Macrophage Transplantation Fails to Improve Repair of Critical-Sized Calvarial Defects.

INTRODUCTION: Over 500,000 bone grafting procedures are performed every year in the United States for neoplastic and traumatic lesions of the craniofacial skeleton, costing $585 million in medical care. Current bone grafting procedures are limited, and full-thickness critical-sized defects (CSDs) of the adult human skull thus pose a substantial reconstructive challenge for the craniofacial surgeon. Cell-based strategies have been shown to safely and efficaciously accelerate the rate of bone formation in CSDs in animals. The authors recently demonstrated that supraphysiological transplantation of macrophages seeded in pullalan-collagen composite hydrogels significantly accelerated wound healing in wild type and diabetic mice, an effect mediated in part by enhancing angiogenesis. In this study, the authors investigated the bone healing effects of macrophage transplantation into CSDs of mice. METHODS: CD1 athymic nude mice (60 days of age) were anesthetized, and unilateral full-thickness critical-sized (4 mm in diameter) cranial defects were created in the right parietal bone, avoiding cranial sutures. Macrophages were isolated from FVB-L2G mice and seeded onto hydroxyapatite-poly (lactic-co-glycolic acid) (HA-PLGA) scaffolds (1.0 × 10 cells per CSD). Scaffolds were incubated for 24 hours before they were placed into the CSDs. Macrophage survival was assessed using three-dimensional in vivo imaging system (3D IVIS)/micro-CT. Micro-CT at 0, 2, 4, 6, and 8 weeks was performed to evaluate gross bone formation, which was quantified using Adobe Photoshop. Microscopic evidence of bone regeneration was assessed at 8 weeks by histology. Bone formation and macrophage survival were compared at each time point using independent samples t tests. RESULTS: Transplantation of macrophages at supraphysiological concentration had no effect on the formation of bones in CSDs as assessed by either micro-CT data at any time point analyzed (all P > 0.05). These results were corroborated by histology. 3D IVIS/micro-CT demonstrated survival of macrophages through 8 weeks. CONCLUSION: Supraphysiologic delivery of macrophages to CSDs of mice had no effect on bone formation despite survival of transplanted macrophages through to 8 weeks posttransplantation. Further research into the physiological effects of macrophages on bone regeneration is needed to assess whether recapitulation of these conditions in macrophage-based therapy can promote the healing of large cranial defects.

Comparative finite element analysis of skull mechanical properties following parietal bone graft harvesting in adults.

INTRODUCTION: Parietal bone grafts are commonly used in cranio-maxillo-facial surgery. Both the outer and the internal layer of the calvarium can be harvested. The bone defect created by this harvesting may induce significant weakening of the skull that has not been extensively evaluated. Our aim was to evaluate the consequences of parietal bone graft harvesting on mechanical properties of the skull using a finite element analysis. METHODS: Finite elements models of the skull of 3 adult patients were created from CT scans. Parietal external and internal layer harvest models were created. Frontal, lateral, and parietal loading were modeled and von Mises stress distributions were compared. RESULTS: The maximal von Mises stress was higher for models of bone harvesting, both on the whole skull and at the harvested site. Maximal von Mises stress was even higher for models with internal layer defect. CONCLUSIONS: Harvesting parietal bone modifies the skull's mechanical strength and can increase the risk of skull fracture, mainly on the harvested site. Outer layer parietal graft harvesting is indicated. Graft harvesting located in the upper part of the parietal bone, close to the sagittal suture and with smooth internal edges and corners should limit the risk of fracture.

How high level of anxiety in Panic Disorder can interfere in working memory? A computer simulation and electrophysiological investigation.

