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Biocompatibility of xenogeneic bone, commercially available coral, a bioceramic and tissue sealant for human osteoblasts

Mary Jo Doherty*, C. S&la&, N. Schwarzs , R.A.B. Mollan*, P.C. Nolan* and D.J. VVilsoni Schools of l Cfinicaf ~edicine/Ori~opaedic Surgery and ‘Biomedical Science/Anatomy, The Queen’s Universify of Be/fast, Belfast 3T9 7BL, UK; The Ludwig Boltzmann fnstitute for Experimental and Clinicaf Traumatotogy, Vienna, Austria

The drawbacks of freshly harvested autologous bone have resulted in the search for an alternative, capable of supporting osteogenic cell growth. This capability was examined in potential bone graft materials by culturing human osteoblasts on each material. Test materials included rat bone, Surgibone’” , Ostilit “, 13iocoral’K and Tisseel’H. After 3 days osteoblasts had adhered to all materials, except Ostilit’ With increasing time the cells multiplied on the materials, to varying extents. Cell affinity was greatest for rat bone and Tisseel ’ Fewer cells attached to Biocoral ” and Surgibone I”. Thus all the materials, with the exception of Ostilit “, were biocompatible for human osteoblasts.

Keywords: Osteoblasts, biocompatibility, bone graft materials

Received 14 October 1993; accepted 1 December 1993

Freshly harvested autologous bone is widely used for grafting in orthopaedic surgery**‘. It does, however, have drawbacks including donor site morbidity and limited availability, particularly in children3. As a consequence, alternative materials to autologous bone are being sought, A variety of bone graft substitute materials are commercially available: human or animal bone derived materials in both mineralized and demineralized forms, bioceramics consisting of hydroxyapatite and/or tricalcium phosphate4 and, more recently, marine corals.

Each of these materials has been tested in vitro and in vivo5-7 with only moderate degrees of success and autologous bone is still considered the best graft material’. The significant difference between autolo- gous bone and these bone graft materials is that autolo- gous bone contains a variety of living cells, including osteoblasts. One approach currently being investigated is the culture of a patient’s own osteoblasts on a bone graft substitute material such that they may be autotransplantedg, thereby constituting a form of ‘self- cell therapy”‘. Such a ‘living graft’ could then be used to increase the rate of bone healing at a graft site by providing a pool of ready differentiated osteoblasts and by lowering the antigenicity of the graft material. One important prerequisite of such a suitable ‘carrier’

Correspondence to Dr D.JI Wilson.

is that it is able to support the attachment and prolifera- tion of osteogenic cells.

So far, osteoblasts (rat and human) have been grown in vitro on bone replacement materials such as synthetic bioactive glass, calcium phosphate powders, ceramics and cora111-‘4. Recently, the mitotic expansion of human osteoblasts in vitro upon deminer- alized human bone matrix has been demons~ated’. Such in vitro assessment of biocompatibility of bone graft materials removes the problems of systemic factors which may be encountered in viva

In the first report of its kind, Begley et al.‘” compared the biocompatibility of several bone graft substitutes for human osteoblasts. The materials tested included different animal bones (mineralized and deminera- lized), coral and an apatite/collagen matrix. The work demonstrated that all the materials, with the exception of mineralized bovine bone (Surgibone ” ), were biocompatible for human osteoblasts. However, the study was not exhaustive in the range of bone graft substitutes examined.

The present investigation expands upon previous work and examines commercially available coral, bioceramic and tissue sealant along with deminera- lized rat bone and demineralized Surgibone “. Biocoral”’ (calcium carbonate) was selected for this study because it is the commercially available form of coral which has undergone purification, shaping techniques and sterilization (whereas only natural

Y’ 1994 Butterworth-Hainemann Ltd 0142-~12/94/156Oi-08

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602 Biocompatibility of bone grafts with human osteoblasts: M.J. Doherty et al.

