Influence of surrounding soft tissues on onlay bone graft incorporation

Influence of surrounding soft tissues on onlay bone graft incorporation Per Alberius, D M D , MD, PhD, a M o n i c a Gordh, D M D , b Lisbeth Lindberg, c and Olof Johnell, MD, PhD, d M a l m t , Sweden LUND UNIVERSITY

Objectives. This investigation aimed to evaluate the influence of three different soft tissue environments on onlay bone graft incorporation into the craniomaxillofacial skeleton and to follow this process immunohistochemically by staining for some bone and cartilage matrix proteins and proteoglycans. Study design. Femoral and tibial bone grafts, either uni- or bicortical, were placed subperiosteally, submuscularly, and intramuscularly in 36 identically sized and aged isogeneic rats. The results were evaluated after 4, 12, and 20 weeks. Results. Corticocancellous grafts showed a more extensive incorporation and more pronounced local resorption. Bicortical grafts produced more resorption of the recipient site, which oftentimes resulted in the graft being almost level with the surrounding bone. The marrow space of both graft types was sealed off by compact bone formation. Intense labeling of tested bone matrix proteins at various parts of the graft-host area was demonstrated. Ultimate graft height was significantly reduced for most groups, but no major differences between groups were registered. Intramuscularly positioned control grafts ultimately showed signs of lacking viability and reduced labeling of cartilage matrix proteins. Conclusions. These findings indicate that either type of graft had its drawbacks and that further studies to enhance integration and size maintenance are necessary to improve overall graft persistence. Immunolabeling may help to identify essential mechanisms m and find markers of graft incorporation. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996;82;22-33)

In craniomaxillofacial surgery, restoration and augmentation of skeletal contour requires use of free onlay bone grafts. The long-term result is often unsatisfactory because of graft resorption and remodeling. Several factors have been identified or suggested that m a y influence the integration of autogeneic grafts and their ultimate maintenance, such as the embryonic origin of the graft, a-4 rate and extent of revascularization. 4q~ structural composition. 4 to biologic differences between graft t y p e s ] 1 rigid fixation, 7 s. 12. 13 divergent local piezoelectric properties. 14 inclusion of perlosteum as a c o m p o n e n t o f the graft,15 graft orientation.4. 15, 16 and contents of local growth factors. 17 Yet the question seems partially unresolved, and reports are contradictory. Previous investigations have thus mainly focused on the graft and its handling. Less attention has been aAssociate Professor. Department of Plastic Surgery, Lund University.

bMaxillofacial Surgeon, Department of Oral Surgery, Centre for Oral Health Sciences. Lund University. CTechnical Assistant. Department of Orthopaedic Surgery, Lund University. dProfessor. Department of Orthopaedic Surgery, Lund University. Received for publication July 25. 1995: returned for revision Oct. 6. 1995: accepted for publication Feb. 7. 1996. Copyright 9 1996 by Mosby-Year Book. Inc. 1079-2104/96/$5.00 + 0 7/12/72581

22

directed toward the recipient milieu. The influence of resorptive or depository recipient surfaces in the craniofacial skeleton 12, 18.19 have been evaluated. Goldstein et al. 2~ recently presented significant effects on graft persistence after experimental alteration of the recipient bed through tissue expansion. Graft placement under muscle tissue has been reported to contribute an advantageous marrow revascularization 21 but also a more extensive resorption of the onlay itself. 22 In contrast, alloplastic materials seem to promote localized bone erosion over the recipient bone surface23-25; this p h e n o m e n o n has been ascribed to the size or composition of the implant, motion. pressure, and placement with respect to the periosteum. The purposes of this study were to explore graft incorporation patterns in three different environments subperiosteally, submuscularly, and intramuscularly and to investigate the potential distribution and progressive spatial alterations o f some bone and cartilage matrix proteins and proteoglycans during graft incorporation in adult rats. MATFRIAL A N D MFTHODS

Animals and anesthesia Thirty-six adult Lewis rats. with a mean weight of 375 gm (standard deviation [SD] 32), were kept under standard laboratory conditions with free access to

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tap water and nutritional support. Anesthesia was provided by an intraperitoneal injection of pentobarbital-sodium (Mebumal; 6 mg/100 gm body weight). The rats were killed 4, 12, and 20 weeks after grafting. Eight additional isogeneic animals of identical size and age were used to obtain donor tissue. The three first animals in this series were not included for further analysis because o f macroscopic nonintegration that was considered to be due to surgical errors. All research protocols were approved by the Animal Ethics Committee of the Lund University, Sweden.

Table I.

Number of animals used for each experimental group and interval Environments Submuscular

Intramuscular

5 9 6

5 6 5

5 6 5

5 3 5

5 6 6

5 6 6

Subperiosteal Unicortical grafts Week 4 Week 12 Week 20 Bicortical grafts Week 4 Week 12 Week 20

