Material properties of the skull layers of the primate parietal bone: A single-subject study.

The outer cortical table of the parietal bone has been commonly used as a calvarial bone graft site for the craniofacial reconstruction. However, little is known about how removing the outer table may affect the function and structure of the inner table, and how the knowledge of the biomechanics and material properties of cortical bones will help the calvarial graft to better integrate into the biological and mechanical functions of its surrounding native tissues. In this study, it was hypothesized that there were significant differences in both density and material properties between inner and outer cortical plates in cranial bones. Twelve cylindrical specimens, including inner-outer layers, of cortical parietal bone of a female baboon were collected. Cortical thicknesses and densities were measured, and elastic properties were assessed using an ultrasonic technique. Results demonstrated remarkable difference in both thickness (t = 8.248, p ≤0.05) and density (t = 4.926, p≤0.05) between inner and outer cortical paired samples. Orthotropic characteristics of the cortical plates were detected as well, these findings suggest that there are differences in biomechanical properties between two surfaces of cranial bones at both tissue and organ levels. How these differences are linked to the stress environments of the inner and outer cranial cortical layers awaits further studies. Further study will greatly enhance our ability to address questions derived from both morphological and craniofacial medicine fields about the development and biomechanics of craniofacial skeletons.

Effect of Induced Membrane on Guided Bone Regeneration in an Experimental Calvarial Model.

OBJECTIVE: The aim of this study was to evaluate the effect of induced membrane on guided bone regeneration and to compare its effect with poly-tetra-flourur-ethylene (PTFE) membrane and collagen membrane. METHODS: Sixteen white Vienna rabbits were used for experiments. Initially 1 defect was created on the parietal bone of all animals and cement was placed inside the defects. After 8 weeks, the bone cements were removed, without damaging the induced membrane formed in the defect cavity. And then 2 more defects were created. All defects were filled with xsenogenic graft materials and were covered with newly formed induced membrane, d-PTFE membrane and collagen membrane. Eight animals were sacrificed at 4th week and other 8 animals were sacrificed at 8th week and all bone specimens were histologically evaluated. RESULTS: New bone formation and bone marrow ratios were significantly higher in induced membrane and d-PTFE membrane group compared to collagen membrane group (P < 0.05) at 4th week. Mature bone ratios were significantly higher in induced membrane and d-PTFE membrane group compared to collagen membrane group (P < 0.05) at 8th week. The best CD31 value was detected with d-PTFE membrane group at 4th week and with induced membrane at 8th week. CONCLUSION: Induced membrane can act as a strong barrier membrane and stimulate bone regeneration. Induced membrane technique can be accepted as a good alternative for the reconstruction of critical size defects in maxillofacial region.

CRISPRai for simultaneous gene activation and inhibition to promote stem cell chondrogenesis and calvarial bone regeneration.

Calvarial bone healing remains difficult but may be improved by stimulating chondrogenesis of implanted stem cells. To simultaneously promote chondrogenesis and repress adipogenesis of stem cells, we built a CRISPRai system that comprised inactive Cas9 (dCas9), two fusion proteins as activation/repression complexes and two single guide RNA (sgRNA) as scaffolds for recruiting activator (sgRNAa) or inhibitor (sgRNAi). By plasmid transfection and co-expression in CHO cells, we validated that dCas9 coordinated with sgRNAa to recruit the activator for mCherry activation and also orchestrated with sgRNAi to recruit the repressor for d2EGFP inhibition, without cross interference. After changing the sgRNA sequence to target endogenous Sox9/PPAR-γ, we packaged the entire CRISPRai system into an all-in-one baculovirus for efficient delivery into rat bone marrow-derived mesenchymal stem cells (rBMSC) and verified simultaneous Sox9 activation and PPAR-γ repression. The activation/inhibition effects were further enhanced/prolonged by using the Cre/loxP-based hybrid baculovirus. The CRISPRai system delivered by the hybrid baculovirus stimulated chondrogenesis and repressed adipogenesis of rBMSC in 2D culture and promoted the formation of engineered cartilage in 3D culture. Importantly, implantation of the rBMSC engineered by the CRISPRai improved calvarial bone healing. This study paves a new avenue to translate the CRISPRai technology to regenerative medicine.