Panic disorder (PD) is characterized by repeated and unexpected attacks of intense anxiety, which are not restricted to a determined situation or circumstance. The coherence function has been used to investigate the communication among brain structures through the quantitative EEG (qEEG). The objective of this study is to analyze if there is a difference in frontoparietal gamma coherence (GC) between panic disorder patients (PDP) and healthy controls (HC) during the Visual oddball paradigm; and verify if high levels of anxiety (produced by a computer simulation) affect PDP's working memory. Nine PDP (9 female with average age of 48.8, SD: 11.16) and ten HC (1 male and 9 female with average age of 38.2, SD: 13.69) were enrolled in this study. The subjects performed the visual oddball paradigm simultaneously to the EEG record before and after the presentation of computer simulation (CS). A two-way ANOVA was applied to analyze the factors Group and the Moment for each pair of electrodes separately, and another one to analyze the reaction time variable. We verified a F3-P3 GC increased after the CS movie, demonstrating the left hemisphere participation during the anxiety processing. The greater GC in HC observed in the frontal and parietal areas (P3-Pz, F4-F8 and Fp2-F4) points to the participation of these areas with the expected behavior. The greater GC in PDP for F7-F3 and F4-P4 pairs of electrodes assumes that it produces a prejudicial "noise" during information processing, and can be associated to interference on the communication between frontal and parietal areas. This "noise" during information processing is related to PD symptoms, which should be better known in order to develop effective treatment strategies.

Biomechanical Studies on Patterns of Cranial Bone Fracture Using the Immature Porcine Model.

This review was prepared for the American Society of Mechanical Engineers Lissner Medal. It specifically discusses research performed in the Orthopaedic Biomechanics Laboratories on pediatric cranial bone mechanics and patterns of fracture in collaboration with the Forensic Anthropology Laboratory at Michigan State University. Cranial fractures are often an important element seen by forensic anthropologists during the investigation of pediatric trauma cases litigated in courts. While forensic anthropologists and forensic biomechanists are often called on to testify in these cases, there is little basic science developed in support of their testimony. The following is a review of studies conducted in the above laboratories and supported by the National Institute of Justice to begin an understanding of the mechanics and patterns of pediatric cranial bone fracture. With the lack of human pediatric specimens, the studies utilize an immature porcine model. Because much case evidence involves cranial bone fracture, the studies described below focus on determining input loading based on the resultant bone fracture pattern. The studies involve impact to the parietal bone, the most often fractured cranial bone, and begin with experiments on entrapped heads, progressing to those involving free-falling heads. The studies involve head drops onto different types and shapes of interfaces with variations of impact energy. The studies show linear fractures initiating from sutural boundaries, away from the impact site, for flat surface impacts, in contrast to depressed fractures for more focal impacts. The results have been incorporated into a "Fracture Printing Interface (FPI)," using machine learning and pattern recognition algorithms. The interface has been used to help interpret mechanisms of injury in pediatric death cases collected from medical examiner offices. The ultimate aim of this program of study is to develop a "Human Fracture Printing Interface" that can be used by forensic investigators in determining mechanisms of pediatric cranial bone fracture.

The Impact of Age Upon Healing: Absolute Quantification of Osteogenic Genes in Calvarial Critical-Sized Defects.

BACKGROUND: The current study was performed to elucidate changes in growth factor expression over time in critical-sized calvarial defects in rats from infancy to skeletal maturity. MATERIALS AND METHODS: Critical-sized parietal defects of 5, 6, and 8 mm were created in postnatal day 6 (P6), postnatal day (P20), and postnatal day (P84) adult rats, respectively. Dura was harvested at 3, 7, or 14 days after surgery, and serial micro-computed tomography imaging was performed through 12 weeks postoperatively. Absolute quantitative polymerase chain reaction was performed for Bone Morphogenic Protein-2 (BMP-2), Fibroblast Growth Factor-2 (FGF-2), Insulin-like Growth Factor-1 (IGF-1), and Transforming Growth Factor-ß1 (TGF-ß). RESULTS: The P6 (6-d-old) rats showed the greatest difference in gene expression between the dura derived from the defect side and the dura derived from the control side, demonstrating significant differences in TGF-ß1, BMP-2, IGF-1, and FGF-2 at various time intervals. Absolute gene expression in the defect dura was highest in the P6 rats and declined with age. Significant differences were noted at limited time points in the P20 rats for TGF-ß1 and BMP-2 as well as in the P84 rats for TGF-ß1. TGF-ß1 was the only gene studied that showed significant differences at postoperative days 3, 7, and 14 in varying age groups. CONCLUSIONS: The P6 rats have a higher osteogenic potential accompanied by a more vigorous alteration in growth factor expression compared with the P20 or P84 rats. Decrease in BMP-2 and FGF-2 as well as relative increase in TGFß-1 messenger RNA were observed in healing defects. These data provide valuable insight into the mechanism of healing of critical-sized defects and may be of use to engineer factor-releasing implants to correct skull defects.