Surgical procedure Isogeneic rats were used for bone harvesting to reduce surgical trauma and somatic stress for the animal to receive the graft and thereby facilitate and assure a safe operative technique. In principle, the bone grafts thus are considered as autogeneic. Surgery was conducted with strict aseptic technique. To prevent variations in technique, the same surgeon performed all operations with an identical protocol. Identically sized bone blocks, 4 m m in diameter, from the femur and tibia were harvested from donors with a trephine mounted in a low-speed dental drill. During bone drilling the surgical field was continuously irrigated with sterile saline solution to reduce thermal and mechanical damage. To obtain unicortical grafts some (Table I) bone blocks were split. Infrequently, the perimeter of the donor bone was too small to allow complete removal of cortical bone, implying that a few grafts were hemicylindrical in appearance rather than bicortical. The exact dimensions of the grafts were monitored with a micrometer and subsequently photographed. With the rats under anesthesia, 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 and the uni- or bicortical grafts were placed at three different regions: 1. After a transverse periosteal incision parallel and anterior to the coronal suture a subperiosteal pocket overlying the parietal bone was created by gentle dissection. After form-to-fit testing a tight pocket was obtained to stabilize the graft. No additional fixation was used. The periosteal incision was closed with resorbable interrupted sutures. 2. On the contralateral side, an incision parallel to the temporal line was made leaving a few millimeters of temporal fascia superiorly and a tight pocket produced between the temporal muscle and the underlying calvarial bone in which the graft was carefully placed. No additional fixation was used. The fascial

incision was closed with resorbable interrupted sutures. 3. A short incision was made over one hip, and the gluteus maximus muscle identified. An intramuscular pocket was prepared allowing placement of the bone graft. The muscle was reapproximated with resorbable interrupted sutures. Unicortical grafts placed on the craniofacial skeleton were constantly positioned with their cancellous portion facing toward the recipient compact bone, and consequently their compact surface was in contact with the surrounding soft tissue. Intramuscular placement was performed to investigate the effects (especially with respect to immunolabeling) of a passive nonosseous environment and for reference purposes. All animals thus received three bone grafts, one in each location. They recovered rapidly, and the postoperative period was uneventful.

Experimental groups The rats were randomized into six groups: (1) unicortical o n l a y to the parietal bone, subperiosteal placement, n = 20; (2) unicortical onlay to the temporal bone, submuscular placement, n = 16; (3) unicortical graft to the gluteal region, intramuscular placement, n = 16; (4) bicortical onlay to the parietal bone, subperiosteal placement, n = 13; (5) bicortical onlay to the temporal bone, submuscular placement, n = 17; (6) bicortical graft to the gluteal region, intramuscular placement, n = 17.

Histology After sacrifice, the bone grafts and recipient bed were carefully excised en bloc without stripping away the soft tissues at intervals described earlier and immediately frozen in isopenthane and stored at - 7 0 ~ C. Sections of 6 lam were prepared with a cryostat. The sections were incubated with rabbit antibodies against proteins prepared from rat bone matrix (the tx62 kDa

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Fig. l. Microphotograph illustrates partial integration and peripheral rounding of margins of subperiosteally positioned unicortical graft after 20 weeks. (PB = parietal bone, G = graft.) (Hematoxylin-eosin stain; original magnification x40.) protein [=a2HS-glycoprotein], bone sialoprotein, and osteopontin) or bovine cartilage matrix (PRELP, i.e., proline arginine-rich end/eucine-rich repeat protein, fibromodulin, COMP, i.e., cartilage oligomeric matrix protein, chondrocalcin). Additional antibodies were directed against the proteoglycans aggrecan (PG-LA) and biglycan (PG-S !) preparedfrom bovine nasal and articular cartilages. The preparation of antibodies has earlier been described. 26 Immunostaining was made with antisera diluted 1/50 to 1/200 from immunized rabbits as previously described. 27 In addition, sections were stained with hematoxylin-eosin, saffranin-O, van Gieson, and antilaminin. In addition, a control specimen without antibody labelling was prepared. The specificity of the immunolabelling was checked as described previously. 26 The analysis focused on the incorporation of the graft, the structural and volumetric changes of the graft, and the reactions of the recipient bed. The graft-host interface was examined, and the relationship was described as either separate, partially incorporated, or fully incorporated. 2~ Separate was defined as the existence of a microscopic gap as seen histologically between the complete length of the graft and the recipient. Partial incorporation was defined as partial bony bridging at the graft-recipient interface. Full incorporation implied complete bony bridging between the graft and the recipient. All histologic examinations were double blinded to the group; they were examined by two observers independently and the results compare& Initial three-dimensional analysis of each graft was easily accomplished by caliper monitoring at the primary operation. At 20 weeks, the dimensions were

much more difficult to measure because of irregular resorption and bony apposition. Furthermore, the intactness of the graft-host region was considered fundamental to correct analysis of the incorporation process, wherefore we preferred not to separate the graft from its recipient bed. The thickness at the midpoint of the graft was calculated from cross-sectional histologic slides at known magnification with the aid of a microscope grid. Only a descriptive report of the volumetric maintenance is given.

Statistics All data are reported as mean • SD. The statistical significance of intragroup differences was calculated by paired t test for pairwise comparison of height maintenance, whereas intergroup differences were calculated with t test for independent samples. A 5% level of confidence was considered to be statistically significant. RESULTS Macroscopic examination found that healing was uncomplicated in all instances with no evidence of infection or graft dislodgement. Surrounding tissues showed a normal appearance.

