Rabbit Cranial Defect Research Articles

Growth factor-loaded scaffolds for bone engineering

The objective of the study presented here was to investigate the bone inductive properties as well as release kinetics of rhTGF-β1- and rhBMP-2-loaded Ti-fiber mesh and CaP cement scaffolds. Therefore, Ti-fiber mesh and porous CaP cement scaffolds were provided with these growth factors and inserted in subcutaneous and cranial implant locations in rats and rabbits. In vitro, a rapid release of rhTGF-β 1 was observed during the first 2 h of the Ti-fiber mesh scaffolds. During this time, more than 50% of the total dose of rhTGF-β 1 was released. Following this initial peak, a decline in the level of rhTGF-β 1 occurred. After 1 week, the entire theoretical initial dose was observed to have been released. This in contrast to the rhTGF-β 1 and rhBMP-2 release of the porous CaP cement scaffolds. Here, no substantial initial burst release was observed. The scaffolds showed an initial release of about 1% after 1 day, followed by an additional marginal release after 1 week. Histological analysis revealed excellent osteoconductive properties of non-loaded Ca-P material. Inside non-loaded Ti-mesh fiber scaffolds, also bone ingrowth occurred. Quantification of the bone ingrowth showed that bone formation was increased significantly in all scaffold materials by administration of rhTGF-β 1 and rhBMP-2. Consequently, we conclude that the release kinetics of growth factors from porous CaP cement differs from other scaffold materials, like metals and polymers. Nevertheless, orthotopic bone formation in a rabbit cranial defect model was stimulated in rhTGF-β 1- and rhBMP-2-loaded CaP cement and Ti-fiber mesh scaffolds compared with non-loaded implants.

Bone inductive properties of rhBMP-2 loaded porous calcium phosphate cement implants in cranial defects in rabbits

In this study, the osteoinductive properties of porous calcium phosphate (Ca-P) cement loaded with bone morphogenetic protein 2 (rhBMP-2) were evaluated and compared with rhBMP-2 loaded absorbable collagen sponge (ACS). Discs with a diameter of 8 mm were loaded with a buffer solution with or without 10 μg rhBMP-2 and inserted in 8 mm full thickness cranial defects in rabbits for 2 and 10 weeks of implantation. Histological analysis revealed excellent osteoconductive properties of the Ca-P material. It maintained its shape and stability during the implantation time better than the ACS but showed no degradation like the ACS. Quantification of the Ca-P cement implants showed that bone formation was increased significantly by administration of rhBMP-2 (10 weeks pore fill: 53.0±5.4%), and also reached a reasonable amount without rhBMP-2 (43.1±10.4%). Remarkably, callus-like bone formation outside the implant was observed frequently in the 2 weeks rhBMP-2 loaded Ca-P cement implants, suggesting a correlation with the presence of growth factor in the surrounding tissue. However, an additional in vitro assay revealed an accumulative release of no more than 9.7±0.9% after 4 weeks. We conclude that: (1) Porous Ca-P cement is an appropriate candidate scaffold material for bone engineering. (2) Bone formation can be enhanced by lyophilization of rhBMP-2 on the cement. (3) Degradation of porous Ca-P cement is species-, implantation site- and implant dimension-specific.

Evaluation of the potential beneficial effects of ostrich eggshell combined with eggshell membranes in healing of cranial defects in rabbits.

Potential beneficial effects of outer and inner ostrich eggshell membranes alone and in combination with the shell powder on bone regeneration in cranial defects of 18 rabbits were investigated using radiological, clinical and histological methods. Three 6 mm wide, half-thickness defects were made in the calvarial bones of each rabbit. One defect remained empty as a control; prepared eggshell membranes were extended across the other two defects; and prepared eggshell powder was inserted inside the membrane in one case. Either alone or in combination with eggshell powder, the outer and inner ostrich eggshell membranes produced little adjunctive effect under the conditions and duration of this experiment.