Use of a Non-Crosslinked Collagen Membrane During Guided Bone Regeneration Does Not Interfere With the Bone Regenerative Capacity of the Periosteum.

PURPOSE: To assess whether the use of a non-crosslinked porcine collagen type I and III bi-layered membrane inter-positioned between the periosteum and a bone defect would interfere with the bone regenerative capacity of the periosteum. MATERIALS AND METHODS: Sixty rats, each with 1 critical-size calvarial defect (CSD; diameter, 5 mm) in the parietal bone, were randomly allocated to 1 of 3 equal-size groups after CSD creation: 1) the periosteum was excised and the flap was repositioned without interposition of a membrane (no-periosteum [NP] group); 2) the flap including the periosteum was repositioned (periosteum [P] group); and 3) a non-crosslinked collagen membrane was inter-positioned between the flap, including the periosteum, and the bone defect (membrane [M] group). Micro-computed tomography, qualitative histology, immunohistochemistry, and reverse transcription real-time quantitative polymerase chain reaction were performed at 3, 7, 15, and 30 days postoperatively. RESULTS: A markedly increased radiographic residual defect length was observed in the NP group compared with the P group at 30 days. The NP group also presented a smaller radiographic bone fill area than the P group at 15 and 30 days and then the M group at 30 days. The P and M groups exhibited considerably greater expression of bone morphogenetic protein-2 and osteocalcin than the NP group at 7 days; expression of transforming growth factor-ß1 was considerably greater in the NP group at 15 days. Further, the P group presented considerably higher gene expression levels of Runx2 and Jagged1 at 7 days and of alkaline phosphatase at 3 and 15 days compared with the M and NP groups. CONCLUSION: Interposition of this specific non-crosslinked collagen membrane between the periosteum and the bone defect during guided bone regeneration interferes only slightly, if at all, with the bone regenerative capacity of the periosteum.

Osteogenesis of Crouzon-Mutated Cells in an Experimental Model.

Crouzon syndrome is an autosomal-dominant congenital disease due to a mutation in the fibroblast growth factor receptor 2 protein. The purpose of this study is to evaluate wound-healing potential of Crouzon osteoblasts and adipose-derived stem cells (ADSCs) in a murine model. Parietal skull defects were created in Crouzon and mature wild-type (WT) CD-1 mice. One group of WT and Crouzon mice were left untreated. Another group was transplanted with both WT and Crouzon adipose-derived stem cells. Additional groups compared the use of a fibrin glue scaffold and periosteum removal. Skulls were harvested from each group and evaluated histologically at 8-week and/or 16-week periods. Mean areas of defect were quantified and compared via ANOVA F-test. The average area of defect after 8 and 16 weeks in untreated Crouzon mice was 15.37 ±â€Š1.08 cm and 16.69 ±â€Š1.51 cm, respectively. The average area of the defect in untreated WT mice after 8 and 16 weeks averaged 14.17 ±â€Š1.88 cm and 14.96 ±â€Š2.26 cm, respectively. WT mice with autologous ADSCs yielded an average area of 15.35 ±â€Š1.34 cm after 16 weeks while Crouzon mice with WT ADSCs healed to an average size of 12.98 ±â€Š1.89 cm. Crouzon ADSCs transplanted into WT mice yielded an average area of 15.47 ±â€Š1.29 cm while autologous Crouzon ADSCs yielded an area of 14.22 ±â€Š3.32 cm. ANOVA F-test yielded P = .415. The fibroblast growth factor receptor 2 mutation in Crouzon syndrome does not promote reossification of critical-sized defects in mature WT and Crouzon mice. Furthermore, Crouzon ADSCs do not possess osteogenic advantage over WT ADSCs.

Guided bone regeneration in osteoporotic conditions following treatment with zoledronic acid.