General histologic findings Unicortical bone gratis--4 weeks. Partial integration of the graft with pronounced periosteal bone formarion at the graft-host interface was found. The grafts demonstrated widespread remodeling along the marrow space with trabecular bone formation and rounding of the cortical margins. Volumetric changes

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seemed small. The morphologic characteristics of the recipient bed were relatively unaffected. No major discrepancies were noted between the subperiosteal and submuscular grafts except for somewhat more marked local resorption and remodeling at the submuscular recipient region. The intramuscular group showed good viability in large parts of the graft and even displayed some bony apposition perivascularly and on Surfaces most adjacent to muscle tissue. A thin layer of rather dense bone was formed sealing off the open marrow space. Resorptive activity was discerned locally all along the peripheral compact bone with resorptive lacunae. Unicortical bone grafts--12 weeks. Graft incorporation showed a similar pattern although integration had progressed. Several grafts, however, exhibited segments separated from the recipient bed by a very thin loose connective tissue layer. Creeping substitution along the cancellous scaffold of the graft had progressed with corticalization of the marrow area and most of the graft area seemed revitalized. Graft size reduction was evident with marked rounding of the graft periphery and perivascular resorptive activity of the external compact layer. At the recipient bed a definite and pronounced local resorption had caused a hollow encompassing the major part of the compact layer. The underlying bone demonstrated remodeling activity. Still no major principal differences between the subperiosteal and submuscular groups were detectable except for a more extensive increase in cancellous bone density and prominent perivascular bone formation in the latter group. Intramuscular grafts were even more resorbed with smoothing of their margins and some perivascular bone remodeling as compared with the remaining groups. Unicortical bone grafts--20 weeks. All grafts were reduced in size and showed partial (Fig. 1) or full bony union with the underlying bone and displayed immature woven bone intermingled with lamellar bone in the interface region. No marrow cells were present. The grafts showed a vital appearance with continuing perivascular osseous formation and remodeling. Their margins were more rounded. At the recipient bed, the same picture was evident as during the preceding interval except for slightly more surface resorption. The development at the subperiosteal (Fig. 1) and the submuscular regions were similar. Intramuscular grafts demonstrated large lacunae and even more resorption. Still osseous formation was evident at restricted areas. Bicortical bone grafts 4 weeks. All grafts demonstrated a lack of integration. A thin acellular connective tissue layer persisted throughout the graft-

Fig. 2. Illustration of effects of massive resorpti,ve process after 12 weeks on bicortical graft positioned in gluteus maximus muscle). Labeling for osteopontin. Arrows indicate the periphery of the graft. (Original magnification •

host interface. There was local trabecular bone formation into the marrow space of the graft and cortical remodeling activity was apparent. The outer margins of the marrow space were bridged by woven bone seemingly emanating from the two cortical bone layers. The recipient bed disclosed marked external resorption, often exposing the underlying marrow to the graft surface. Local remodeling activity was apparent. More resorption was noted for the subperiosteal recipient bed than for those of the submuscular group, although remodeling seemed far more prominent in the latter one, even leading to integration in one animal, Intramuscular grafts demonstrated signs of revitalization, remodeling, and peripheral bone formation at the marrow space. Graft volume appeared seemingly unaltered. Bicortical bones grafts--12 weeks. Some grafts were partially integrated. The grafts had retained their

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ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY July 1996 the original surface of the recipient bed, that is, the graft that was partly resorbed was protruding minimally relative to the recipient adjacent bone. The submuscularly positioned grafts differed relative to those of subperiosteal placement in that submuscular graft integration was more pronounced and further that more extensive remodeling was apparent at the graft-host interface and local spots of advanced resorption were observed adjacent to the muscle surface on the outer aspect of the graft. The intramuscular grafts showed a mixed pattern of lacking viability, resorption, and, at certain areas, restricted peripheral bone formation and perivascular bone production. Volumetric loss seemed extensive.

Specific histologic findings

Fig. 3. Fibromodulin labeling in the soft tissues adjacent to a submuscularly placed graft--after 20 weeks. (G = bone graft, ST = soft tissues.) (Original magnification x300.)

main morphologic characteristics and structure although the external marrow was sealed off by newly formed bone making the grafts appear circular. The cortical surface disclosed some bone deposition intermingled with resorptive lacunae. At the recipient bed a striking external resorption was evident with remodeling going on beneath; less resorption was noted in the submuscular recipient site as compared with the subperiosteal. The intramuscularly placed grafts were fairly structurally intact but showed signs of lacking internal viability and marked resorption (Fig.2) around the complete surface of the compact layers. Bicortical bone grafts--20 weeks. Partial to full graft incorporation was encountered. The grafts had attained a circular configuration, and their cortical layers were thinner. The recipient site disclosed marked resorption, sporadically leading to a loss of two thirds of the bones width. Occasionally the outer compact layer of the graft was seen almost level with

The appearance, main localizations, and a semiquantitative grading of the macromolecules at the intervals studied are demonstrated in Tables II, III, and IV. Cartilage matrix proteins (Table II). PRELP and chondrocalcin stained most intensely by 12 weeks, primarily marking the connective tissue in general and the resorption lacunae of the graft, respectively. Antibodies against fibromodulin labeled mostly the graft-host interface, its surrounding area (Fig. 3), and the adjacent periosteai bone; the intensity did not decrease during the period investigated. Interestingly, both onlay placements of the bicortical grafts implied rather intense labeling of the diploic space of the recipient side. COMP labeled distinctly very infrequently. Intramuscular grafts displayed less labeling than the remaining groups. Bone matrix proteins (Table III). The 62 kDa protein and osteopontin showed intense labeling in the bone tissue in general, that is, both the graft and recipient site, at all intervals. Labeling also revealed the 62 kDa protein to be localized in the connective tissue surrounding the graft with most activity concentrated to the contact area between the graft and connective tissue at week 4. The perivascular region of the graft canaliculi exhibited a most profound labeling for both bone sialoprotein and osteopontin (Fig. 4). The pericanalicular areas of the periosteal bone for both subperiosteally placed uni- and bicortical grafts were unlabeled at week 4 for the 62 kDa, bone sialoprotein, and osteopontin proteins. The intramuscular bone grafts appeared roughly similar in labeling patterns as compared with remaining groups (Fig. 2). Proteoglycans (Table IV). Labeling for both biglycan and aggrecan was weak or absent for most intervals studied. By week 12, biglycan labeling was noted primarily in the resorption lacunae of the graft.