Poly(propylene fumarate) and Poly(DL-lactic-co-glycolic acid) as Scaffold Materials for Solid and Foam-Coated Composite Tissue-Engineered Constructs for Cranial Reconstruction

This pilot study investigates the osseointegration of four types of critical-size (1.5-cm diameter) rabbit cranial defect (n = 35) bone graft scaffolds. The first is a solid poly(propylene fumarate)/beta-tricalcium phosphate(PPF/beta-TCP) disk; the three remaining constructs contain a PPF/beta-TCP core coated with a 1-mm resorptive porous foam layer of PPF or PLGA [poly(DL-lactic-co-glycolic acid)], and bone marrow. Animals were killed at 6, 12, and 20 weeks. There was no evidence of a foreign body inflammatory response at any time during the study. Histomorphometric analyses of new bone formation sorted lineal and areal measures of new bone into three cranial layers (i.e., external, middle, and internal). Statistical analyses revealed significantly more bone in the PLGA foam-coated constructs than in the PPF foam-coated constructs (p < 0.03). No implant fixation was used; there is no strength at time 0. Twenty percent of all explants were tested for incorporation strength with a one-point "push-in" test, and failure ranged from 8.3 to 34.7 lb. The results of this study support the use of PPF as a biocompatible material that provides both a structural and osteogenic substrate for the repair of cranial defects.

Investigation of platelet-rich plasma in rabbit cranial defects: A pilot study

Purpose: The purpose was to evaluate the effect of platelet-rich plasma (PRP) on bone healing. Materials and Methods: Fifteen rabbits were included in this randomized, blinded, prospective pilot study. Four equal 8 mm diameter cranial bone defects were created and immediately grafted with autogenous bone, PRP alone, autogenous bone and PRP, and no treatment as a control. The defects were evaluated by digital subtraction radiography with step-wedge calibration, histology, and histomorphometric analysis performed at 1, 2, and 4 months. Results: The results showed a significant increase in histomorphometric bone area and radiographic bone density in both bone and bone and PRP samples as compared with the control and PRP alone. No ignificant increase in bone formation was seen with the addition of PRP to autogenous bone. No significant difference in bone formation was seen between defects treated with PRP alone and control sites. Conclusions: No significant improvement, radiographically or histomorphometrically, was seen with the addition of PRP in bone formation in noncritical sized defects in the rabbit cranial model. However, bone and bone and PRP showed a histomorphometric tendency toward increased bone formation at 1, 2, and 4 months. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:1176-1181, 2002

Effect of perforations in burr hole covers on cranial bone regeneration in rabbits

The reconstruction of bone defects is a significant problem in cranio-maxillofacial surgery. In an effort to avoid the known disadvantages of autogenous bone grafting, alternatives have been investigated. Bone substitute materials, degradable or nondegradable, aim at facilitating bone regeneration, while they take over load-bearing functions for a period of time. In this study, the healing of cranial defects in rabbits was assessed using polylactide guiding covers with and without perforations. Bilateral circular cranial defects were produced in 16 New Zealand white rabbits. The defects were covered with extruded and laser cut polylactide burr hole covers, each animal receiving a perforated burr hole cover on one side and a nonperforated one on the contralateral side. Bone seeking fluorochromes were administered at regular intervals. After an observation period of 8 weeks the amount of bone regeneration in the area of the defects was quantified from contact radiographs, and the dynamics of bone formation were assessed by fluorescence microscopy. Stained sections were used to analyze morphologic differences. No signs of adverse tissue reactions or osteolysis were observed. A bone-guiding function was observed for both covers with or without perforations. Intracranial tissue herniation into the defect hindered the regeneration process. Wide intraindividual and interindividual variation became apparent and average defect filling was only 40% within the 8-week observation period. In this model the perforated covers offered no advantage over nonperforated covers. It can be concluded from this study, that the use of external burr hole covers alone does not guarantee a full thickness regeneration of the cranial defect, but it provides a guiding function for promotion of structured bone regeneration.

Bone formation in transforming growth factor beta-1-coated porous poly(propylene fumarate) scaffolds.