OBJECTIVES: To evaluate new bone formation in calvarial critical size defects (CSD) under dense polytetrafluoroethylene (d-PTFE), microporous membranes for guided bone regeneration (GBR) in healthy, osteoporotic and osteoporotic treated with zoledronic acid (ZA) rats. METHODS: Forty-eight, female, 6-month old Wistar rats were included in the study. Osteoporosis was induced by ovariectomy (OVX) and calcium-deficient diet in 32 rats. Sixteen OVX rats were treated with a single dose of Zolendronic Acid (ZA) (OZ), while 16 OVX rats received no treatment (O). The remaining 16 rats were sham-operated and used as healthy controls (C). At 6 weeks following osteoporosis induction, two 5 mm CSD were created in the parietal bones and one of them was treated with a double d-PTFE membrane. The healing periods were 30 and 60 days. New bone formation (NB) was assessed by qualitative and quantitative histological analysis. RESULTS: After 30 days of healing, NB (mean% (95% CI)) was 78.9% (21), 93.1% (9.3) and 84.2% (26.9) in the membrane treated defects and 18.8% (24.1), 27.1% (7.9) and 31% (38.8) in the untreated defects of group O, OZ and C, respectively. After 60 days of healing, NB was 78.3% (14.4), 95.8% (9) and 90.1% (26.1) in the membrane treated defects and 10.8% (17.4), 51.6% (39.4) and 15.7% (12.1) in the untreated defects of group O, OZ and C, respectively. Hierarchical analysis of variance showed that treatment with ZA (P = 0.001) and the use of membrane (P = 0.000) significantly increased new bone formation while presence of osteoporosis may have reduced new bone formation (P = 0.028). CONCLUSION: d-PTFE membranes for GBR promote bone healing in osteoporotic and healthy rats. Treatment with ZA may improve new bone formation in osteoporotic rats.

Effect of Wnt3a delivery on early healing events during guided bone regeneration.

OBJECTIVE: To evaluate the effect of recombinant Wnt3a delivery on the bone regeneration potential following application of the guided bone regeneration (GBR) principle. MATERIALS AND METHODS: A critical-size calvarial defect was created on each parietal bone of 14 Wistar strain rats. One defect was used as the test side and was treated with a collagen sponge carrying 2.0 µg of recombinant Wnt3a protein, whereas the contralateral side served as sham-operated control. Both defects were covered at both the extracranial and intracranial aspects with ePTFE non-resorbable membranes, according to the GBR principle. Following healing periods of 4 and 7 days, qualitative histological and histomorphometric evaluation of undecalcified sections was performed in subgroups of seven animals. The primary outcome parameter was the mean percentage of defect closure in the test and control defects. RESULTS: At 4 days of healing, a network of coagulum and fibrin was observed and initial signs of granulation tissue formation were evident with no apparent differences between the test and control groups. At 7 days of healing, the test group presented newly formed woven bone, originating from the borders of the defect, as opposed to the control group, whereby woven bone formation was not observed in any of the specimens. CONCLUSIONS: The delivery of mouse recombinant Wnt-3a protein in combination with GBR may promote woven bone formation in critical-size calvarial defects at 7 days of healing.

Comparative Study Between Mesenchymal Stem Cells Derived from Bone Marrow and from Adipose Tissue, Associated with Xenograft, in Appositional Reconstructions: Histomorphometric Study in Rabbit Calvaria.