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Volume 82. Number 1 Table II. Time-related distribution of cartilage matrix protein PRELP

Unicortical grafts Week 4 Subperiosteal Submuscular Intramuscular Week 12 Subperiosteal Submuscular Intramuscular Week 20 Subperiosteal Submuscular Intramuscular Bicortical grafts. Week 4 Subperiosteal Submuscular Intramuscular Week 12 Subperiosteal Submuscular Intramuscular Week 20 Subperiosteal Submuscular Intramuscular

Fibromodulin

COMP

Chondrocalcin

0 0 0

+;RS-g,PB.G-rs +:RS-g,G-co,Ci 0

0 0 0

(+);G-rl(p) (+);G-r/ 0

++:C ++:C (+);G-p

++:PB.G-p.Cs ++'PB.G-rs,p (+):G-rs:p

(+);G-rl(p) 0 0

++;G-rl(p),d +;G-rl 0

+;Ci:G-rl(p) 0 (+):G-rl

+:Cs ++:PB.Ci (+);G-rs

(+);G-fl(p) 0 0

+;G-rl(p) +;G-rl(p) +;G-rs

0 0 0

+:Ci(RS-g.PB) +:Ci.PB 0

0 0 0

+;G-fl(p) (+);G-rl(p) +;G-rl(p)

++'C ++:C 0

~+ ~:PB /+/:Ci.PB 0

0 0 0

+;G-rl(p) ++;G-fl 0

(+);C +:Co.G-c 0

+:d. ~Ci,PB) +:d.(RS-g,1 ,Ci,PB) +'G-co:Cs

0 0 (+);G-rl(p)

+;G-rs +;G-rl,(PB) +;G-rl

RS, Recipient site-g (at graft region), -1 (laterally); d, diploic space; PB, periosteal bone; G, graft-c (centrally, implies bone marrow space for bicortical grafts), -p (peripherally), -rs (at RS, implies bone marrow space for unicortical grafts), -co (complete); rl (resorption lacunae or canaliculi); C (connective tissue, -i (at graft host interface), -s surrounding the graft, o = no staining, (+) = nouspecific labeling, + = moderate labeling, ++ = intense labeling.

Laminin (Table IV). Antilaminin staining demonstrated a late appearance of blood vessels; these were most abundant in the graft-host interface region and the surrounding connective tissue. Height measurements Graft height reduction from week 4 to 20 was dramatic in most groups (mean percentage height lost: unicortical grafts, subperiosteal 43.9%, p = 0.015; submuscular 41.3%, p = 0.018; intramuscular 57.2%, p = 0.000; bicortical grafts, subperiosteal 39.2%, p = 0.045; submuscular 27.8%, NS; intramuscular 46.2%, p = 0.009). A significant intergroup difference was only registered when comparing bicortical submuscular to unicortical intramuscular grafts (p = 0.034). DISCUSSION

Some factors have been emphasized to minimize bone-graft resorption12: the vascularity of the recipient bed, the physiologic stress placed on the graft, and the graft-to-recipient bone contact. The purpose of this study was to obtain more details on these matters by exploring autogeneic onlay bone graft behav-

ior in different environments in a rat nonosteogenic model. Intramuscular placement was routinely included in the experimental design to control for the reactions of a graft lacking direct contacts with a bone surface. Isogeneic bone graft donors were used to reduce host animal suffering, postoperative morbidity, and operation time. Considering a more or less identical interanimal genetic composition, the grafts can be considered autogeneic. Tibial and femoral grafts were preferred relative to membranous skull grafts because of their rich content of bone marrow, large size, easy accessability, and acceptable possibilities to standardize the graft volume. Furthermore, the partition of the bone to accomplish unicortical corticocancellous grafts was easily accomplished. Rigid fixation of the graft to the recipient bed was not technically feasible. However, the dissected tight subperiosteal and submuscular pockets allowed nonflexible graft positioning. Bone grafting to the craniofacial area is a fundamental clinical tool that enables skeletal reshaping and reconstruction. Cortical bone largely dies after disruption of its blood supply except for a superficial thin layer of cells. 28 Cancellous bone, on the other

28

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July 1996 Table III. Extent and main localizations of bone matrix proteins at the intervals studied et62 kDa Unicortical grafts Week 4 Subperiosteal Submuscular Intramuscular Week 12 Subperiosteal Submuscular Intramuscular Week 20 Subperiosteal Submuscular Intramuscular Bicortical grafts Week 4 Subperiosteal Submuscular Intramuscular Week 12 Subperiosteal Submuscular Intramuscular Week 20 Subperiosteal Submuscular Intramuscular

Bone sialoprotein

Osteopontin

++;G-p,Cs ++;Cs ++;G-p,Cs

+;G-rl ++;G-rl ++;G-rl

++;PB,G-rl,rs ++;G-rl ++;G-rl,Cs

++;Cs ++;Cs +;G-co,Cs

++;G-rl,p (+);G-rs (+),G-rl

++;G-rl,PB +;PB,G-rl +;G-rl

++;Cs +;G-p,Cs +;G-p,rl

++;G-rl +;G-rl +;G-rl

+;G-rl,Cs +;G-rl ++;G-rl

++;Cs ++;G-c,Cs,RS-g +;G-co

(+);G-rl,p +;G-rl (+);G-rl

++;G-rl ++;G-rl,PB +;G-rl,co

++;Cs ++;G-c,Cs ++;G-co

(+);G-rl +;G-rl ++;G-rl

+;G-rl(p) +;G-rl ++;G-rl

++;Cs ++;Cs,G-c ++;G-rl,Cs

+;G-rl ++;G-rl ++;G-rl

++;G-rl ++;G-rl(p) ++;G-rl

RS, Recipient site-g (at graft region), -1 (laterally); d, diploic space; PB, periosteal bone; G, graft-c (centrally, implies bone marrow space for bicortical grafts), -p (peripherally), -rs (at RS, implies bone marrow space for unicortical grafts), -co (complete); rl (resorption lacunae or canaliculi); C (connective tissue, -i (at graft host interface), -s surrounding the graft, o = no staining, (+) = nonspecific labeling, + = moderate labeling, ++ = intense labeling.

