes (Figure 3b, c). Exceptionally, one specimen exhibited calcification at 2 wk based on the von Kossa stain analysis. Blocks of SCG complex encapsulated in fibrous tissue remain after 36 and 52 wk implantation (Figure SC).

Analysis by transmission electron microscopy reveals the presence in macrophages of fibrous exogen- ous material, related to the sepiolite fibres. This is observed for all the specimens and the three materials tested (Figure 4~). Regarding the control sites, abundant deposition of collagen is observed at the non-bone filled areas (Figure 4b). The scanning electron microscopy has been used to illustrate the bone-cellular reaction interface, as shown in Figure 5.

Radiographical analysis

No inflammatory reaction was found at the non- Radiology has been considered for the evaluation of implanted control sites (except the initial one due to osseous formation’*“. The precision of this methodo- the surgical procedure), as can be observed in logical approach depends on both the mineralization

Biomaterials 1995. Vol. 16 No. 8


Figure 3 Observation by light microscopy of implantation of sepiolite-collagen glutaraldehyde treated (SCG) complex. (a) Control, 2 weeks. SCG complex at 2 weeks (b) and 9 months (c). (Original magnification: x40; m, material; b, bone margins; g, granulomatous infiltrate; f, fibrous tissue; c, connective tissue.)

rate and the thickness of the new bone. Therefore, thin or poorly mineralized osseous tissue might be radiolo- gically undetectable, which implies a limitation for this technique. In spite of this, such a methodology has given valuable information.

The defects observed at z wk implantation time have an irregular and oval morphology, which indicates that a radiographically detectable osseous activity exists (Figure 6). While bone commonly grows at only one side in the control areas, a continuous border growth is observed at the implantation sites (Figure 6a, b). In some defects, the area shows a radiodensity compar- able to that corresponding to normal bone (Figure 6b).

In order to quantify the reparative process, all the radiographs were analysed as described above. The data obtained are given in Table 1 and represented in Figure 7. These data show great variability, which has also been observed in a similar model for the study of collagen implants’“, 14. Mean values of bone filling greater than 66% are observed from 12 wk implantation. The SC and SCG groups show low or negative values, the latter meaning that an increase in the

Figure 4 Transmission electron micrographs of implantation of sepiolite-collagen (SC) complex and empty control. a, SC complex at 4 weeks, arrows show limits of a macrophage (n, cellular nucleus; m, material; I, lipidic drop). b, Control at 2 weeks. (Original magnification: x4800.)

initial defect area (resorption) took place. Such a phenomenon may be in relation to the inflammatory and phagocytic activity that occurs in the implantation area. The Mann-Whitney U test detects statistically significant differences between the control group and the SC and SCG groups (except by the first periods of time). No differences were seen between the two experimental SC and SCG groups. Differences between the control and the sepiolite groups are less consistent.

DISCUSSION

Materials showing the ability to support mineral formation, with osteoinductive, osteoconductive or filling properties, have been extensively investigated



Figure 5 Scanning electron micrograph of sepiolite- collagen complex implants. a, 8 weeks (original magnification x800) (bar = 10pm). b, Detail of 5a. (Original magnification: x3000; b, bone; r, reaction.)

in order to heal bone defects. Polymeric materials”, ceramics and bioactive glassl”, demineralized bone”, and collagen-based biomaterials17 are of interest in some areas of the biomedical field, such as orthopaedic surgery or periodontologyr8. Taking into account the characteristics of the SC complexeslP8, we have studied their biocompatibility and behaviour upon osseous implantation. The response observed is similar for the three materials tested and consists of a foreign

Figure 6 Radiographs of . . . .~. calvaria. a, 6 months; b, 9

montns. Irregular aetects can be seen. A greater bone filling is observed in the control sites (inferior right). In b, the control defect shows a radiodensity nearly equal to normal bone.

body reaction. The initial acute inflammatory reaction, common for most biomaterials, is related to both the surgical injury and the presence of the material itself. The resorption dynamics of sepiolite and SC complex resembles that observed in soft tissue implants’, although slower. For the SCG complex, the dynamics are also similar, persisting in the material 1 year after implantation and becoming encapsulated by fibrous tissue. Calcification is considered a negligible event on the osseous implantation of SC complexes, since it has been observed only in one specimen (2 wk) of SCG complex. No stimulatory action of the collagen component of the complexes has been observed on new bone formation, in contrast with the results described after implantation of poly(hexaethy1 methacrylate)-collagen into bone, where the collagen component of this composite seems to be important in bone defect healing17.