This study determined the bone growth into pretreated poly(propylene fumarate) (PPF) scaffolds implanted into a subcritical size, rabbit cranial defect. PPF scaffolds were constructed by using a photocrosslinking-porogen leaching technique. These scaffolds were then either prewetted (PPF-Pw), treated with RF glow-discharge (PPF-Gd), coated with fibronectin (PPF-Fn), or coated with rhTGF-beta1 (PPF-TGF-beta1). One of each scaffold type was then placed into the cranium of nine rabbits. The rabbits were sacrificed after 8 weeks, and the scaffolds were retrieved for histological analysis. The most bone formation was present in the PPF-TGF-beta1 implants; the newly formed bone had a trabecular appearance together with bone marrow-like tissue. Little or no bone formation was observed in implants without rhTGF-beta1. These histological findings were confirmed by image analysis. Bone surface area, bone area percentage, pore fill percentage, and pore area percentage were significantly higher in the rhTGF-beta1-coated implants than in the noncoated implants. No statistical difference was seen between the PPF-Fn, PPF-Pw, or PPF-Gd scaffolds for these parameters. Quadruple fluorochrome labeling showed that in PPF-TGF-beta1 implants bone formation mainly started in the interior of a pore and proceeded toward the scaffold. We conclude that (a) PPF-TGF-beta1 scaffolds can indeed adequately induce bone formation in porous PPF, and (b) PPF scaffolds prepared by the photocrosslinking-porogen leaching technique are good candidates for the creation of bone graft substitutes.

Cranial reossification with absorbable plates.

The purpose of this study was to examine the effect of Lactosorb absorbable plates on bone healing across cranial bone defects in the rabbit skull. Two 10-mm diameter parietal skull defects were created in each of 20 rabbits, with one defect being placed on either side of the sagittal suture. In 10 rabbits, an absorbable plate was placed across both the inner and outer cortices of the left defect, and in the other 10 rabbits, an absorbable plate was placed across the outer cortex only of the left defect. The right defect always served as the control side, with no plate being placed across it. Rabbits were killed an average of 25 weeks postoperatively. Areas of reossification in the experimental and control defects of each rabbit were then measured, examined histologically, and compared. Growth across defects spanned by one plate was also compared with growth across defects spanned by two plates. Histologic and statistical analyses revealed no significant differences in reossification between the control and experimental defects in each animal and between the defects spanned by one versus two plates. This study suggests that these copolymer absorbable plates neither inhibit nor facilitate reossification across 10-mm diameter rabbit cranial defects.

Ostrich eggshell as a bone substitute: a preliminary report of its biological behaviour in animals – a possibility in facial reconstructive surgery

The aim of this study was to assess the biological behaviour of an implant of ostrich eggshell in various animal models of facial bone reconstruction. The implant was first bioassayed in a rat muscle pouch (n =10), and then tested as an interpositional graft in rat (n =10) and rabbit (n =5) cranial defects. It was finally used as an onlay graft on rabbit mandibles (n =5). Animals were killed after two months in the bioassay, three months in the interpositional model, and six months in the onlay model. The specimens were studied by contact radiography and standard histological techniques. All animals showed normal wound-healing. In the bioassay, the implants produced only a minimal inflammatory reaction. In the interpositional model, the implants maintained a good contour, but there was no sign of graft-remodelling. In the onlay model, the grafts were stable and partly osteointegrated. The onlay graft model gave the most promising results. Because ostrich eggshell is inexpensive and has good mechanical properties, it deserves further study. Long-term studies will clarify its possible role in maxillofacial surgery.

Evaluation of a novel malleable, biodegradable osteoconductive composite in a rabbit cranial defect model

The ceramic form of calcium phosphate osteoconductive material such as hydroxyapatite is brittle, non-malleable and non-degradable, and these mechanical properties limit its clinical application in calvarium reconstruction. To improve these properties, we developed a malleable, biodegradable osteoconductive composite composed of tricalcium phosphate particles bound by a gelatin which is set by glutaraldehyde mediated cross-linking. The composite was implanted into a 15 × 15 mm full-thickness, calvarial defect in 20 rabbits for up to 3 months. Twelve rabbits were left unreconstructed as controls. Specimens were retrieved at 2 weeks, 1, 2 and 3 months. Five reconstructed and 3 unreconstructed rabbits were examined for each time period. The assessment included a series of post operative gross examinations, radiographs and histologic evaluations. We are able to demonstrate that this composite is (1) biocompatible, with little tissue reaction; (2) osteoconductive, with progressive growth of new bone into the calvarial defect; (3) biodegradable, with progressive replacement of the composite by new bone, acellular matrix and bone-like material. Replacement of this composite by new bone is postulated to occur by a combination of osteoconduction and biodegradation. These results indicate that further experimental research to combine this malleable, biodegradable, osteoconductive composite with an osteoinductive agent such as bone morphogenetic protein may generate new biomaterial for full-thickness calvarial defect reconstruction.

Evaluation of a novel osteogenic factor, bone cell stimulating substance, in a rabbit cranial defect model.

