Failure of onlay bone grafts to integrate over the calvarial suture: observations in adult isogeneic rats

Journal of Cranio-MaxillofacialSurgery (1996) 24, 251 255 © 1996 European Association for Cranio-Maxillofacial Surgery

Failure of onlay bone grafts to integrate over the calvarial suture: observations in adult isogeneic rats P. Alberius 1, M. Gordh 2

1Department of Plastic Surgery (Head." Magnus ftberg, M.D., Ph.D.), MAS, Malm6, Sweden, 2Department of Oral Surgery (Head." Jan Rosenquist, D.M.D., Ph.D.), Centre for Oral Health Sciences, University of Lund, Sweden SUMMARY. Bone grafting constitutes an important tool in cranio-maxillofacial skeletal reconstruction and augmentation. Much effort has been directed to improve graft survival and volumetric maintenance. The effects of the sutural tissue proper on graft incorporation has not yet been explored. The purpose of this report was to analyze the effects of positioning an onlay graft over a non-growing sutural region. Twelve adult rats received femoral or tibial uni- or bieortieal grafts placed over the temporal suture. The findings were assessed by routine microscopy and immunohistochemistry after 4, 12, and 20 weeks. The sutural tissue expanded between the graft and the host bed in an umbrella-like pattern, which locally inhibited graft incorporation. Of the tested cartilage and bone proteins and proteoglycans, labelling was distinct only for osteopontin and fihromodnlln, indicating a moderate remodelling activity in the area. The importance and consequences of the findings are discussed.

INTRODUCTION

growth, but the consequences of such a manoeuver over a non-growing, yet not fused, suture on graft integration are unknown. The purpose of this report was to analyze the effects on integration of positioning an onlay graft over a non-growing sutural region.

Bone grafts are routinely used in cranio-maxillofacial surgery to augment skeletal contour, bridge bony defects, and stabilize bone segments. Multiple investigations have tried to explore various aspects of autogeneic and allogeneic bone graft incorporation to optimize graft survival and volumetric maintenance, and interest also has been directed toward some alloplastic materials. Furthermore, the influence of a growing recipient site, in terms of resorptive or depository growth patterns, on onlay graft survival has attracted attention (Zins et al. 1984; Fasano et al. 1989). Recently, our group has presented a series of experiments examining the role of the tissue environment and the effects of cortical perforations to enhance graft take (Alberius et al. 1996a,b,c; Gordh et al. 1996). To the best of our knowledge, no analysis of the outcome of graft positioning over a suture has been undertaken. The effects of premature isolated or syndromic sutural closure have been characterized extensively, and various sutural manipulations have been used to understand the biology of sutural behaviour. Also, the healing of defects involving the sutural area has been investigated. For example, in an attempt to elucidate the effects on growth and sutural regeneration after wide osteotomies in the craniofacial skeleton, Alberius et al. (1990) compared healing of defects encompassing the coronal suture and the parietal bone in rabbits. Only minor divergences in healing pattern without any adverse effects on sutural growth were observed. Naturally, graft positioning over a growing suture would impede further local

MATERIALS AND METHODS Animals and anaesthesia Twelve adult Lewis rats, with a mean weight of 419 (SD 36)g, were kept under standard laboratory conditions with free access to tap water and nutritional support. Anaesthesia was provided by an intraperitoneal injection of Mebumal (6mg/100g body weight). The rats were sacrificed 4 (n=6), 12 (n = 3), and 20 (n= 3) weeks after grafting. Three additional isogeneic animals of identical size and age were used to obtain donor tissue. Isogeneic rats were used for bone harvesting to reduce surgical trauma and somatic stress for the animal to receive the graft. In principal, the bone grafts are considered as autogeneic. All research protocols were approved by the Animal Ethics Committee of Lund University, Sweden. Surgical procedure Surgery was conducted with a strict aseptic technique. To prevent variations in technique, the same surgeon performed all operations and used an identical protocol. 251

