Non-viral-mediated gene therapy approaches for bone repair.

OBJECTIVES: Bone repair strategies continue to be developed for alternatives to autografting, allogeneic implants of banked bone, and other bone substitutes. Efforts have included the delivery of potent growth and/or differentiation factors and the use of gene therapy. For bone regeneration, gene therapy is the delivery, uptake and expression of DNA that has been localized to a wound bed. The objective of the current study is to investigate methods to enhance non-viral-mediated means of gene uptake and expression for use in bone regeneration. METHODS: Several types of DNA-polymer complexes, either applied directly to baby hamster kidney (BHK) cells, or released from a porous, resorbable gene-activated matrix (GAM), were evaluated in vitro for their ability to transfect cells with a circular plasmid DNA construct expressing green fluorescent protein. Complexes included conjugates containing a lipophilic reagent, liposomes, poly-ethyl-oxazoline, and poly-ethyleneimine (PEI). Data were subjected to analysis of variance and Fisher's protected least significant difference for multiple comparisons with significance established at p < 0.05. RESULTS: Transfection efficiencies of the liposome and PEI complexes improved in vitro when released from resorbable GAMs. The lipophilic reagent FuGene 6 demonstrated abundant uptake and expression in the initial 1- and 2-day evaluation periods. In contrast, the DNA-liposome and PEI GAM complexes demonstrated a sustained release, uptake and expression by the BHK cells at the 2-, 4-, and 7-day, and 4- and 7-day evaluation intervals, respectively. CONCLUSION: GAM technology appears to improve the functional stability and release duration of incorporated DNA-polymer complexes in the present in vitro studies. The ongoing objective of our research is to develop a localized treatment to improve the uptake and expression of plasmid DNA by non-viral-mediated gene therapy.

A comparative analysis of the microarchitecture of cortical membranous and cortical endochondral onlay bone grafts in the craniofacial skeleton.

Previous work in this laboratory established that an onlay bone graft's survival is determined primarily by its relative cortical and cancellous composition rather than its embryologic origin. A volumetric analysis of external bone graft resorption, however, does not explain the internal microarchitectural changes that may be occurring as these grafts become incorporated. To expand the knowledge of bone graft dynamics beyond volumetric parameters, a better understanding of the internal processes of bone graft remodeling is needed. In this comparative study of cortical onlay bone graft microarchitecture, the authors propose to show that cortical onlay bone grafts undergo measurable internal microarchitectural changes as they become incorporated into the surrounding craniofacial skeleton. In addition, the authors propose to further demonstrate similarities between the internal microarchitecture of cortical onlay bone grafts of different embryologic origin over time. Twenty-five adult New Zealand White rabbits were used for this study. They were divided into two groups of eight animals and one group of nine. The groups were killed at 3, 8, and 16 weeks. Cortical membranous and endochondral bone grafts were placed subperiosteally onto each rabbit's cranium. In addition, five ungrafted cortical endochondral and membranous bone specimens were used as controls. Microcomputed tomography (MCT) scanning and histomorphometric analysis were performed on all of the specimens to obtain detailed information regarding the microarchitecture of the cortical bone grafts. The parameters of bone volume fraction, bone surface area to volume, mean trabecular number, and anisotropy were used to give quantitative information about a bone's micro-organization. The results showed that there is no statistically significant difference between the cortical endochondral and the cortical membranous bone grafts for bone volume fraction, bone surface to volume, mean trabecular number, and anisotropy measurements for all time points. There were, however, statistically significant differences when comparing the control and 3-week groups to the 16-week group for all parameters. The advanced MCT technology and histomorphometric techniques proved to be effective in providing a qualitative and quantitative ultrastructural comparison of cortical endochondral and membranous onlay bone grafts over time. In this study, a statistically significant change in the internal microarchitecture of cortical onlay bone grafts of different embryologic origins was seen as they were remodeled and resorbed at all time points. Specifically, the onlay cortical bone grafts developed a less dense, more trabecular, and less organized internal ultrastructure. In addition, no difference in the three-dimensional ultrastructure of cortical endochondral and membranous bone was found. These results challenge some of the currently accepted theories of bone-graft dynamics and may eventually lead to a change in the way clinicians approach bone-graft selection for craniofacial surgery.

Subjective and objective assessment of the temporalis myofascial flap in previously operated temporomandibular joints.