coral was used in the previous study15). It is reported by its manufacturers to be biologically active, resorb- able, osteoconductive and osteophilic, in that it attracts osteogenic cells and enhances bone neoforma- tion. The bioceramic Ostilit”’ is a biphasic material composed of 60% tricalcium phosphate and 20% hydroxyapatite, and was examined because a trical- cium phosphate containing material has not been previously examined for human osteoblast biocompat- ibility. Tisseel” (fibrin sealant) is currently used in Europe to achieve haemostasis, seal/glue tissue and support wound healing. It has been used in bone grafting where it is thought to enhance bone healing, although some controversy remains16. For example, it has been reported to stimulate and accelerate repara- tive osteogenesis1’-20, but other authors argue that it is not advantageous in bone grafting, causing decreased growth and remodelling of bone transplants and less bone formation in grafts21~22. Demineralized Surgibone and demineralized rat bone were included to act effectively as controls, since it has been shown that they become populated with human osteoblasts within 15 days of culture”. In addition, mineralized rat bone was studied to compare its biocompatibility to the demineralized form.

In summary, the aims of this study were to determine the biocompatibility of each graft material for human osteoblasts, thereby indicating whether they would be suitable as carrier systems for osteoblasts in bone graft surgery. In addition to cell attachment, the growth of osteoblasts on the materials was examined after 3, 5 and 6 d in culture to ascertain if the materials promoted cell proliferation.

SERIALS AND HODS

The materials used, which were kindly donated by the manufacturers, included:

1.

2.

3.

4.

5.

Unilab Surgibone!“‘ (UNILAB, Inc. Mississauga, Ontario, Canada), which was provided in the mineralized form. It is sterile, processed, mature bovine bone with a mineral composition equivalent to hydroxyapatite, and was demineralized according to the Reddi and Huggins protocolz3. Biocoral ” (INOTEB, Saint Gonnery, France) in the form of 2 x 2.3 cm3 granules of sterile calcium carbonate. Ostilit ” (R3, Howmedica, London, UK), bioceramic granules consisting of 80% tricalcium phosphate and 20% hydroxyapatite (sized between 1.4 and 2.5 mm). Tisseel”“/tissue sealant (Immuno, Sevenoaks, Kent, UK), a two-component system consisting of a sealer protein solution and a thrombin solution. Rat bone (mineralized and demineralized), a complex, insoluble substance composed of type I collagen and a heterogeneous group of non-collagen- ous proteinsZ4.

Rat bone matrix was obtained as described previously by Begley et a1.15. Cleaned, dissected rat femora and tibia were demineralizedz3. Briefly, clean pieces of bone were extracted with absolute ethanol,

further extracted with anhydrous ethyl ether and then dried overnight at 37°C. Hydrochloric acid (0.5~) was used to demineralize the bone pieces (cut to approxi- mately 1-2 mm3). Similarly, Surgibone was deminera- lized. Completeness of demineralization was assessed by electron probe microanalysis using a JEOL Super- probe 733.

Osteoblast culture

Human trabecular bone fragments (approximately 1-2 cm”) were obtained at total hip replacement operations, After washing, bone fragments were placed in sterile flat bottom flasks and cultured in 5ml of media composed of Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Gibco, UK), penicillin, streptomycin, fungizone, L- glutamine and pyruvatez5. Culture flasks were incubated at 37°C in an atmosphere supplemented with 5% CO, and fed with complete changes of medium twice weekly. The osteoblastic phenotype of the cells was assessed by the tests previously reportedI’ and included spectrophotometric alkaline phosphatase assay, Western blotting of secreted bone proteins and culture mineralization when fed medium supplemented with ~-glycerophosphate and ascorbic acid.

Seeding of osteoblasts onto the test materials

Each material (previously sterilized in 70% ethanol for 12 h, in ~ngizone for 72 h and washed repeatedly in Hank’s buffered saline solution) was placed in a separate well of a 24-multiwell plate so that the material covered the base of the well. A small area of each well was left uncovered to allow assessment of cell plating efficiency by phase microscopy. Six wells of each material were set up to provide duplicate samples for each material at each of the time points. Confluent human osteoblast cultures were passaged by trypsin-EDTA and were seeded into each well on top of the implant materials (which remained at the bottom of the wells) at a seeding density of 12 500 cells/cm’. The day of plating was considered as the first day of culture. The test samples were removed from wells at days 3, 5 and Q, fixed in 3% glutaraldehyde in 0.1~ cacody- late buffer (pH7.4), and left for 12 h at 4°C. The samples were dehydrated in a graded series of alcohols, critical point dried, mounted on stubs and coated with 40nm of gold in a Polaron scanning electron microscopy (SEM) sputter coating system. The samples were examined and photographed using a JEOL WINSEM JSM-6400 scanning electron microscope.

