Marco T. Amarante

A 1-year study of osteoinduction in hydroxyapatite-derived biomaterials in an adult sheep model: part I.

The study presented here investigated hydroxyapatite biomaterials implanted in soft-tissue sites in adult sheep to determine whether these materials are osteoinductive and whether the rate of osteoinduction can be increased by manipulating the composition and porosity of the implants. For the study, 16.8-mm x 5-mm discs were prepared from mixtures of hydroxyapatite and beta-tricalcium phosphate. Five mixtures of hydroxyapatite-ceramic and hydroxyapatite-cement paste forms were studied: 100 percent hydroxyapatite-ceramic (Interpore), 60 percent hydroxyapatite-ceramic, 100 percent hydroxyapatite-cement paste, 60 percent hydroxyapatite-cement paste, and 20 percent hydroxyapatite-cement paste. Biomaterials were implanted in subcutaneous and intramuscular soft-tissue pockets in 10 adult sheep. Cranial bone grafts of equal dimension were implanted as controls. One year after implantation, the volume of all biomaterials and bone grafts was determined from a computed tomographic scan, and porosity and bone formation were determined using backscatter electron microscopy. Cranial bone and the 20 percent hydroxyapatite-cement paste implants demonstrated significant volume reduction in all sites after 1 year (p < 0.001). No significant difference in volume of the remaining four biomaterials was found. There was no significant change in pore size in the ceramic implants (range, 200 to 300 micro) and in the cement-paste implants containing 60 percent hydroxyapatite or more (range, 3 to 5 nm). Pore size in the cement-paste implants containing 20 percent hydroxyapatite increased significantly with resorption of the tricalcium-phosphate component, reaching a maximum of 200 to 300 micro in the periphery, where the greatest tricalcium-phosphate resorption had occurred. Both ceramic biomaterials demonstrated lamellar bone deposition within well-formed haversian systems through the entire depth of the implants, ranging from a mean of 6.6 percent to 11.7 percent. There was minimal bone formation in the cement-paste implants containing 60 percent hydroxyapatite or more. In contrast, cement-paste implants containing 20 percent hydroxyapatite demonstrated up to 10 percent bone replacement, which was greatest in the periphery of the implants where the greatest tricalcium-phosphate resorption had occurred. This study confirms the occurrence of true osteoinduction within hydroxyapatite-derived biomaterials, when examined using backscatter techniques. In this study, the rate of osteoinduction was greatest when a porous architecture was maintained, which was best achieved in ceramic rather than cement-paste forms of hydroxyapatite. Porosity and resultant bone formation in cement-paste implants can be improved by combining hydroxyapatite with a rapidly resorbing component, such as tricalcium phosphate.

A 1-year study of osteoinduction in hydroxyapatite-derived biomaterials in an adult sheep model: part II. Bioengineering implants to optimize bone replacement in reconstruction of cranial defects.