Although several authors reported that cranial defects usually heal by means of a fibrous scar and not by bone1g220, in the present study a significant amount of osseous regeneration is found. As demonstrated by Frame’l, the type of healing of a cranial defect by either bone or fibrous tissue depends on the defect size. The discrepancy between the results obtained in this study and others reportedl’-” may be due to the

Table 1 Bone filling in experimental and control defects evaluated by radiology

Time (wk) Percentage of radiopaque area

Control Sepiolite SC complexa SCG complexb

2 -4.577f 4.086 (4) 12.207 zt 13.219 4 43.662 zt 20.511 (5) 36.267 f 10.329 8 37.089 zt 9.562 (3) 41.080f 16.921 12 66.784 f 24.152 (6) 39.296* 22.526 24 85.681 f 2.151 (3) 34.742 zt 19.533 36 92.253 f 6.718 (3) 25.587 -f 28.461 52 78.404 & 11.319 (3) 59.389 * 37.173

(3) -12.324 f- 17.643 (4) -18.028 f 14.977 (3) -15.023 f 16.095 (5) 7.394 f 21.764

I:; -20.657 25.117 zt f 15.909 16.279 (3) 15.493 f 18.887

(4) 4.754 * 19.100 (4) (5) 8.451 zt 28.314 (5) (3) 6.338 zt 25.615 (3) (6) 10.446 zt 21,.250 (6)

I:; -0.176 18.310 zt f 34.037 11.591 (4) (4) (4) -6.162 f 23.801 (4)

Data correspond to the means and standard deviations. In each case, the number of specimens used IS indicated in parentheses

“SC complex = sepiolite-collagen complex.

%CG complex = sepiofite-collagen glutaraldehyde treated complex.

Biomaterials 1995. Vol. 16 No. 8


%RA-S %RA-SCG

loo Ix, ._____ - ___._._ _ __._____._.____.._ __ __.._..

60 30

60 30

40 40

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0 0

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-40 -40

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loo loo ; . . . . . . I_. . . . . __..*. __.._. _-?--.__.._. . .._....

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-40 -4o/ , t W) o IO 20’ so 40 50 60

/ 0 IO x so 4) SO 60 t(W

Figure 7 Bone filling in experimental and control defects evaluated by radiology. Means and standard deviations expressed in Table 7 are represented. (%RA, percentage of radiopaque area; S, sepiolite; SCG, 0.5% glutaraldehyde-treated sepiolite- collagen complex; SC, sepiolite-collagen complex; C, control; t, time in weeks; *, statistically significant difference with respect to the control.)

different defect sizes used, which could be over or under the critical size for each type of animal.

The bone growth pattern, including excrescence formation at the defect margins, is probably due to the presence of the material itself, preventing to some extent bone filling and healing of the defect. In other cases bone can overcome the obstacle building a thin layer starting from either the outer or the inner face of the cranial vault, and resulting in a bridge. This kind of growth pattern has also been reported in empty control and experimental defects for other materials using a similar animal mode113~14~21.

The materials tested do not produce any toxic effect or necrosis, and they allow bone activity. Nevertheless, SC and SCG complexes do not exhibit any enhancing effect on osseous regeneration in this experimental model with the defect size employed. However, they facilitate a certain degree of osseous resorption. In spite of the clearly different behaviour of SC and SCG complexes regarding resorption, no differences in bone filling are observed in this model. This may be due to the fact that the reaction to both materials is high enough that the bone dynamics hinder any differences from appearing.

CONCLUSIONS

Implantation of SC complexes in created rat calvaria defects does not produce any toxic effect or necrosis. They exhibit a resorption dynamics similar to that observed for implantation in soft tissues. Therefore, SC complexes do not exhibit, in this particular experimental model, any enhancing effect on osseous regeneration

when compared with the evolution of control defects. However, resorption of SC complexes is followed by osseous regeneration, thus allowing the healing of bone defects.

ACKNOWLEDGEMENT

The authors are indebted to Dr M. A. Aragoncillo for her skillful assistance in the microscopy studies.

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implantation of sepiolite-collagen complexes in surgically created rat calvaria defects

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