PURPOSE: This study analyzed the use of bone marrow-derived mesenchymal stem cells and adipose tissue-derived stem cells, associated with xenograft, in appositional reconstructions in rabbit calvaria using histomorphometry. MATERIALS AND METHODS: Fifteen New Zealand rabbits, weighing 3.5 to 4.0 kg and aged between 10 and 12 months, were randomly divided into three groups. Appositional bone reconstruction situations were created in the calvaria of the animals using titanium cylinders, fitted with titanium occlusive caps. Bone decortication was performed to promote bleeding. Inside the cylinders, only xenograft was positioned in the control group (CG; n = 5); xenograft combined with mesenchymal bone marrow-derived stem cells was positioned in group 1 (G1; n = 5), and a xenograft combined with adult mesenchymal stem cells derived from adipose tissue was positioned in group 2 (G2; n = 5). After 56 days, all rabbits were euthanized and their parietal bones processed for histomorphometric analysis, and the following parameters were evaluated: newly formed bone; residual graft particles; soft tissue; vital bone titanium contact, also called the level of osseointegration; and the level of bone volume contained inside the cylinders, also called the internal bone volume. RESULTS: The histomorphometric study revealed the following for CG, G1, and G2: newly formed bone of 18.96% ± 9.00%, 27.88% ± 9.98%, and 22.32% ± 7.45%; residual graft particles of 28.43% ± 2.44%, 23.31% ± 3.11%, and 27.58% ± 3.98%; soft tissue of 52.61% ± 10.80%, 50.23% ± 8.72%, and 49.90% ± 8.76%; vital bone titanium contact of 4.98% ± 4.30%, 34.91% ± 7.82%, and 20.87% ± 5.43%; and internal bone volume of 88.36% ± 25.97%, 98.73% ± 19.05%, and 98.52% ± 19.87%, respectively. No statistical difference between groups for newly formed bone, residual graft particles, soft tissue, and internal bone volume (P > .05) were verified. CONCLUSION: Regarding vital bone titanium contact, it was observed that the use of bone marrow mesenchymal stem cells, when compared with the adipose mesenchymal stem cells, showed the highest level of osseointegration, and both of them obtained superior levels to the xenograft alone.

Parietal Bone Thickness and Vascular Diameters in Adult Modern Humans: A Survey on Cranial Remains.

Cranial bone thickness varies among modern humans, and many factors influencing this variability remain unclear. Growth hormones and physical activity are thought to influence the vault thickness. Considering that both systemic factors and energy supply influence the vascular system, and taking into account the structural and biomechanical interaction between endocranial vessels and vault bones, in this study we evaluate the correlation between vascular and bone diameters. In particular, we tested the relationship between the thickness of the parietal bone (which is characterized, in modern humans, by a complex vascular network) and the lumen size of the middle meningeal and diploic vessels, in adult modern humans. Our results show no patent correlation between the thickness of parietal bone and the size of the main vascular channels. Values and distributions of the branching patterns, as well as anatomical relationships between vessels and bones, are also described in order to provide information concerning the arrangement of the endocranial vascular morphology. This information is relevant in both evolutionary and medical contexts. Anat Rec, 299:888-896, 2016. © 2016 Wiley Periodicals, Inc.

Enhanced Activation of Canonical Wnt Signaling Confers Mesoderm-Derived Parietal Bone with Similar Osteogenic and Skeletal Healing Capacity to Neural Crest-Derived Frontal Bone.

Bone formation and skeletal repair are dynamic processes involving a fine-tuned balance between osteoblast proliferation and differentiation orchestrated by multiple signaling pathways. Canonical Wnt (cWnt) signaling is known to playing a key role in these processes. In the current study, using a transgenic mouse model with targeted disruption of axin2, a negative regulator of cWnt signaling, we investigated the impact of enhanced activation of cWnt signaling on the osteogenic capacity and skeletal repair. Specifically, we looked at two calvarial bones of different embryonic tissue origin: the neural crest-derived frontal bone and the mesoderm-derived parietal bone, and we investigated the proliferation and apoptotic activity of frontal and parietal bones and derived osteoblasts. We found dramatic differences in cell proliferation and apoptotic activity between Axin2-/- and wild type calvarial bones, with Axin2-/- showing increased proliferative activity and reduced levels of apoptosis. Furthermore, we compared osteoblast differentiation and bone regeneration in Axin2-/- and wild type neural crest-derived frontal and mesoderm-derived parietal bones, respectively. Our results demonstrate a significant increase either in osteoblast differentiation or bone regeneration in Axin2-/- mice as compared to wild type, with Axin2-/- parietal bone and derived osteoblasts displaying a "neural crest-derived frontal bone-like" profile, which is typically characterized by higher osteogenic capacity and skeletal repair than parietal bone. Taken together, our results strongly suggest that enhanced activation of cWnt signaling increases the skeletal potential of a calvarial bone of mesoderm origin, such as the parietial bone to a degree similar to that of a neural crest origin bone, like the frontal bone. Thus, providing further evidence for the central role played by the cWnt signaling in osteogenesis and skeletal-bone regeneration.