The present study investigated hydroxyapatite biomaterials implanted in critical-size defects in the calvaria of adult sheep to determine the optimal bioengineering of hydroxyapatite composites to facilitate bone ingrowth into these materials. Five calvarial defects measuring 16.8 mm in diameter were made in each of 10 adult sheep. Three defects were filled with cement paste composites of hydroxyapatite and beta-tricalcium phosphate as follows: (1) 100 percent hydroxyapatite-cement paste, (2) 60 percent hydroxyapatite-cement paste, and (3) 20 percent hydroxyapatite-cement paste. One defect was filled with a ceramic composite containing 60 percent hydroxyapatite-ceramic, and the fifth defect remained unfilled. One year after implantation, the volume of all biomaterials was determined by computed tomography, and porosity and bone replacement were determined using backscatter electron microscopy. Computed tomography-based volumetric assessment 1 year after implantation demonstrated that none of the unfilled cranial defects closed over the 1-year period, confirming that these were critical-size defects. There was a significant increase in volume in both the cement paste and ceramic implants containing 60 percent hydroxyapatite (p < 0.01). There was no significant change in volume of the remaining cement paste biomaterials. Analysis of specimens by backscatter electron microscopy demonstrated mean bone replacement of 4.8 +/- 1.4 percent (mean +/- SEM) in 100 percent hydroxyapatite-cement paste, 11.2 +/- 2.3 percent in 60 percent hydroxyapatite-cement paste, and 28.5 +/- 4.5 percent in 20 percent hydroxyapatite-cement paste. There was an inverse correlation between the concentration of hydroxyapatite and the amount of bone replacement in the cement paste for each composite tested (p < 0.01). Bone replacement in 60 percent hydroxyapatite-ceramic composite (13.6 +/- 2.0 percent) was not significantly different from that in 60 percent hydroxyapatite-cement paste. Of note is that the ceramic composite contained macropores (200 to 300 microm) that did not change in size over the 1-year period. All cement paste composites initially contained micropores (3 to 5 nm), which remained unchanged in 100 percent hydroxyapatite-cement paste. Cement paste implants containing increased tricalcium phosphate demonstrated a corresponding increase in macropores following resorption of the tricalcium phosphate component. Bone replacement occurred within the macropores of these implants. In conclusion, there was no significant bone ingrowth into pure hydroxyapatite-cement paste (Bone Source, Stryker-Leibinger Inc., Dallas, Texas) in the present study. The introduction of macropores in a biomaterial can optimize bone ingrowth for reconstruction of critical-size defects in calvaria. This was demonstrated in both the ceramic composite of hydroxyapatite tested and the cement paste composites of hydroxyapatite by increasing the composition of a rapidly resorbing component such as beta-tricalcium phosphate.

Cyanoacrylate fixation of the craniofacial skeleton : an experimental study

This study examined the feasibility of achieving bone fixation of the upper facial skeleton using n-butyl-2-cyanoacrylate. We compared the fixation obtained with this adhesive to that obtained with plates and screws in an animal model. The stability of fixation of both osteotomies and onlay grafts was studied in six minipigs. Three osteotomies were performed on each side of the skull. Additionally, a parietal split-thickness autograft was fixated to the nasal bone as an onlay. Plates and screws were used on the right and adhesive on the left. The animals were killed at 6 weeks, and both sides were compared biomechanically and histologically. Clinical assessment showed stability of all fragments. No statistical difference was found in the comparison of the maximum torque to failure between analogous plated and glued sides. Radiographic cephalometry revealed no significant displacement of the fragments. Histologic analysis demonstrated bony union. In this study, n-butyl-2-cyanoacrylate was as effective as plates and screws in the fixation of surgically created osteotomies of the upper facial skeleton.

Craniofacial skeletal fixation using biodegradable plates and cyanoacrylate glue.

&NA; This study examined the feasibility of fixation of craniofacial bone using Lactosorb biodegradable plates adhered to bone with butyl‐2‐cyanoacrylate adhesive (Histoacryl) in a pig. The stability and bone‐healing characteristics of this rigid fixation method were studied and compared with standard rigid fixation using metal plates and screws on osteotomy sites in the frontal bones and infraorbital rims. Rectangular osteotomies (2.0 × 3.0 cm) were performed on the right and left sides of the frontal bone and wedge‐shaped osteotomies (1.5 × 1.7 cm) were made on the left and right infraorbital rims in seven Yorkshire pigs. Metal plates were applied with screws to the osteotomies on one side, and the other side was fixed with a biodegradable plate and butyl‐2‐cyanoacrylate. The animals were sacrificed at 8 weeks, and both sides were compared biomechanically and histologically. Radiographic, biomechanical, and histologic analyses were performed to evaluate skeletal stability, contour, accurate positioning of bony fragments, bone healing, and maximum torque to failure of the repair sites. Clinical and radiographic observations demonstrated stability of the bone fragments without any evidence of displacement. According to Students t test for paired data, no statistical difference was found in the maximum torque to failure of fragments fixed with biodegradable plates and glue compared with those fixed with metal plates and screws (p > 0.05), whether or not a gap existed at the osteosynthesis site. Although the sample size was small, no differences were noted between the two types of treatment groups. This study demonstrates that rigid internal fixation of osteotomized cranial bone fragments using biodegradable plates and butyl‐2‐cyanoacrylate is as effective as metal plate and screw fixation in this animal model.