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Identical sized bone blocks, 4 mm in diameter, from the femur and tibia were harvested from donors using a trephine mounted in a low speed dental drill. During bone drilling the surgical field was continuously irrigated with sterile saline to reduce thermal and mechanical damage. No periosteum was left attached to the graft. Under anaesthesia, a paramedian skin incision was made from the occipital to the frontal region. The cranial vault and the temporal fascia and muscle were then exposed. After a periosteal incision parallel and posterior to the coronal suture, a subperiosteal pocket overlying the temporal suture was created by gentle dissection. When possible, the temporal crest was removed by grinding. After form-to-fit testing, a tight pocket was obtained to stabilize the graft. No additional fixation was used. The periosteal and skin incisions were closed with resorbable interrupted sutures. All animals recovered rapidly and the postoperative period was uneventful.

primarily to bone include bone sialoprotein (having a distribution essentially restricted to the osteoblasts and involved in the onset of mineralization), 62 kDa protein (which appears early in osteogenesis and is considered to diminish the resorptive process) and osteopontin (considered to anchor osteoclasts to the bone surface and inhibit nucleation). The large aggregating proteoglycan (aggrecan) is a cartilage molecule. Moreover, cartilage contains relatively large amounts of a fragment released from procollagen II on cleavage of the procollagen molecule prior to fibril assembly, which is referred to as chondrocalcin (participating in the focal calcification process). Fibromodulin (believed to participate in the regulation of collagen fibril formation), biglycan (binds to collagen and possibly related to the control of fibre growth and organization, as well as attachment of other matrix constituents), and PRELP (considered to promote cell attachment) are macromolecules common to many connective tissues.

Histology

RESULTS

After sacrifice, the bone grafts and recipient bed were carefully excised en bloc without stripping away the soft tissues, at intervals described above and immediately frozen in isopenthane and stored at -70°C. Sections of 6 ~tm were prepared using a cryostat. The sections were incubated with rabbit antibodies against proteins prepared from rat bone matrix (the 62 kDa protein, bone sialoprotein, and osteopontin) or bovine cartilage matrix (the 58 kDa protein i.e., PRELP, fibromodulin, chondrocalcin). Additional antibodies were directed against the proteoglycans aggrecan (PG-LA) and biglycan (PG-S1) prepared from bovine nasal and articular cartilages. The preparation of antibodies has been described earlier (Hulth et al. 1993). Immunostaining was performed with antisera from immunized rabbits diluted 1/50 to 1/200 as previously described (Klareskog et al. 1982). Additionally, sections were stained with haematoxylin-eosin, saffranin-O, van Gieson, and anti-laminin. Also, a control specimen without antibody staining was prepared. The specificity of the immunostaining was checked as described previously (Hulth et al. 1993). In particular, the graft-host interface and its relationship to the temporal suture was examined and the extent of integration evaluated. All histological examinations were performed by two observers independently and then compared.

Presentation of proteins analyzed For a detailed review of the proteins tested, see reviews by Heineg~rdand Oldberg(1989) and Young et al. (1992). Information on the function, specific localization, and regulation of these newly characterized macromolecules in bone tissue is presently limited. Briefly, proteins with a distribution restricted

Macroscopically, healing was uncomplicated in all instances with no evidence of infection or graft dislodgement. Surrounding tissues showed a normal appearance. Microscopically, graft incorporation was incomplete after 4 weeks, but thereafter progressed and seemed complete after 12 weeks except for the perisutural area. Generally, the suture tissue proper expanded in an umbrella-like pattern into the graft-host interface, and seemed to inhibit bony integration locally (Figs. 1 and 2). Hence, the connective tissue penetrated along the inner aspect of the graft for varying distances. This soft tissue appeared structurally identical at the grafthost interface and the suture. Fibromodulin was the only cartilage protein demonstrated in the actual region. It was distributed all along the suture and its bony margins and extended slightly along the endocranial surface. Fibromodulin labelling was most pronounced for weeks 4 and 12, while that of week 20 was weaker. Osteopontin was the only bone protein tested that was labelled clearly. It was most distinct in the bone producing areas adjacent to the suture, primarily perivascularly, and less and very diffusely in the suture itself (Fig. 1). Osteopontin labelling was most apparent during week 4 after which it declined markedly. Neither proteoglycan seemed present in the sutural tissue during the period investigated. Laminin, a component of the basement membrane of the vessel wall, was widely distributed in the connective tissue between the graft and the host bed at week 12. After 20 weeks, the labelling at the surrounding connective tissue was similar to the graft-host interface. No such labelling was observed at week 4.