RESULTS

Initially, the materials were examined without osteoblasts to assess their surface topographical features. The rat bone and demineralized rat bone had both smooth and rough surfaces; the smooth surfaces where pitted by empty lacunae. The rough surfaces occurred at the edges of the bone where it had been

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Biocompatibility of bone grafts with human osteoblasts: M.J. Doherty et al. 603

fractured (Figure ~a). The demineralized Surgibone was characteristic of trabecular bone, containing smooth surfaced trabeculae with roughened areas where fracturing had occurred (Figure ~b). The surface of Biocoral had raised, roughened, blunt ended spicules, with flatter surfaces between spicules (Figure 1~). The tissue sealant surface was predominantly undulating

and smooth (Figure Id), whereas the surfaces of the bioceramic granules were highly contoured roughened with raised areas (Figure le).

After 3 d in culture the mineralized rat bone samples exhibited only a sparse population of osteoblasts (Figure ~a). The number of cells had increased by day 5, the cells exhibiting contact with each other via

Figure 1 Scanning electron micrographs illustrating the various surface topographies of the graft materials examined. a, Scanning electron micrograph of mineralized rat bone showing smooth surfaces, which exhibited small pits representing empty lacunae, and rough surfaces where the bone had been fragmented. b, The demineralized Surgibone was characteristic of trabecular bone containing smooth surfaced trabeculae and roughened areas where fracturing had occurred. c, An electron micrograph demonstrating the raised, roughened, blunt ended spicules of Biocoral. The flattened surface beneath the spicules contained pores. d, Tissue sealant had a predominantly smooth surface exhibiting undulations and occasional furrows. e, The highly contoured, roughened surface of Ostilit.

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604 Biocompatibility of bone grafts with human osteoblasts: M.J. Doherty et al.

filopodial processes, and by day 9 an almost complete layer of osteoblasts coated the surface of the bone (Figure Zb).

The number of cells that populated the deminera- lized rat bone by day 3 was significantly greater than that on the mineralized form, with most of the surfaces

of the demineralized bone being coated with osteoblasts (Figure ZC). Again, these cells had prolifer- ated by days 5 and 9, and formed connections with adjacent neighbouring cells via cellular extensions (Figure 24.

Few osteoblasts were found to be attached to the

Figure 2 Scanning electron micrographs showing the coverage of human osteoblasts on rat bone, demineralized rat bone and demineralized Surgibone after 3 and 9d in culture. a, A sparse population of osteoblasts was present on the mineralized rat bone after 3d in culture. b, An almost complete layer of osteoblasts coated the surface of mineralized rat bone after 9d in culture. The cells exhibited contact with each other via filopodial processes. c, Most of the surfaces of demineralized rat bone were coated by osteoblasts by day 3. d, The cells had proliferated and formed intercellular connections on the deminer- alized rat bone by day 9. e, Few osteoblasts were attached to the surface of demineralized Surgibone by day 3. 1, By day 9 the number of osteoblasts on the demineralized Surgibone had increased, but large areas of the material remained unpopulated with cells.

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Biocompatibility of bone grafts with human osteoblasts: M.J. Doherty et al. 605

surface of demineralized Surgibone at day 3 (Figure Ze), although by day 5 the number of cells had increased. By day 9 these cells had proliferated and spread on the surfaces of demineralized Surgibone, but large areas of the material remained unpopulated with cells. The cells present were isolated from each other and exhibited few intercellular connections (Figure of).

At day 3 osteoblasts were observed on the surfaces of a few Biocoral granules (Figure 3a), but there were also granules that were devoid of cells. The number of cells did not appear to increase between days 3 and 5, although by day 9, cells were more numerous, apart from the granules that still had no cells adhering to their surfaces. Few intercellular connections were observed between the osteoblasts (Figure 3b).