hand, contains many osteogenic cells that are more easily nourished, by diffusion and survive direct transfer within the host. 29 A tendency for superficial osteocytes to survive and contribute to bone formation has been observed by several investigators, 3~ although the contribution of the endosteal lining cells, and possibly the stromal cells of the marrow, seems to be most important. 31 Two recipient milieus at the craniofacial skeleton--subperiosteally at the parietal region and under the temporal muscle at the temporal f o s s a - - w e r e tested and compared with a control environment in a relatively passive environment, intramuscularly over the hip. The intervals were chosen to reflect a dynamic healing process with most interest focused on the long-term result in consideration of the late result being most critical clinically. Furthermore they are identical to those selected for studies in our laboratory on both orthopedic and craniofacial subjects. One technique not previously used in this context to our knowledge is immunohistochemical labeling with antibodies used against various bone and cartilage proteins and proteoglycans that are considered essential to bone growth, mineralization, and resorp-

tion. These macromolecules may be synthesized locally or by cells in other tissues. Bone tissue is composed of a unique combination of noncollagenous proteins that distinguish it from other connective tissues.33, 34 Proteins with a distribution restricted primarily to bone include bone sialoprotein, a 6 2 kDa protein, and osteopontin. The large aggregating proteoglycan (aggrecan) is a cartilage-specific molecule. Moreover, cartilage contains relatively large amounts of a fragment released from procollagen II on cleavage of the procollagen molecule before fibril assembly, which is referred to as chondrocalcin. 35 Fibromodulin, biglycan, and PRELP are macromolecules common to many connective tissues. Information on the structure, function, specific localization, and regulation of these newly characterized macromolecules in bone tissue is essential and presently limited. To date, it is important to identify the stage of development and localization of the appearance of individual macromolecules, and in the future, perform quantitative evaluations that at present are impossible to undertake because of variable penetration of tissue sections by antibodies and variable presentation of epitopes. Thus, new details of the in-

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Table IV. Immunolabeling patterns of proteoglycans tested and laminin Biglycan Unicortical grafts Week 4 Subperiosteal Submuscular Intramuscular Week 12 Subperiosteal Submuscular Intramuscular Week 20 Subperiosteal Submuscular Intramuscular Bicortical grafts Week 4 Subperiosteal Submuscular Intramuscular Week 12 Subperiosteal Submuscular Intramuscular Week 20 Subperiosteal Submuscular Intramuscular

Aggrecan

Laminin

0 t + ~:Ci 0

0 0 0

+;C-s +;C-s,G-rs +;C-s,G-rl

++:Ci.G-rl +:Cs.~PB) +:G-rl

0 0 0

++;C-s,G-rs ++;G-rs,C-s ++;C-s,G-rs

+;Cs,G(p).-rl 0 0

+:G-rl 0 0

+;G-rs,C-s +;G-rs,rl +;G-rl

(+);PB,G-p 0 0

0 0 0

(+);C-s-i,G-c (+);C-s-i,RS-g (+);C-s,G-rl

(+);G-rl ++;G-rl,(RS-g) 0

0 0 0

++;C-s-i,G-p +;C-s-i,G-rs +;C-s,G-rl

(+);PB (+);G-rl,PB 0

0 0 +;G-rl

++;G-rs,PB ++;G-c-rs,C-s +;G-rl

RS, Recipient site-g (at graft region), -1 (laterally); d, diploic space; PB, periosteal bone; G, graft-c (centrally, implies bone marrow space for bicortical grafts), -p (peripherally), -rs (at RS, implies bone marrow space for unicortical grafts), -co (complete); rl (resorption lacunae or canaliculi); C (connective tissue, -i (at graft host interface), -s surrounding the graft, o = no staining, (+) = nonspecific labeling, + = moderate labeling, ++ = intense labeling.

tricate integration process of a bone graft may be detected that are not revealed by routine microscopic techniques, and hopefully markers indicating such integration will be identified. Detailed reviews of the proteins and proteoglycans studied have been published elsewhere 33, 34 and are beyond the scope of this report. In a previous study, 36 we investigated the early regenerative process of healing skull fractures and defects in rats with the same technology. The labeling pattern of the macromolecules for the mutual weeks 4 and 12 was similar to this study. Macroscopically, integration of all grafts appeared stable after 4 weeks. On the contrary, a diversified picture was seen microscopically. Corticocancellous grafts were partially incorporated at 4 weeks; they displayed a loose connective tissue network intermingled with distinct bony connections between the graft and recipient bed, a process that was clearly illustrated by fibromodulin labeling. Fibromodulin is considered to participate in the regulation of collagen fibril formation and remodeling. 37 A complete bony union was not observed until 20 weeks. Bicortical grafts, on the other hand, disclosed lack of integration at 4 weeks, partial integration after 12 and 20 weeks,

and only rarely was a full incorporation identified by 20 weeks. Speculatively, there may be a slight advantage to placing a cancellous bone surface facing the recipient site over that of compact bone, but this was not evaluated specifically. At present, it seems premature to draw any definite conclusions on this subject. However, a separate study on this issue with a more appropiate experimental design has just been completed and will be presented shortly. The intervening connective tissue labeled rathe1 intensely for fibromodulin, especially before full integration. No major differences were established between the submuscular and subperiosteal placements except for week 20 for bicortical grafts when a bettel integration was displayed for the former group. In general, the surrounding connective tissue labeled markedly for PRELP (week 12), the oL62 (all intervals) protein and laminin (see following). The functions of the two former proteins is somewhat obscure. PRELP binds to many connective tissues and is believed to promote cell attachment. 33 The cr kD~ protein is synthesized exclusively in the liver, appears early in osteogenesis, and is considered to restrict the resorptive process. 3s