PURPOSE: This study evaluated the subjective and objective findings in patients who had undergone temporomandibular joint (TMJ) reconstruction with a temporalis myofascial flap. All joints had previously been reconstructed with alloplastic, allogeneic, or autogenous material. PATIENTS AND METHODS: Twenty-three consecutive patients who underwent 28 temporalis myofascial flap procedures were assessed subjectively and objectively preoperatively and at an average of 36 months postoperatively. Panoramic radiographs, magnetic resonance imaging (MRI), or coronal computed axial tomography scans (CT scans) were performed on all patients preoperatively to evaluate for joint disease. A visual analog scale (VAS) was used to assess pain preoperatively and postoperatively. Patients also reported their use of pain medication, ability to function, diet, complications, and overall satisfaction. Preoperative and postoperative objective assessment consisted of an evaluation of range of motion, deviation on opening, joint noise on function, and cosmesis. RESULTS: On preoperative radiographic examination, 24 of 28 joints showed signs of bony degeneration, including cortical erosion, condylar flattening, and joint space alterations. Four joints showed evidence of ankylosis. The average preoperative maximal interincisal opening (MIO) was 23.7 mm, and the postoperative average was 32.3 mm (P<.05). Preoperatively, all patients displayed one or more objective clinical signs of joint disease such as joint noise on function, deviation on opening, limited mouth opening (less than 20 mm), or limited excursions (less than 2 mm). Postoperatively, 65% displayed one or more of these signs, a significant reduction (P<.05). Preoperatively, the average pain score was 8.2 on the VAS, and postoperatively the average pain score was 3.4 (P<.0005). Fifteen patients used less pain medication postoperatively, 7 used the same amount, and 1 patient used more. Thirteen patients were very satisfied with the overall results of the surgery, 4 were satisfied, and 4 were not satisfied. Two patients were satisfied with their increased function but were not satisfied with their pain reduction. All were satisfied with their cosmetic appearance. Minor complications after the procedure included 3 patients who had preauricular paresthesia and 1 who had an intraoperative dura mater exposure without sequelae. Two patients had postoperative superficial suture infections, and 2 noted hearing changes that were found to be clinically insignificant by audiologic examination. CONCLUSION: The temporalis myofascial flap is an autogenous graft that has the advantages of close proximity to the temporomandibular joint, minimal surgical morbidity, and successful clinical results. It was found to be a valuable option for TMJ reconstruction in joints in which alloplastic, allogeneic, or autogenous materials have previously been placed unsuccessfully.

The ultrastructure and resorptive pattern of cancellous onlay bone grafts in the craniofacial skeleton.

The authors' laboratory has shown cancellous onlay bone grafts to resorb faster than cortical grafts. To understand the nature of cancellous bone grafts beyond volumetric measurements, a temporal analysis of the internal microarchitecture of these grafts was performed. Their hypothesis is that the forces of remodeling and resorption cause cancellous onlay bone grafts to develop a denser, more interconnected, and a more mechanically stable microarchitecture. Twenty-five adult New Zealand White rabbits were used in this study and were divided into three groups. Microcomputed tomography (MCT) was performed on all cancellous bone grafts to obtain detailed information regarding the microarchitecture of the cancellous bone. Bone graft specimens were examined histologically, and histomorphometric analysis was also performed. Their results show that cancellous onlay bone grafts develop a higher bone volume fraction, mean trabecular thickness, connectivity, and degree of anisotropy. Furthermore, cancellous onlay bone grafts developed a lower bone surface area-to-volume ratio and mean trabecular separation. The unique combination of MCT technology and histomorphometric techniques proved to be effective in providing a qualitative and quantitative ultrastructural analysis of cancellous onlay bone grafts over time. The authors were able to show changes in the internal microarchitecture of cancellous onlay bone grafts as they were remodeled and resorbed. Specifically, they found the cancellous onlay bone grafts to develop a more dense, less trabecular, more organized, and more interconnected internal ultrastructure over time. Their findings have helped to provide a reproducible description of the temporal sequence of changes in bone microarchitecture, revascularization, and internal remodeling.

Investigation of the influences of biomechanical force on the ultrastructure of human sagittal craniosynostosis.

This study presents comparisons of the ultrastructure of synostotic and open portions of synostotic sagittal sutures using histomorphometry, scanning electron microscopy, and microcomputed tomography. By using stereologic and histomorphometric analysis, this study proposes to demonstrate evidence of the influence of biomechanical force on the suture during the process of sagittal craniosynostosis. Finally, we propose to link the pathologic changes transforming normal suture fusion to craniosynostosis with concurrent changes in the polarity of suture fusion initiation. Seven infants (four boys and three girls) with sagittal craniosynostosis, ranging in age from 1.4 to 4.8 months (mean = 3.0 months), underwent sagittal synostectomies. The synostotic bone specimens were sectioned into three regions: an open suture, partial synostosis, and complete synostosis. Microcomputed tomographic and scanning electron microscopic scanning as well as histomorphometry was performed on all specimens to obtain detailed qualitative and quantitative information regarding the trabecular microarchitecture of the synostosed suture. Microcomputed tomographic analysis determined the bone volume fraction, trabecular thickness, trabecular separation, bone surface to bone volume ratio, and anisotropy for all specimens. Our results showed significant differences in all of these quantitative measurements when comparing the complete synostotic suture with the open portion of the synostotic sutures (p < 0.05). Microcomputed tomographic stereologic analysis showed evidence of the influence of biomechanical force on the synostotic and open portions of the synostotic sutures. Results of scanning electron microscopy show a definite qualitative difference in the trabecular pattern of the partial and complete synostotic suture when compared with the open portion of the synostotic sagittal suture. In this study, we performed both qualitative and quantitative comparisons of the ultrastructure of the complete synostotic and nonsynostotic sagittal sutures using stereologic and histomorphometric techniques. We also demonstrated evidence of the influence of biomechanical force on the synostotic sagittal suture. Finally, we established a link between the pathologic changes transforming normal suture fusion to craniosynostosis and concurrent changes in both the vector and direction of suture fusion initiation.