John Howship (1781-1841) and growing skull fracture: historical perspective.

In the late 18th and early 19th centuries, Dr. John Howship, a pioneering British surgeon, described the clinical features and pathophysiology of various surgical disorders of the human body. His critical contributions to pediatric neurosurgery came in 1816 when he first described the features of an important childhood condition following head trauma, what he referred to as parietal bone absorption. This condition as depicted by Dr. Howship was soon to be christened by later scholars as traumatic cephalhydrocele, traumatic meningocele, leptomeningeal cyst, meningocele spuria, fibrosing osteitis, cerebrocranial erosion, and growing skull fracture. Nevertheless, the basic features of the condition as observed by Dr. Howship were virtually identical to the characteristics of the above-mentioned disorders. This article describes the life and accomplishments of Dr. Howship and his contributions to the current understanding of growing skull fracture.

Statistical analysis of biomechanical properties of the adult sagittal suture using a bending method in a Japanese forensic sample.

This study examined the mechanical properties of the adult sagittal suture compared with surrounding parietal bones using bending tests and investigated the association between the mechanical properties of the suture and age. We used the heads of 116 Japanese cadavers (76 male cadavers and 40 female cadavers) of known age and sex. A total of 1160 cranial samples, 10 from each skull, were collected. The samples were imaged using multidetector computed tomography, and the sample thickness at the center of each sample (ST) was measured. The failure stress of each sample (FS) was measured by a bending test, and the ratio of failure stress to the square of sample thickness (FS/ST(2)) was calculated. Statistical analyses revealed that the FS and FS/ST(2) values were significantly lower at all suture sites than at all bone sites regardless of sex. There were not significant but slight positive correlations between age and FS and FS/ST(2) values at any suture site in male samples. In female samples, age had significant positive correlations with FS and FS/ST(2) values at the middle suture sites, whereas there were not significant but slight positive correlations between age and FS and FS/ST(2) values at the edges of the suture. Statistical analyses also demonstrated that FS and FS/ST(2) values were significantly greater in male samples than in female samples at the middle suture sites. These findings suggest that the bending strength of the adult sagittal suture is significantly lower than that of surrounding parietal bones. Therefore, avoiding direct impact on cranial sutures may be important for preventing skull fractures and severe complications that can cause death. The results of this study also revealed that the bending strength of the middle sagittal suture significantly increases with age in only female samples, whereas the bending strength is significantly higher in male samples than in female samples at the middle suture sites, indicating the possibility of sex difference in the bony interdigitation of the sutures during childhood.

Efficacy of bone healing in calvarial defects using piezoelectric surgical instruments.

OBJECTIVE: This study compared bone healing following the use of 2 piezoelectric surgery units or conventional mechanical cutting with carbide and diamond drills to explore their future applications for bone surgery. METHODS: Subcritical-size (approximately 1.5-2 mm) calvarial defects were created in the parietal bones of adult mice. Following defect standardization, a full-thickness semicircular defect was created on the parietal bones of 12 mice divided into 4 groups: carbide bur, Surgystar, diamond bur, and Piezoelectric System. Hard tissue healing was assessed using micro-computed tomography at 1 day, 2 weeks, 4 weeks, and 8 weeks after surgery. RESULTS: At 4 weeks, the Surgystar group and Piezoelectric System group showed a significant difference from the carbide group. The Surgystar and Piezoelectric System groups did differ from the diamond group. At 8 weeks, the Surgystar and Piezoelectric System groups differed significantly from the carbide and diamond groups. The fraction of healing results over the 8 weeks demonstrated that the Surgystar group had a significantly higher bone healing percentage than did the carbide group (P = 0.001) and the diamond group (P = 0.026), but it did not differ significantly from the Piezoelectric System group (P = 0.420). CONCLUSIONS: The Surgystar and Piezoelectric System are suitable for bone osteotomy and provide faster bone healing in comparison with mechanical instrumentation.

Negative pressure technology enhances bone regeneration in rabbit skull defects.