A novel osteogenic factor, bone cell stimulating substance (BCSS), was recently isolated from bovine bone. Unlike the bone morphogenetic proteins, which are generally 20- to 40-kDa glycoproteins belonging to the transforming growth factor-beta superfamily, BCSS is a 2.5-kDa, water-soluble polypeptide. This study evaluated the osteogenic potential of BCSS in combination with coralline-derived porous hydroxyapatite to reconstruct a large, nonhealing cranial defect in the rabbit. Twenty-four rabbits underwent a 16 x 20 mm full-thickness (extradural) excision of the parietal bones and were divided into six groups of four rabbits each. Group 1 through 5 were reconstructed with 16 x 20 x 1.5 mm hydroxyapatite implants treated with: (1) 20 osteogenic units (424 micrograms) BCSS, (2) 8 osteogenic units (170 micrograms) BCSS, (3) 4 osteogenic units (85 micrograms) BCSS, (4) 0 osteogenic units (424 micrograms) inactive BCSS analog, or (5) left untreated. Group 6 was left unreconstructed. Implants were harvested at 12 weeks and analyzed for percentage of lamellar bone formation by a computerized microscope video image analysis system. Groups reconstructed with BCSS-treated hydroxyapatite implants demonstrated more bone ingrowth than did the control hydroxyapatite groups not treated with BCSS (inactive BCSS analog or untreated). Linear regression dose-response analysis indicated an average 3.2 percent increase in bone ingrowth for a 10-U increase in BCSS (p = 0.043). The unreconstructed control group demonstrated no healing. With a molecular mass of only 2.5 kDa and its identity not completely known, BCSS is a novel factor that seems to possess osteogenic potential similar to that of previously investigated osteogenic proteins.

Temporal dynamics of healing in rabbit cranial defects using guided bone regeneration

Purpose: The objective of this study was to histologically evaluate the early stages of bone regeneration using rabbit calvaria defects in conjunction with guided tissue regeneration. Materials: A semilunar cutaneous-periosteal flap was raised on the forehead of four rabbits exposing the top of the skull. A standardized transosseous skull defect (≥ 15 mm in diameter) was made in the area of the right parietal bone with a rotating round bur. Care was taken not to damage the underlying dura. A flat expanded polytetrafluoroethylene (ePTFE) membrane was placed to cover the defect. The membrane was tightly adapted, extending at least 4 mm onto intact bone, and the flap was sutured. One, 2, 3, and 5 weeks later, the specimens were removed and processed using standard, undecalcified, hard-tissue histologic techniques. Contact radiographs were also taken. Results: Bone growth increased with time, starting at the borders of the defect. At 1 week, trabeculae of woven bone grew into the highly vascularized loose connective tissue occupying the defect. Two weeks postsurgery, isolated islands of new bone were detected in this connective tissue. Subsequently, neighboring small islands merged to form large islands. In later stages, the primary trabeculae of woven bone were reinforced by layers of regularly deposited lamellar bone. Conclusion: Rabbit calvaria defects treated by guided tissue regeneration heal by ingrowth of woven bone from the defect margins and by formation of bony islands within the defect area. Bone healing showed the histophysiological characteristics of intramembranous bone.

A Multiphase System Bone Implant for Regenerating the Calvaria

The regeneration of bone with a biodegradable, biocompatible carrier and osteo-regenerative proteins may be possible if the carrier has the appropriate architecture to support osteoconduction and prevent soft-tissue prolapse into ablated bone segments. Using radiomorphometry and histomorphometry, our group assessed individually and combined the components of a multiphase system bone implant (MSI) in rabbit cranial defects (critical-size defects: CSDs): two disks of biodegradable polymer (PLG) and demineralized bone matrix (DBM), a recognized osteo-regenerative material. We used 48 rabbits evenly divided between four treatment groups and two time periods. Six weeks after treatment, both DBM and PLG had significant positive effects on bone regeneration (based on a three-factor analysis of variance, p < 0.05). These effects were synergistic when the components were combined (MSI). Defects treated with DBM or PLG alone continued to regenerate bone through 12 weeks. By this time, bone within the defects treated with MSI appeared to be maturing and consolidating. We conclude that the concept of placing osteo-regenerative factors between two biodegradable disks for regenerating segments of calvaria is viable.

Morphometric assessment of fibrin glue in rabbit cranial bone defects

Morphometric assessment of fibrin glue in rabbit cranial bone defects