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ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY July 1996

Fig. 4. Perivascular bone formation and remodeling external to cortical layer in unicortical graft placed subperiosteally--4 weeks. Osteopontin labeling. Note the predominant intense labeling with sparse labeling around the vascular channels, probably representing ongoing activity and former bone formation, respectively, (PB = parietal bone, NB = newly formed bone. (Original magnification xl00.) Concerning the recipient bed of unicortical grafts, no obvious reactions were observed after 4 weeks except for limited fibromodulin and biglycan (both binding to collagen) labeling of the connective tissue at the graft-host region. At 12 and 20 weeks a fairly pronounced locally restricted resorption was demonstrated with remodeling of the underlying bone tissue as demonstrated by fibromodulin and the definite but unspecific labeling obtained by the oL62 kDa protein and osteopontin. Osteopontin is believed to anchor osteoclasts to the bone surface and to inhibit crystal formation, 33 thereby indicating remodeling of the bone tissue. The bicortical grafts caused an earlier appearing resorptive process exposing the recipient bed marrow as early as 4 weeks after grafting with simultaneous intense remodeling of the bed mainly for the subperiosteally placed grafts. By 20 weeks, the resorption was quite dramatic, in some specimens even leading to the graft being almost level with the adjacent bone surface. Here fibromodulin distinctly labeled the underlying diploic space. The volumetric maintenance and early remodeling of any bone graft is of fundamental importance to the success of the surgical intervention. Early loss of onlay graft volume implies great risks for a second procedure. Remodeling is a complex simultaneous process of resorption and bony accretion. The resorption cavities produced depends on early revascularization (see following); osteoclasts may be present but are not central to this process. 28, 39 In this series, corticocan-

cellous grafts displayed early widespread remodeling along the marrow space and some trabecular bone formation peripherally, but no major volumetric changes were registered. After 12 weeks, a corticalization of the marrow was apparent I with loss of volume, primarily localized to the cancellous part of the graft. The graft seemed partially revitalized; the process appeared more or less Complete after 20 weeks. Furthermore, at that interval volumetric loss seemed arrested. Bicortical grafts displayed early trabecular bone production emanating from the cortical margins of the graft into the marrow space with sealing off of the intervening marrow space with woven bone. Interestingly, this process developed into a reshaping of the graft to a more circular configuration. This study did not explore any difference of membranous and enchondral bone formation. Hardesty and Marsh 4 proposed a mechanism for differences found between the volume maintenance of bone grafts of membranous and enchondral origin on the basis of their content of cortical and cancellous bone. All grafts used in this study were harvested from long bones, but a certain variance in cortical to cancellous bone ratio may have been present. Pilot data (not shown) suggest, however, that this minimally influenced the conclusions we have drawn. As expected, the cortical bone manifested a slow revitalization and less resorption (mostly localized peripherally) than the cancellous portion of the graft. The analysis of the remodeling and revitalization processes was greatly

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facilitated and improved by the illustrative labeling of bone sialoprotein (having a distribution essentially restricted to the osteoblasts and involved in the onset of mineralization4~ osteopontin, the oL62 kDa protein, chondrocalcin (participating in the focal calcification process35), and to some extent biglycan. The process of creeping substitution and the perivascular bone formation were clearly illustrated by bone sialoprotein and osteopontin labeling. Especially, fibromodulin was labeled in the adjacent periosteal bone formation. With respect to the site of grafting, more extensive local resorptive and remodeling activities were observed for submuscularly positioned uni- and bicortical grafts. Ermis and Poole 22 also noted more resorption of iliac onlays grafted to the rabbit mandibular rami covered by muscle. This implies that intermittent and more intensive pressure probably stimulates a more rapid graft revitalization and that such grafts are more exposed to repetitive trauma leading to structural disintegration and loss of volume. Graft survival is also partly dependent on whether the recipient site is a resorptive or a depository growth field as emphasized by Zins et al. 19 and Fasano et al., 18 although this influence is smaller in the mature animal.12 In the present study, both onlay areas were depository, and the animals were adult. which excludes this issue as affecting the observed results. Intramuscularly placed grafts retained their viability in large parts of both unl- and bicortical grafts. Perivascular and peripheral bone formation and apposition were demonstrated locally, whereas other areas of the same graft showed peripheral resorption. Interestingly, with the use of the radioactive microsphere technique, Fisher and Wood 21 described better medullary vascularization and less marrow necrosis for bone segments underlying muscle compared with those beneath a cutaneous flap. Our finding that the marrow space generally sealed off from the surrounding connective tissue by newly produced compact bone emphasizes the dynamics of the bone tissue and its responsiveness to changes of component exposure to the surrounding tissue; the marrow seems to prefer a homogenous cortical bone coverage. Moreover, after 12 and 20 weeks, resorption was successively more pronounced, but some osteogenesis was still apparent. Bicortical grafts showed less vitality for the latter two intervals. Remarkedly, less labeling of cartilage matrix proteins was noted, whereas the bone matrix proteins labeled similarly or slightly less compared with the subperiosteal and submuscular implantation sites.