At the first time point, osteoblasts were present on most of the tissue sealant surfaces (Egure 3~). The osteoblasts formed bridges spanning adjoining uneven areas (Figure 3d). The cells proliferated and had increased in number by day 5. By day 9 post-plating a layer of flattened polygonal cells was present, covering almost the complete surface of the tissue sealant (Figure 3e).

Typically the cells that grew on the materials exhibited numerous pseudopodial and filopodial extensions anchoring the cells to the surface of the material (~jgu~ 3f). In general, the osteoblasts had successfuIly seeded onto and mitotically expanded to varying degrees on all the materials, with the exception of the bioceramic. No osteoblasts were detected on the bioceramic samples examined by scanning electron microscopy at any of the time intervals studied.

DISCUSSION

The results of this study have established two important prerequisites for a bone graft substitute material. Firstly, not only should the material be able to support osteoblast attachment but it should also promote proliferation of the attached cells. Secondly, a bone material is more suited to bone cell attachment than a bioceramic or a coral, and cell attachment and proliferation is faster on demineralized bone as opposed to the mineralized form.

Previous work suggests that attachment of cells to a substrate may be influenced by surface topography with particular types of cells preferring specific surfaces, e.g. fibroblasts show a preference for smooth surfaces over rough ones whilst macrophages like rough surfaces’“. In the present study human osteoblasts adhered to both rough and smooth surfaces of rat bone (mineralized and demineralized), to porous surfaces of demineralized Surgibone, to the blunt ended spicules of Biocoral and to the predominantly smooth surface of tissue sealant. This attachment achieved by cellular processes suggests that the cells tend to attach regardless of the topography of the material. Such observations are in accord with those of Jones and Boydez7, who showed that migratory osteoblasts adhere to any firm substrate presented in vitro. In the case of materials to be used in bone grafting, porosity may be important to allow fluid

circulation, bone ingrowth and mechanical stability at the implantation sitez8.

Following cell attachment, division and growth of the cells should occur, and although surface topogra- phy had no apparent affect on cell attachment, it may affect the shape and orientation of the cells. By observing the cells on the materials at intervals up to 9 d it was shown that proliferating cells on the smooth surfaces lay flat and in close contact with the materials’ surfaces (rat bone both mineralized and demineralized and tissue sealant), whereas on the more porous materials cells appeared suspended across the pores by their processes (demineralized Surgibone and Biocoral). As a result, the cells were not in close contact with the underlying substratum and a low number of cells were observed on these two materials. It has been demonstrated that cell attachment regulates cell proliferation, in that a cell which adheres easily to a substrate and becomes flattened against it promotes cell division. In contrast, poorly attached cells retain a cuboidal morphology and are less mitotically activezg.

A factor which may affect the growth of cells on the bone graft substitutes in vivo is the presence of alloan- tigens, and consequently the use of xenogeneic bone has met with only limited success. For example, Upton et ~1.~’ used xenogeneic demineralized bone implants to correct bony defects in the fingers, where they induced osteogenesis and promoted healing. Xenogeneic implants have many advantages, includ- ing the avoidance of harvesting procedures and a potentially unlimited supply of banked material. However, the suitability of xenogeneic bone grafts is questionable because they are reported to be rapidly resorbed3’.

Demineralization prevents rapid resorption3*32 and is also reported to remove alloantigens but preserve the morphogenic proteins in bone matrix3”. However, complete demineralization results in the loss of the structural rigidity of bone. This problem may be overcome by determining the optimal degree of demineralization that promotes cell attachment wi~out a signi~cant loss of the material’s structural strength. It remains to be determined what degree of demineralization is required for bone to be used in bone grafting sites where weight-bearing is an important consideration.

The experiments performed here demonstrated that human osteoblasts grew on both demineralized xenogeneic material, i.e. Surgibone and rat bone, and are in accordance with the work of Begley et ~1.‘~. In addition, the cells also grew on the mineralized form of the rat bone but not to the same extent. Within 3d of culture the human osteoblasts had formed an almost complete layer on the demineralized rat bone, whereas there was only a sparse population of osteoblasts on the mineralized rat bone, suggesting that the cells preferred a demineralized surface. This is not to say that all demineralized materials are populated equally well by osteoblasts, as this study demonstrated that demineralized rat bone was better populated with osteoblasts than the demineralized Surgibone.