BACKGROUND: Bone is a slowly regenerating tissue influenced by various physiological processes, including proliferation, differentiation, and angiogenesis, under the control of growth factors. Shortening this healing time is an important and popular clinical research focus in orthopedics. Negative pressure can stimulate angiogenesis, improve blood circulation, promote granulation tissue growth and accelerate tissue wound healing. We sought to determine whether negative pressure could reduce bone healing time in a rabbit cranial defect model. METHODS: Four symmetrical holes (diameter, 3.5 mm) were drilled into the skulls of 42 New Zealand white rabbits, with two holes in each parietal bone. For each rabbit, the two sides were then randomly assigned into experimental and control groups. Using negative pressure suction tubes, experimental holes were treated with -50 kPa for 15 minutes, four times per day, whereas the control holes remained untreated. After 4 weeks, the negative pressure suction tubes were removed. At 2, 4, 6 and 8 weeks, three-dimensional (3D) reconstruction computed tomography (CT), X-ray radiopacity, and two-photon absorptiometry were used to evaluate new bone formation. Histological changes were determined by hematoxylin and eosin (H.E) staining. At weekly intervals until 6 weeks, the mRNA expression levels of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2 were evaluated by RT-PCR. A paired student's t-test was employed to compare X-ray radiopacity and bone density measurements between the experimental and control groups. RESULTS: 3D-reconstruction CT showed that new bone regeneration in the experimental group was greater than that in the control group at 4 and 6 weeks. At these time points, the experimental group presented with higher X-ray radiopacity and increased bone density (P < 0.05) as compared with the control group. Cartilage islands and new bone were observed by H.E staining at 2 weeks in the experimental group. By 6 weeks, the new bone had matured into lamellar bone in the experimental group. RT-PCR results showed that VEGF and BMP-2 were highly expressed in the experimental group as compared with control. CONCLUSIONS: Intermittent negative pressure can promote the regeneration of bone possibly by enhancing the expression of VEGF and BMP-2.

Micro-CT observation of angiogenesis in bone regeneration.

OBJECTIVES: Restoration of an adequate blood supply is essential for the bone healing process and is key to the success of bone augmentation procedures. In this study, we evaluated angiogenesis in rat calvarial flat bone defects using in vivo microfocus computed tomography (micro-CT). MATERIALS AND METHODS: Twenty rats were used. The calvarium was exposed and calvarial bone defects of critical (5-mm diameter) and non-critical (2.7-mm diameter) sizes were prepared. Bone regeneration and angiogenesis were evaluated by image analysis using micro-CT and histological examination. RESULTS: Critical- and non-critical-sized calvarial bone defects showed bone regeneration and angiogenesis around the midsagittal suture. Critical-sized calvarial bone defects showed approximately 1.2% reossification of the original surgical defect, whereas the non-critical-sized defects showed approximately 43.3% reossification at day 28. Furthermore, angiogenesis was observed later in the critical-sized calvarial bone defects (about 38.2%), whereas angiogenesis was observed early in the non-critical-sized calvarial bone defects (about 75.5%) at day 28. New blood vessel networks were observed around defects of both sizes. CONCLUSIONS: Angiogenesis preceded bone regeneration around critical- and non-critical-sized calvarial bone defects. Angiogenesis led to full bone formation in non-critical-sized defects.

Reharvested cranial particulate bone graft ossifies inlay calvarial defects.

Particulate bone graft (PBG) heals calvarial critical-size defects and is procured from the cranium with a hand-driven bit and brace. The donor sites ossify, and thus PBG potentially could be reharvested from the original areas. The purpose of this study was to determine if PBG obtained from a healed donor site is effective for inlay cranioplasty. A 17 × 17-mm critical-size defect was created in the parietal bones of 8 rabbits and treated with either no implant (group 1) or PBG harvested from the frontal bone (group 2). In 4 animals (group 3), a parietal defect was not created initially; PBG was harvested from the frontal bone and then discarded. Sixteen weeks later after the PBG donor sites had healed, a 17 × 17-mm parietal defect was made and filled with PBG reharvested from the previous donor area. Animals underwent micro-computed tomography 16 weeks after inlay cranioplasty. Critical-size defects in controls (group 1) exhibited partial ossification (35.1% ± 10.5%) compared with those treated with PBG (group 2) (99.1% ± 1.5%) or reharvested PBG (group 3) (99.3% ± 1.5%) (P = 0.02). No difference was found between groups 2 and 3 (P = 0.69). Bony thickness was similar in defects implanted with PBG (1.8 mm ± 1.1 mm) or reharvested PBG (2.1 mm ± 0.5 mm) (P = 0.68). Particulate bone graft reharvested from healed donor sites ossifies inlay cranial defects. Because the donor area for PBG is of partial thickness and less than critical size, reparative osteogenesis theoretically allows an unlimited supply of autologous bone for inlay cranioplasty using PBG.