Volume maintenance of onlay bone grafts in the craniofacial skeleton: micro-architecture versus embryologic origin.

The superior volume maintenance of membranous over endochondral bone has been shown in several studies and provides the basis for its preferred clinical use as an onlay grafting material in the craniofacial skeleton. The scientific rationale for this seeming embryologic advantage, however, has never been proven. Our hypothesis is that the pattern of onlay bone graft resorption is primarily determined by a graft's micro-architecture (relative cortical and cancellous composition) rather than its embryologic origin (membranous versus endochondral). Twenty-five adult New Zealand, White rabbits were used for this study. Eight animals were killed at 3 weeks, eight animals at 8 weeks, and nine animals at 16 weeks. Three graft types were placed onto each rabbit cranium: cortical bone graft of membranous origin and cortical and cancellous bone graft of endochondral origin. Fluorochrome markers were injected into all living rabbits at 1, 6, and 14 weeks. Microcomputed tomography scanning was performed on all of the bone grafts to determine postsacrifice volumes and to obtain detailed information regarding the bone graft's trabecular architecture. In addition, specimens were examined histologically. Volume analysis showed a statistically greater resorption rate in the cancellous endochondral bone graft than in either the endochondral or membranous cortical bone grafts (p < 0.05) for all time points. In addition there was no significant difference in the resorption rates between the endochondral and membranous cortical bone grafts. A post-test power analysis (alpha = 5 percent) of the volume data comparing the two types of cortical bone grafts showed that a difference in resorption of 8.9 percent would be detected with a 90-percent probability. Previous studies, which have shown a seeming superiority of membranous over endochondral bone grafts, used composite grafts composed of both cortical and cancellous portions. By separating these components, we have shown that cortical bone grafts maintain their volumes significantly better than cancellous bone grafts. In addition, we found no statistical difference in the resorption rates between the two cortical onlay bone grafts of different embryologic origins, a finding that has never been previously published. From our results, we believe cortical bone to be a superior onlay grafting material, independent of its embryologic origin. We believe these results challenge the currently accepted theories of bone graft dynamics and may lead to a change in the way clinicians approach bone graft selections for craniofacial surgery.

Biomechanical study of sternal closure using rigid fixation techniques in human cadavers.

BACKGROUND: We believe rigid plate fixation may be superior to wire fixation in sternal closure, as rigid fixation used in the craniofacial skeleton has shown greater stability, lower postoperative pain, and accelerated bone healing. We hypothesize that sterna fixed with titanium plates are more stable mechanically than sterna fixed with wires. METHODS: The sterna from human cadavers were used in this two-phased study. Phase I compared wires to four-hole titanium straight plates. Phase II compared wires to four-hole titanium custom H plates. The sterna were tested biomechanically using all fixation methods. RESULTS: Phase I showed no statistically significant difference in the stiffness or lateral displacement between the wired and straight plated sterna. Phase II showed a statistically significant greater stiffness (p < 0.05) and less lateral displacement (p < 0.05) in the custom plated sterna over the wired sterna. CONCLUSIONS: Our results showed that custom titanium H plates were superior to wire fixation. Furthermore, our results established the importance of plate configuration in sternal fixation. Our study may have beneficial clinical implications, as decreased motion at the sternotomy site could mean less postoperative pain, a decreased incidence of infection, and accelerated bone healing.

Use of scanning electron microscopy in the evaluation of craniosynostosis.

The cause of craniosynostosis continues to elude researchers. Although several studies have looked at the ultrastructure of normal suture closure, no previous studies have examined the microarchitecture of the synostotic suture. Our goal was to assess the scanning electron microscope (SEM) as a viable and useful tool in examining craniosynostosis. Our hypothesis is that the SEM is a powerful analytical tool that can evaluate nonsynostotic, partial synostotic, and complete synostotic cranial sutures. We analyzed the cranial suture of 3 human infants with nonsyndromic sagittal craniosynostosis. The specimens were separated into three groups, which included regions of partial and complete synostosis and a region of open suture. Histological examination provided cellular and tissue data about craniosynostosis, whereas the SEM provided detailed information regarding the trabecular microarchitecture of the synostosed suture. The SEM produced quality images of complete and partially synostotic sutures and open sutures. At low magnification, the SEM characterized the general bony microarchitecture of cranial sutures in a manner different from, but complementary to, standard histological sections. At higher magnification, the SEM allowed us a look at the cellular population of craniosynostotic sutures in a way that surpasses standard light microscopy. The SEM is an excellent tool for the study of craniosynostosis and has proved invaluable in our ability to evaluate the microarchitecture and cellular population of the fusing suture. We believe we have proven our hypothesis by demonstrating the SEM to be a powerful analytical tool that can evaluate nonsynostotic, partial synostotic, and complete synostotic cranial sutures.