Failure of onlaybone grafts to integrate over the calvarial suture

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F i g . 1 - Photomicrograph showing the sutural tissue expanding below the graft (arrow), 12 weeks. Osteopontin weakly labels both newly formed bone and original bone tissue and illustrates the remodelling activity of the area. Trabecular connection (partly labelled) between graft and host bed indicated (arrowheads). Original magnification a, x 16; b, x 40.

DISCUSSION Previous investigators have demonstrated that arterial blood flow changes (Oudhof and van Doorenmaalen, 1983) and functional stresses are important for maintenance of the normal sutural ligament in the growing individual (Moss 1957, Watanabe et al., 1957, Nash

and Kokich 1985). Premature immobilization of the sutural region, on the other hand, by means of free periosteal transplants (Alhopuro et al., 1973), inlay (Giblin and Alley, 1944, Watzek et al., 1982) or onlay (StenstrOm and Thilander, 1967, Butow, 1990) bone grafting, or cyanoacrylate adhesives (Persson et al., 1979, Foley and Kokieh, 1980) implies profound

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Journal of Cranio-Maxillofacial Surgery

Fig. 2 - The suture (arrow) spreading in to the graft-host interface. Note that the suture contacts the surrounding soft tissue (arrowhead), 20 weeks. G = graft, R = recipient bed. Htx. Original magnification x 40.

structural changes. However, in the adult skeleton such growth restrictions are of less functional importance. In previous experiments, we observed that parts of bone grafts overlying such fibrous articulations in adult animals were locally non-integrated, which may theoretically influence graft survival and volumetric maintenance. This study aimed to explore this issue. The connective tissue emanating from the sutural tissue was observed to spread in an umbrella-like pattern between the graft and its recipient bed. The constant feature of impeded bony integration in this area seems to imply that the presence of sutural tissue entails an obstacle to complete bony incorporation. This is very interesting considering the understanding of premature sutural synostosis. Obviously, some osteoinhibitive factor, which may present itself even in the adult sutural tissue, was reactivated in the ectocranial extension of the suture. Also, this implied sutural tissue expansion outside of the ordinary sutural area. This phenomenon requires further experimental study. The immunolabelling aimed to explore the possible occurrence and localization of several proteins essential to various stages of osteogenesis. However, only moderate labelling activity was disclosed as compared with the situation of the growing suture (Alberius and Johnell, 1990). The only proteins being distinctly visualized were osteopontin, which is considered to anchor osteoclasts to the bone surface and inhibit crystal formation (Heinegdrdand Oldberg, 1989), and fibromodulin, which is believed to participate in the regulation of collagen fibril formation (Hedbom and Heinegdrd, 1989). This implies that a moderate bone remodelling activity was at hand during the period studied. The illustrated passive atmosphere probably served to further delay graft incorporation locally.

The revascularization pattern observed corroborates our previous findings on onlay graft integration (Alberius et al., 1996a,b) and does not contribute any new data. Again, the late appearance of vessels in this process stresses the importance of stable fixation of the graft not to further jeopardize this delicate but important process. In conclusion, this study points to the importance of correctly selecting the optimal host bed of an onlay graft and, further, indicates that the sutural tissue may locally inhibit osseous incorporation.

Acknowledgements We thank Professor D. Heineg~trd, Department of Medical and Physiological Chemistry, Lund University, for supplying the antibodies, Professor Olof Johnell, Department of Orthopaedic Surgery, Malta6 General Hospital for valuable discussions, and Lisbeth Lindberg for technical assistance.

References Alberius, P., O. Johnell: Immunohistochemical assessment of cranial suture development in rats. J. Anat. 173 (1990) 61-68 Alberius, P., S. Isaksson, B. Klinge, S. Sj6gren, J. JOnsson." Regeneration of cranial suture and bone plate lesions in rabbits. Implications for positioning of osteotomies. J. Cranio-Max-Fac. Surg. 18 (1990) 271-279 Alberius, P., M. Gordh, L. Lindberg, O. Johnell: Influence of surrounding soft tissues on onlay bone graft incorporation. A study in the adult rat skull. Oral Snrg. Oral Med. Oral Path. 1996a Alberius, P., M. Gordh, L. Lindberg, O. Johnell: Assessment of marrow exposure to uni- and bicortical onlay bone graft incorporation in the adult rat skull. Submitted: J. Oral Maxillofac. Surg. 1996a Alberius, P., M. Gordh, L. Lindberg, O. Johnell: Experimental evaluation of combining graft and host bed cortical perforations. Submitted: Br. J. Plast. Surg. 1996c Alhopuro, S., R. Ranta, V. Ritsil~: Growth of the rabbit snout after bony fusion of the frontonasal suture achieved by means of a free periosteal transplant. Proc. Finn. Dent. Soc. 69 (1973) 166-167