Changes in biomechanical strain and morphology of rat calvarial sutures and bone after Tgf-ß3 inhibition of posterior interfrontal suture fusion.

Craniofacial sutures are bone growth fronts that respond and adapt to biomechanical environments. Little is known of the role sutures play in regulating the skull biomechanical environment during patency and fusion conditions, especially how delayed or premature suture fusion will impact skull biomechanics. Tgf-ß3 has been shown to prevent or delay suture fusion over the short term in rat skulls, yet the long-term patency or its consequences in treated sutures is not known. It was therefore hypothesized that Tgf-ß3 had a long-term impact to prevent suture fusion and thus alter the skull biomechanics. In this study, collagen gels containing 3 ng Tgf-ß3 were surgically placed superficial to the posterior interfrontal suture (IFS) and deep to the periosteum in postnatal day 9 (P9) rats. At P9, P24, and P70, biting forces and strains over left parietal bone, posterior IFS, and sagittal suture were measured with masticatory muscles bilaterally stimulated, after which the rats were sacrificed and suture patency analyzed histologically. Results demonstrated that Tgf-ß3 treated sutures showed less fusion over time than control groups, and strain patterns in the skulls of the Tgf-ß3-treated group were different from that of the control group. Although bite force increased with age, no alterations in bite force were attributable to Tgf-ß3 treatment. These findings suggest that the continued presence of patent sutures can affect strain patterns, perhaps when higher bite forces are present as in adult animals.

Enhancement of human adipose-derived stromal cell angiogenesis through knockdown of a BMP-2 inhibitor.

BACKGROUND: Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone. METHODS: Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry. RESULTS: Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2-releasing scaffolds significantly enhanced vascular signaling in the defect site. CONCLUSIONS: Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.

Evaluation of skull strength following parietal bone graft harvest.

BACKGROUND: Parietal bone grafts are commonly used in craniomaxillofacial surgery. The primary aim of this study was to quantify the loss of strength following monocortical parietal bone graft harvest. The secondary aim was to establish a correlation between strength and thickness of calvaria. METHODS: Thirty fresh human cadaver heads (nonfrozen, unembalmed heads) were used for this study. Loss of strength was determined by comparing the maximum impact resistance of bone on the donor side versus the intact side, using a precalibrated pendulum Charpy impact testing machine. Thickness was measured using a surgical navigation system with optoelectronic tracking. RESULTS: Loss of strength at the donor site was 36 percent (p=0.0000000001) for a 40 percent loss of thickness. Although correlation between these two parameters is rather moderate (r=0.46), it is highly significant (p<0.0001). CONCLUSIONS: Although loss of strength is quite significant, serious complications at the donor site are rare. As shown in this study, these risks are nonnegligible. However, because of strong legal pressure, surgeons must carefully weigh the risks incurred by the patient against the expected benefits, whether immediate or deferred. Therefore, the patient should receive well-documented information before such monocortical parietal bone graft harvest is performed.

Fluctuating asymmetry and developmental instability in sagittal craniosynostosis.