The revascularization pattern has been considered vital for the success of the bone grafting procedure. The nonvascularized bone graft is largely nonviable and serves primarily as an inert scaffolding for the ingrowth of host tissues. Trueta 41 and Albrektsson 5 have demonstrated that the ossification process is vascular-dependent. Generally, intensive resorption preceeds osteogenesis in cortical bone, whereas cancellous bone primarily heals by accretion and considerably later by bone resorption. However, divergent views as to the importance of the appearance of t h e blood vessels relative to the resorptive process have been presented. Some investigators suggest that early revascularization implies less volumetric loss, 6 whereas others are convinced of the opposite. 8-1~We investigated the labeling characteristics of laminin, an important component of the basement membrane, which forms an intervening layer between epithelial cells and the underlying stroma. Here the resumption of blood circulation can be appreciated. A similar pattern of laminin labeling for the uni- and bicortical grafts with most labeling localized to the surrounding soft tissue component or at the interface between the graft and its recipient site was observed; this corroborates earlier descriptions. 6 By 12 weeks, a more intensive labeling was demonstrated both at the interface region and the resorption lacunae of the graft: the latter finding supports reports of more time-consuming vascular penetration through the cortical layer.9. m.42 This might influence the reparative processes as demonstrated by the increased labeling of PRELP. fibromodulin, chondrocalcin, and biglycan. CONCLUSIONS This study demonstrated the following: 9 the bicortical grafts appeared to retain volume to a better extent than corticocancellous grafts, seemingly dependent on the cortical bone being more resistant to resorption: 9 the integration of the unicortical grafts was superior, probably related to the marrow of the graft being in contact with the recipient site; 9 the bone marrow space of both the uni- and bicortical grafts was sealed off from the surrounding connective tissue by compact bone emanating from the cortical margins of the graft: 9 resorption of the recipient bed was most pronounced at sites that received bicortical grafts, presumably because of the increased external pressure resulting from the robust structure of the graft in the restricted pocket: 9 differences between submuscular and subperiosteal graft placement were distinct, which shows

32

Alberius et al.

improved integration, more extensive remodeling, and more resorption for the former group; 9 intramuscular control grafts demonstrated similar adaptations to their environment as remaining groups, but immunolabeling indicated a more passive atmosphere; volumetric bone loss was pronounced as compared with grafts in contact with a bone surface; 9 several bone (o~62 kDa, bone sialoprotein, osteopontin) and cartilage (PRELP, fibromodulin, chondrocalcin) matrix proteins as well as the proteoglycan biglycan were demonstrated during various phases and at different structural parts of the graft-host area during the integration process. An ongoing series of studies seeks to provide further data to be used in the evaluation of graft incorporation. We thank Professor D. Heineg~rd for supplying the antibodies, M. T. E. Fahlman, MSc, for language revision, and J. E. Blomqvist, DDS, for statistical advice. REFERENCES 1. Peer LA. The fate of autogenous human bone grafts. Br J Plast Surg 1951;3:233-43. 2. Smith JD, Abramson M. Membranous vs endochondral bone autografts. Arch Otolaryngol 1974;99:203-5. 3. Zins JE, Whitaker LA. Membranous versus endochondral bone: implications for craniofacial reconstruction. Plast Reconstr Surg 1983;72:778-84. 4. Hardesty RA, Marsh JL. Craniofacial onlay bone grafting: a prospective evaluation of graft morphology, orientation, and embryonic origin. Plast Reconstr Surg 1990;85:5-14. 5. Albrektsson T. Repair of bone grafts: a vital microscopic and histologic investigation in the rabbit. Scand J Plast Reconstr Surg 1980;14:1-12. 6. Kusiak JF, Zins JE, Whitaker LA. The early revascularization of membranous bone. Plast Reconstr Surg 1985;76:510-6. 7. Phillips JH, Rahn BA. Fixation effects on membranous and endochondral onlay bone graft revascularization and bone deposition. Plast Reconstr Surg 1990;85:891-7. 8. Lin KY, Bartlett SP, Yaremchuk MJ, Fallon M, Grossman RF, Whitaker LA. The effect of rigid fixation on the survival of onlay bone grafts: an experimental study. Plast Reconstr Surg 1990;86:449-56. 9. Sullivan WG, Szwajkun PR. Revascularization of cranial versus iliac crest bone grafts in the rat. Plast Reconstr Surg 1991;87: 1105-9. 10. Chen NT, Glowacki J, Bucky LP, Hong H-Z, Kim W-K, Yaremchuk MJ. The roles of revascularization and resorption on endurance of craniofacial onlay bone grafts in the rabbit. Plast Reconstr Surg 1994;93:714-22. 11. Alberius P, Dahlin C, Linde A. Role of osteopromotion in experimental bone grafting to the skull: a study in adult rats using a membrane technique. J Oral Maxillofac Surg 1992; 50:829-34. 12. LaTrenta GS, McCarthy JG, Breitbart AS, May M, Sissons HA. The role of rigid fixation in bone-graft augmentation of the craniofacial skeleton. Plast Reconstr Surg 1989;84:57888. 13. Phillips JH, Rahn BA. Fixation effects on membranous and endochondral onlay bonegraft resorption. Plast Reconstr Surg 1988;82:872-7. 14. Wilkes GH, Kernahan DA, Christenson M. The long-term survival of onlay bone grafts: a comparative study in mature and immature animals. Ann Plast Surg 1985;15:374- 8.