Begley et ~1.~~ previously demonstrated that human

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606 Biocompatibility of bone grafts with human osteoblasts: M.J. Doherty et al.

osteoblasts populate natural coral in vitro. The results of this study show that the processing of coral for commercial purposes does not interfere with its ability to support osteoblast growth. However, the type of coral chosen for bone grafting operations is important as they have different porosities, are resorbed at differ-

ent rates and affect the rate at which new bone is formed34.

To date, the mechanism whereby tissue sealant affects bone healing remains unclear. This study indicates that tissue sealant exhibits a high biocom- patibility with human osteoblasts, although it was

Figure 3 Scanning electron micrographs demonstrating the coverage of human osteoblasts on Biocoral and Ostilit at 3 and 9d post-culture. a, At day 3 the Biocoral granules were only sparsely covered by osteoblasts. b, Cells were more numerous on the Biocoral granules by day 9 apart from the granules that still had no cells adhering to their surfaces. Few intercellular connections were observed between the osteoblasts. c, By day 3 osteoblasts were present in large numbers on the tissue sealant surfaces, forming an interdigitating sheet of cells. d, At the same time point, osteoblasts covering uneven areas of the tissue sealant formed bridges spanning adjoining surfaces. e, By day 9 post-plating a layer of flattened polygonal cells was present, covering almost the entire surface of the tissue sealant. f, Typically the cells that grew on the materials exhibited numerous pseudopodial and filopodial extensions anchoring the cells to the surface of the material.

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Biocompatibility of bone grafts with human osteoblasts: M.J. Doherty et al. 607

not designed to act as a graft substitute material per se but to be packed in around she graft material to hold it in place. It has already been used as a carrier for antibiotics3” and it is possible that its use could be extended to include a delivery system for bone growth factors which could enhance hone formation at the graft site.

These experiments have defined further properties required of a bone graft substitute material if it is to he considered bio~ompatib~e for human osteoblasts. Not only must the material support cell attachment but it must also allow proliferation of the cells. All the materials tested, except the bioceramic, are biocompatible with human osteoblasts. However, this study has been able to separate the more biocompat- ible materials (tissue sealant, rat bone and deminera- lized rat bone) from the less biocompatible ones (demineralized Surgibone, commercial coral and bioceramic). A further refinement would be to quanti- tate the number of cells attached to the surfaces of these graft substitutes, in particular the materials which are more biocompatible with human osteoblasts.

Support from the Arthritis and Rheumatism Council, UK is gratefully acknowledged. The authors wish to thank Dr D. Hankey for his assistance in tissue culture.

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IONIZINGRADIATIONANDPOLYMERS-IRaP 94

NOVEMBER 14~~ - 19~~, 1994

This first international symposium will take place in the French Department Guadaloupe, located in the French West Indies, close to the Saint-Fraqois village. It is addressed to scientists from research and industrial applications interested in assessing the present state and future trends in radiation polymer science, using ionising radiations such as electrons, gamma rays, photons and fast heavy ions with the aim of understanding, modifying and creating advanced materials. Its primary objective is to provide an interdisciplinary platform in this rapidly diversifying field forming a bridge between physical and chemical aspects of in-ad&ion treatment from basis to applications and to accelerate the transfer of knowledge to other fields such as biological and medical research.

The program consists of keynote lectures, invited and contributed talks, posters. Topics will be:

a Basic physical and chemical processes of interaction 0 Surface and bulk modifications of polymers a Advanced materials 0 Industrial applications

Particular attention is given to new materials which can find applications in medicine, biology, biotechnology and environmental protection.

For more information and inscription, please contact: A. Le M&I, Chairman of lRaP 94 Organiziig Committee, DRECAM/SRSlM/LPI, CEA - CE Saclay, F-91191 Gif sur Yvette Cedex, France. Tel: (33) 169 085 485 or Fax: (33) 169 089 600.

Biomaterials 1994, Vol. 15 No. 6