OBJECTIVE: To determine whether premature sagittal craniosynostosis is associated with developmental instability in the skull by analyzing fluctuating asymmetry in skull shape. DESIGN: Cranial shape was quantified by collecting coordinate data from landmarks located on three-dimensional reconstructions of preoperative computed tomography (CT) images of 22 children with sagittal craniosynostosis and 22 age-matched controls. A fluctuating asymmetry application of Euclidean distance matrix analysis (EDMA) was used to quantify and compare asymmetry in cranial shape using these landmark data. RESULTS: In contrast to expectations, the sagittal craniosynostosis group did not show a statistically significant increase in the overall level of fluctuating asymmetry relative to the control group. However, we discerned statistically significant localized increases in fluctuating asymmetry in the sagittal craniosynostosis group at pterion and the anterior clinoid processes (alpha = .05). We also determined a significant correlation of fluctuating asymmetry values between the two groups (r = .71). CONCLUSIONS: We conclude that there is no evidence of a role for system-wide developmental instability in the etiology of nonsyndromic sagittal craniosynostosis. However, the localized evidence of asymmetry at the anterior clinoid processes in the sagittal synostosis group suggests an association with the tracts of dura mater that attach there.

Biomechanical and histologic evaluation of the Norian craniofacial repair system and Norian Craniofacial Repair System Fast Set Putty in the long-term reconstruction of full-thickness skull defects in a sheep model.

BACKGROUND: The purpose of this study was to investigate the long-term (12 months) strength and osteoconductive properties of two forms of carbonated calcium phosphate cements (i.e., the Norian Craniofacial Repair System and Norian Craniofacial Repair System Fast Set Putty) and to compare these two bone cement forms to an autogenous cranial bone graft in a full-thickness skull defect adult sheep model. METHOD: Twenty-six sheep were assigned to one of eight groups (n = 3 per group). A 4.5-cm2 full-thickness defect was created in the right and left parietal bones. Reconstruction was performed with a full-thickness cranial bone autograft, the Craniofacial Repair System, or Fast Set Putty. Skull samples were harvested at day 1, 6 months, and 12 months. Biomechanical testing was performed using a vertical drop test. RESULTS: Although the Craniofacial Repair System and Fast Set Putty osseointegrated fully, there was little osteoconduction at 12 months. The Craniofacial Repair System was the weakest reconstruction, presenting the lowest peak force transmission and the highest displacement at 12 months. Fast Set Putty showed significantly higher values for peak force transmission and lower displacement when compared with the Craniofacial Repair System. CONCLUSIONS: The Craniofacial Repair System progressively lost strength over the course of this study. Fast Set Putty demonstrated greater strength and rigidity than the Craniofacial Repair System. Both implants had limited bone ingrowth from defect borders, but both cements osteointegrated completely. Bone grafts regained biostructural characteristics and strength similar to those of intact bone and clinically performed the same in this sheep model.

An immunolocalization study of tissue inhibitors of metalloproteinase-1 of bone graft healing on parietal bone.

This immunolocalization study was performed to investigate the temporal and spatial expression of tissue inhibitors of metalloproteinase (TIMP) 1 within endochondral and intramembranous bone grafts during the early stages of healing, in the hope of gaining a better understanding of the mechanisms of bone graft healing, which could influence the choice of bone graft used. Twenty-seven adult New Zealand White rabbits were used as the experimental model. Autogenous bone grafts taken from the cranial bone (intramembranous in origin) and the femur (endochondral in origin) were grafted into skull defects created on either side of the parietal suture. Rabbits were killed on days 1 to 9 postgrafting, and the bone graft alone was harvested for immunolocalization of TIMP-1. In endochondral bone grafts, TIMP-1 was expressed on days 1 to 3, followed by a period of absence until days 8 and 9. Intramembranous bone grafts did not express TIMP-1 until days 6 to 9. The timing and location of TIMP-1 expression coincided with osteogenesis, which indicates a role for TIMP-1 in preserving newly formed bone during the initial stages of graft healing. The differential temporal expression of TIMP-1 in endochondral and intramembranous bone grafts suggests that bone graft type plays an important role in influencing the healing process mediated by the host tissues. The earlier expression of TIMP-1 in endochondral bone grafts could be the reason for delayed vascularization of defects containing these grafts, whereas the delayed expression of TIMP-1 in intramembranous bone grafts could allow earlier vascularization of the intramembranous bone grafts.

Correlation between micro-computed tomography and histomorphometry for assessment of new bone formation in a calvarial experimental model.