A 1-year Study of Hydroxyapatite-derived Biomaterials in an Adult Sheep Model: Iii. Comparison with Autogenous Bone Graft for Facial Augmentation

Background: The present study investigates onlay bone grafts and implants in a large-animal (sheep) model to determine whether there are composite biomaterials that can maximize long-term facial augmentation when compared with conventional bone grafts. Methods: Facial augmentation was performed in 10 adult sheep. First, 16.8 × 5-mm disks were prepared from autogenous calvarial bone, hydroxyapatite ceramic, ceramic composite of 60 percent hydroxyapatite and 40 percent β-tricalcium phosphate (60 percent hydroxyapatite ceramic), and hydroxyapatite cement paste. Facial recipient sites were the body of the mandible (depository), the maxillary region (resorptive), and the frontal bone (depository). The volume of all bone grafts and implants was determined using computed tomographic scans, and the amount of bone formation was measured by means of backscatter electron microscopy 1 year postimplantation. Results: Cranial bone graft demonstrated a highly significant reduction in volume in all sites studied. Other than a slight decrease in volume of hydroxyapatite cement paste disks applied to the maxillary region, there was no significant change in volume of the biomaterials implanted in any of the remaining recipient sites. Bone replacement was greatest in hydroxyapatite ceramic (23.9 percent) followed by 60 percent hydroxyapatite ceramic (16.4 percent) and least with hydroxyapatite cement paste (4.2 percent). Minimal differences in bone replacement were noted between recipient sites. Conclusions: This study demonstrates that the volume maintenance of onlay hydroxyapatite composites is highly predictable, whereas that of cranial bone graft is unpredictable. Minimal differences were seen in bone replacement within biomaterials between “depository” and “resorptive” facial recipient sites. Ceramic forms of onlay hydroxyapatite implants demonstrated significantly greater bone replacement than did the cement paste forms of hydroxyapatite.

Long-term remodeling of vascularized and nonvascularized onlay bone grafts: a macroscopic and microscopic analysis.

The present study was performed to compare vascularized and nonvascularized onlay bone grafts to investigate the potential effect of graft-to-recipient bed orientation on long-term bone remodeling and changes in thickness and microarchitectural patterns of remodeling within the bone grafts. In two groups of 10 rabbits each, bone grafts were raised bilaterally from the supraorbital processes and placed subperiosteally on the zygomatic arch. The bone grafts were oriented parallel to the zygomatic arch on one side and perpendicular to the arch on the contralateral side. In the first group, vascularized bone grafts were transferred based on the auricularis anterior muscle, and in the second group nonvascularized bone grafts were transferred. Fluorochrome markers were injected during the last 3 months of animal survival, and animals were killed either 6 or 12 months postoperatively. The nonvascularized augmented zygoma showed no significant change in thickness 6 months after bone graft placement and a significant decrease in thickness 1 year after graft placement (p < 0.01). The vascularized augmented zygoma showed a slight but statistically significant decrease in thickness 6 months after graft placement (p < 0.003), with no significant difference relative to its initial thickness 1 year after graft placement. In animals killed 6 months after bone graft placement, both the rate of remodeling and the bone deposition rate measured during the last 3 months of survival were significantly higher in the vascularized bone grafts compared with their nonvascularized counterparts (p < 0.02). By 1 year postoperatively, there were no significant differences in thickness, mineral apposition rate, or osteon density between bone grafts oriented perpendicular and parallel to the zygomatic arch. These findings indicate that the vascularity of a bone graft has a significant effect on long-term thickness and histomorphometric parameters of bone remodeling, whereas the direction of placement of a subperiosteal graft relative to the recipient bed has minimal effect on these parameters. In vascularized bone grafts, both bone remodeling and deposition are accelerated during the initial period following graft placement. Continued bone deposition renders vascularized grafts better suited for the long-term maintenance of thickness and contour relative to nonvascularized grafts.