Failure of onlaybone grafts to integrate over the calvarial suture Butow, K.-W.: Craniofacial growth disturbance after skull base and associated suture synostosis in the newborn Chacma baboon: a preliminary report. Cleft Palate J. 27 (1990) 241-251 Fasano, D., G. Gasparini, V. Menoni, F Bertoni, P. Baeehin# The fate of onlay membranous bone grafts in different facial recipient sites. Eur. J. Plast. Surg. 12 (1989) 160-166 Foley, W. or., K G. Kokieh: The effects of mechanical immobilization on sutural development in the growing rabbit. J. Neurosurg. 53 (1980) 794-801 Giblin, N., A. Alley: Studies in skull growth. Coronal suture fixation. Anat. Ree. 88 (1944) 143-153 Gordh, M., P. Alberius, L. Lindberg, O. Johnell: Onlay bone graft orientation and incorporation relative to cortical perforations of the host bed. An experimental study in rats. Submitted: Otolaryngol. Head Neck Surg. 1996 Hedbom, E., D. Heinegdrd: Interaction of a 59-kDa connective tissue matrix protein with collagen I and collagen II. J. Biol. Chem. 264 ~(1989) 6898-6905 Heinegdrd, D., A. Oldberg: Structure and biology of cartilage and bone matrix non-collagenous macromolecules. FASEB J. 3 (1989) 2042-2051 Hulth, A., O. Johnell, L. Lindberg, D. Heinegdrd: Sequential appearance of macromolecules in bone induction in the rat. J. Orthop. Res. 11 (1993) 367-378 Klareskog, L., U. Forsum, A. Wigren, H. Wigzell: Relationship between HLA-DR expressing cells and T-lymphocytes of different subsets in rheumatoid synovial tissue. Scand. J. Immunol. 15 (1982) 501-507 Moss, M. L.: Experimental alteration of sutural area morphology. Anat. Rec. 127 (1957) 569-589 Nash, S. B., V. G. Kokich: Evaluation of cranial bone suture autotransplants in the growing rabbit. Acta. Anat. 123 (1985) 39-44

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Oudhof, H. A. J., W. J. van Doorenmaalen: Skull morphogenesis and growth: Hemodynamic influence. Acta. Anat. 117 (1983) 181 Persson, K. M., W. A. Roy, J. A. Persing, G. T. Rodeheaver, H. R. Winn: Craniofacial growth following experimental craniosynostosis and craniectomy in rabbits. J. Neurosurg. 50 (1979) 187-197 Stenstr6m, S. J., B. L. Thilander: Facial skeletal growth after bone grafting to surgically created premaxillomaxillary suture defects: an experimental study on the guinea pig. Plast. Reconstr. Surg. 40 (1967) 1-12 Watanabe, M., D. M. Laskin, A. G. Brodie: The effect of autotransplantation on growth of the zygomaticomaxillary suture. Am. J. Anat. 100 (1957) 319-329 Watzek, G., F Grundsehober, H. Plenk Jr., J. Esehberger: Experimental investigations into the clinical significance of bone growth at viscerocranial sutures. J. Max-Fac. Surg. 10 (1982) 61-79 Zins, J. E., J. F Kusiak, L. A. Whitaker, D. H. Enlow: The influence of the recipient site on bone grafts to the face. Plast. Reconstr. Surg. 73 (1984) 371-379 Young, M. F , J. M. Kerr, K Ibaraki, A.-M. Heegaard, P. G. Robey: Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. Clin. Orthop. 281 (1992) 275-294 Per Alberins D.M.D., M.D., Ph.D. Department of Plastic Surgery MAS S-205 02 Malm6 Sweden

Paper received 15 January 1996 Accepted 7 May 1996