Conventional histologic or histomorphometric evaluation provides clear evidence of the bone healing process. However, the sample preparation process is tedious and destructive, and the three-dimensional (3D) anisotropic information of the bone trabeculae is compromised. Micro-computed tomography (microCT) has been introduced as an alternative to these traditional evaluation methods. microCT is noninvasive and provides a faster approach to evaluate and quantify cancellous bone. Most previous studies that used microCT have focused on studying trabecular structures of cancellous bone. In this study, we used microCT to analyze the micro-architecture of the regenerated membranous bone using a rabbit cranial defect model. Two 1 cm diameter circular bony defects were created in 12 New Zealand white rabbits. Specimens were harvested at 6 weeks and 12 weeks after surgery and were scanned using a MicroCT machine (Skyscan 1072, Aartselaar, Belgium). The specimens were then sectioned and stained with Goldner's trichrome. Bone volume density (BV/TV), bone surface density (BS/BV), and trabecular thickness (TbTh) were determined from histomorphometric and two-dimensional (2D) and 3D microCT analysis. Pearson's correlation coefficient (gamma), paired t-tests, and intraclass correlation coefficients from measurements between the 2D and 3D microCT and histomorphometry were calculated. There were very strong positive correlations of BV/TV between histomorphometric and 2D or 3D microCT measurements. Correlation between histomorphometric and 2D microCT measurements for BS/BV was moderate, whereas correlation between histomorphometric and 3D microCT measurements was weak. Weak correlations in TbTh among the three methods were found. In conclusion, the present study suggests that, in evaluating micro-architectures in regenerated bones, the correlation between measuring methods vary according to the features measured.

Applications of an athymic nude mouse model of nonhealing critical-sized calvarial defects.

Calvarial bone defects are a common clinical scenario in craniofacial surgery. Numerous approaches are used to reconstruct skull defects, and each possesses its own inherent disadvantages. This fact underscores the opportunity to develop a novel method to repair osseous defects in craniofacial surgery. Recent literature strongly suggests that cell-based therapies in the form of regenerative medicine may be a developing paradigm in reconstructive surgery. Although numerous studies have probed osteoprogenitor cells from mice, few have explored the biology of human cells in the setting of osteogenesis in an equally rigorous manner. This study proposes a nude mouse model of critical-sized calvarial defects to study the in vivo biology of human osteoprogenitor cells. Critical-sized 4.0-mm calvarial defects were created in nude mice (n = 15) with a custom trephine drill bit outfitted to a dental drill handpiece. During the craniotomy, the dura mater was spared from injury. Gross inspection, routine histology, and micro-computed tomographic scanning were performed at 2, 4, 8, and 16 weeks postoperatively. There was no calvarial healing in any of the animals by 16 weeks. The dura mater remained intact in all subjects. Gross, histologic, and radiographic assays confirmed these findings. Although several studies have implanted human osteoprogenitor cells in vivo in various animal models, few have documented the appropriate controls or conditions necessary to support the potential to translate benchtop findings into clinical applications. We propose in this study that the nude mouse critical-sized calvarial defect model will be valuable with increasing investigations with human osteoprogenitor cells.

Sustained release and osteogenic potential of heparan sulfate-doped fibrin glue scaffolds within a rat cranial model.

This paper explores the potential therapeutic role of the naturally occurring sugar heparan sulfate (HS) for the augmentation of bone repair. Scaffolds comprising fibrin glue loaded with 5 microg of embryonically derived HS were assessed, firstly as a release-reservoir, and secondly as a scaffold to stimulate bone regeneration in a critical size rat cranial defect. We show HS-loaded scaffolds have a uniform distribution of HS, which was readily released with a typical burst phase, quickly followed by a prolonged delivery lasting several days. Importantly, the released HS contributed to improved wound healing over a 3-month period as determined by microcomputed tomography (microCT) scanning, histology, histomorphometry, and PCR for osteogenic markers. In all cases, only minimal healing was observed after 1 and 3 months in the absence of HS. In contrast, marked healing was observed by 3 months following HS treatment, with nearly full closure of the defect site. PCR analysis showed significant increases in the gene expression of the osteogenic markers Runx2, alkaline phosphatase, and osteopontin in the heparin sulfate group compared with controls. These results further emphasize the important role HS plays in augmenting wound healing, and its successful delivery in a hydrogel provides a novel alternative to autologous bone graft and growth factor-based therapies.