Regulation of osteogenesis and survival within bone grafts to the calvaria: The effect of the dura versus the pericranium

Background: The present study evaluates the isolated role of dura and pericranium in the survival of fresh (osteoblasts viable) and frozen (osteoblasts nonviable) bone grafts. Methods: Bilateral craniectomies were performed in 48 mature rabbits. On one side, bone was replaced immediately; on the contralateral side, it was flash-frozen before replacement. Animals were randomized into four groups by placement of Silastic barriers adjacent to bone grafts, as follows: (1) control (no barriers); (2) dural barrier; (3) pericranial barrier; and (4) double (dural and pericranial) barriers. Fluorescein labels were injected at specified intervals, with animals euthanized after 1 or 10 weeks. Results: After 1 week, fresh grafts without dural barriers demonstrated greater fluorescein labeling on the dural than on the pericranial surface (p < 0.05); in contrast, fresh grafts without pericranial barriers had no statistical difference in fluorescein labeling between pericranial and dural surfaces. After 10 weeks, the new bone area was greater in fresh than in frozen grafts (p < 0.05). Total new bone area and dural-side new bone were greater in grafts without dural barriers (p < 0.001); this was not seen in grafts without pericranial barriers. Pericranial new bone was greatest in fresh grafts without a pericranial barrier (p < 0.001); this was not seen in frozen grafts. Conclusions: The dura and pericranium each contributed to osteogenesis, although dural contact was more effective. Maintenance of dural contact enhanced osteogenesis through the entire graft, whereas pericranial contact enhanced osteogenesis only on the pericranial surface of fresh grafts. These data suggest dura is largely responsible for cranial graft survival.

Effects of laser versus scalpel tenolysis in the rabbit flexor tendon

&NA; The use of surgical lasers has been shown to decrease adhesion formation as compared with scalpel control groups in various surgical procedures. The potential benefits of laser technology have not been assessed in the treatment of adherent tendons. The current study was designed to first develop a reliable and reproducible model for consistent adhesion formation following flexor tendon trauma. The second goal was to compare the effects of laser tenolysis procedures on tendon gliding with those of traditional scalpel tenolysis. In phase I. the adhesion‐induction model utilized bilateral standardized crush‐abrasion injuries to the hind limb digital flexor tendons of New Zealand White rabbits. Following 4 weeks of immobilization, the animals were sacrificed, and peritendoneal adhesions were assessed biomechanically. A significantly higher maximal force was required to extract the adherent tendons from the foot as compared with nontraumatized control tendons. In phase II. six groups of animals underwent the same standardized tendon trauma. Four weeks later the rabbits were randomly assigned to undergo either CO2 laser or holmium: YAG laser tenolysis on one foot. Scalpel lysis was used on the contralateral foot and served as an intraindividual control. Biomechanical assessment was performed at 1. 2, and 4 weeks following tenolysis. Significantly less force was required to extract the treated tendons at 1 and 2 weeks following holmium: YAG laser tenolysis when compared with scalpel or CO2 laser tenolysis. After 4 weeks, differences between holmium: YAG and CO2 laser and scalpel treatment were no longer significant. Extracted tendons were pulled apart to failure, and no difference in breaking strength was noted between groups. We conclude that holmium: YAG laser tenolysis results in easier tendon gliding as compared with scalpel or CO2 laser tenolysis at early time points. Laser tenolysis does not affect intrinsic tendon strength.

Biomechanical Evaluation of the Canine and Porcine Models for Experimental Craniofacial Surgery

This investigation compared the variation of the biomechanical properties of canine and porcine craniofacial bones in homotypical (same site in opposite sides of an animal) and heterotypical (same site in different animals) sites. Biomechanical analysis is a reliable method to assess bone healing, because fracture repair correlates closely with the changes in biomechanical properties. Paired bone fragments were harvested in nine dogs and nine minipigs from each side of the skull from three different sites—the frontal bone, the supraorbital rim, and the zygomatic arch—and submitted to torque to failure. Maximum torque, stiffness, and toughness were recorded and comparative analysis performed. A normal range of variation between paired craniofacial bones in two useful animal models is provided. The results showed that the variability between homotypic left and right sides was not significant, whereas the variability between heterotypic sites in separate animals was. Maximum torque was the most reliable of the three parameters considered, because the data fell over a much narrower range. We conclude that the use of the contralateral side is a valid control in experimental procedures that may alter the biomechanical properties of one side.