ORAL SURGERY ORAL MEDICINE ORAL PATHOLOGY July 1996 15. Knize D. The influence of periosteum and calcitonin on onlay bone graft survival. Plast Reconstr Surg 1974;53:190-9. 16. Thompson N, Casson JA. Experimental onlay bone grafts to the jaws: a preliminary study in dogs. Plast Reconstr Surg 1970;46:341-9. 17. Finkelman RD, Eason AL, Rakijian DR, Tutundzhyan Y, Hardesty RA. Elevated IGF-II and TGF-13 concentrations in human calvarial bone: potential mechanism for increased graft survival and resistance to osteoporosis. Plast Reconstr Surg 1994;93:732-8. 18. Fasano D, Gasparini G, Menoni V, Bertoni F, Bacchini P. The fate of onlay membranous bone grafts in different facial recipient sites. Eur J Plast Surg 1989;12:160-6. 19. Zins JE, Kusiak JF, Whitaker LA, Enlow DH. The influence of the recipient site on bone grafts to the face. Plast Reconstr Surg 1984;73:371-9. 20. Goldstein J, Mase C, Newman MH. Fixed membranous bone graft survival after recipient bed alteration. Plast Reconstr Surg 1993;91:589-96. 21. Fisher J, Wood MB. Experimental comparison of bone revascularization by musculocutaneous and cutaneous flaps. Plast Reconstr Surg 1987;79:81-90. 22. Ermis I, Poole M. The effects of soft tissue coverage on bone graft resorption in the craniofacial region. Br J Plast Surg 1992;45:26-9. 23. Robinson M, Shuken R. Bone resorption under plastic chin implants. Oral Surg Oral Med Oral Pathol 1969;27:116-8. 24. Jobe R, Iverson R, Vistnes L. Bone deformation beneath alloplastic implants. Plast Reconstr Surg 1973;51:169-73. 25. Wellisz T, Lawrence M, Jazayeri MA, Golshani S, Zhou ZY. The effects of alloplastic implant onlays on bone in the rabbit mandible. Plast Reconstr Surg 1995;96:957-63. 26. Hulth A, Johnell O, Lindberg L, HeinegSxd D. Sequential appearance of macromolecules in bone induction in the rat. J Orthop Res 1993;11:367-78. 27. Klareskog L, Forsum U, Wigren A, Wigzell H. Relationship between HLA-DR expressing cells and T-lymphocytes of different subsets in rheumatoid synovial tissue. Scand J Immunol 1982;15:501-7. 28. Ham AW. Some histophysiological problems peculiar to calcified tissues. J Bone Joint Surg Am 1952;34:701-28. 29. Burwell RG. The fate of bone grafts. In: Apley AG, ed. Recent advances in orthopaedics. Chap. 6. London: J & A Churchill Ltd, 1969;115-207. 30. Burwell RG. Studies in the transplantation of bone: VII. The fresh composite homograft-antograft of cancellous bone. J Bone Joint Surg Br 1964:46:110-40. 31. Craig Gray J, Elves MW. Early osteogenesis in compact bone isografts: a quantitative study of the contributions of the different graft cells. Calcif Tissue Int 1979;29:225-37. 32. Heslop BF, Zeiss IM, Nisbet NW. Studies of transplantation of bone: I. a comparison of autologous and homologous bone implants with reference to osteocyte survival, osteogenesis, and host reaction. Br J Exp Pathol 1960;41:269-87. 33. Heineggtrd D, Oldberg A. Structure and biology of cartilage and bone matrix noncollagenons macromolecules. FASEB J 1989;3:2042-51. 34. Young MF, Kerr JM, Ibaraki K, Heegaard A-M, Robey PG. Structure, expression, and regulation of the major noncollagenous matrix proteins of bone. Clin Orthop 1992;281: 275-94. 35. Poole AR, Pidoux I, Reiner A, Choiu H, Rosenberg LC. Association of an extracellular protein (chondrocalcin) with the calcification of cartilage in endochondral bone formation. J Ceil Biot 1982;98:54-62. 36. Alberius P, Johnell O. Repair of intramembranous bone fractures and defects in rats: Immunolocalization of bone and cartilage proteins and proteoglycans. J Craniomaxillofac Surg 1991;19:15-20. 37. Hedbom E, Heineg~d D. Interaction of a 59-kDa connective

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Volume 82, Number 1 tissue matrix protein with collagen I and collagen II. J Biol Chem 1989;264:6898-905. 38. Wendel M, Heineg~d D, Franzrn A. A major non-collagenous 62 kDa protein from rat bone mineralized matrix is identical to pp63, a phosphorylated glycoprotein from liver. Matrix 1993;13:331-99. 39. Pappas AM, Beisaw NE. Bone transplantation: correlation of physical and histologic aspects of graft incorporation. Clin Orthop 1968;61:79-91. 40. Hunter GK, Goldberg HA. Nucleation of hydroxyapatite by bone sialoprotein. Proc Natl Acad Sci U S A 1993;90: 8562-5.

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41. Trueta J. The role of the vessels in osteogenesis. J Bone Joint Surg Br 1963;45:402-18. 42. Bartlett SP, Lin KY, Yaremchuk MJ, Fallon M, Grossman RS, Whitaker LA. Membranous versus endochondral bone. [Letter] Plast Reconstr Surg 1991;87:1145. Reprint requests: Per AIberius, DMD, MD, PhD Department of Plastic Surgery MAS S-20502 Malmr, Sweden

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