Differences in biomechanical properties and thickness among frontal and parietal bones in a Japanese sample.

The aim of this study was to assess the mechanical properties and thickness of adult frontal and parietal bones. The heads of 114 Japanese cadavers (78 male cadavers and 36 female cadavers) of known age and sex were used. A total of 912 cranial samples, 8 from each skull, were collected. Samples were imaged using multidetector computed tomography to measure sample thickness. The fracture load of each sample was measured using a bending test with calculation of flexural strength. Statistical analyses demonstrated no significant bilateral difference in either the mechanical properties or thickness of frontal or parietal bones. The mechanical properties and thicknesses of frontal bones were significantly greater than those of parietal bones regardless of sex. Therefore, the skull may have a great ability to resist frontal impacts compared with parietal impacts. In female samples, parietal bones were found to have a more uniform structure when compared with male samples. Male parietal bones were found to be thicker at medial sites than at lateral sites. This study also revealed parietal bones at lateral sites in female samples were thicker than in male samples. No strong association was observed between age and flexural strength of frontal or parietal bones. However, the fracture load was negatively correlated with age most likely due to the reduction of thickness.

Cellular and molecular characterization of a novel primary osteoblast culture from the vertebrate model organism Xenopus tropicalis.

Osteogenesis is the fundamental process by which bones are formed, maintained and regenerated. The osteoblasts deposit the bone mineralized matrix by secreting large amounts of extracellular proteins and by allowing the biochemical conditions for the nucleation of hydroxyapatite crystals. Normal bone formation requires a tight control of osteoblastic activity, and therefore, osteoblasts represent a major focus of interest in biomedical research. Several crucial features of osteogenesis can be readily recapitulated using murine, avian and fish primary and immortalized osteoblastic cultures. Here, we describe a novel and straightforward in vitro culture of primary osteoblasts from the amphibian Xenopus tropicalis, a major vertebrate model organism. X. tropicalis osteoblasts can readily be extracted from the frontoparietal bone of pre-metamorphosing tadpole skulls by series of gentle protease treatments. Such primary cultures efficiently proliferate and can conveniently be grown at room temperature, in the absence of CO2, on a variety of substrates. X. tropicalis primary osteoblasts express well-characterized genes known to be active during osteogenesis of teleost fish, chick, mouse and human. Upon differentiation, such cultures mineralize and activate DMP1, an osteocyte-specific gene. Importantly, X. tropicalis primary osteoblasts can be efficiently transfected and respond to the forced activation of the bone morphogenetic protein pathway by increasing their nuclear levels of phospho-Smad. Therefore, this novel primary culture is amenable to experimental manipulations and represents a valuable tool for improving our understanding of the complex network of molecular interactions that govern vertebrate bone formation.

Xenograft impregnated with bone marrow mononuclear fraction for appositional bone regeneration in rabbit calvaria: a clinical and histomorphometric study.

PURPOSE: This study investigated the combination of a bone marrow mononuclear fraction with a bone xenograft material in an appositional bone regeneration technique. MATERIALS AND METHODS: Twelve New Zealand rabbits were randomly divided into two groups of six animals each. Bone reconstruction situations were created using titanium cylinders; these were filled with xenograft in group 1 animals and xenograft enriched with bone marrow mononuclear fraction in group 2 animals. Two cylinders were adapted onto the calvaria of each animal. Bone marrow aspirate was obtained from the tibia of every animal. After 8 weeks, the animals were sacrificed and the parietal bone and cylinders were fixed in 10% formalin for analysis of clinical measurement of the bone volume formed inside the cylinders and histomorphometric evaluation of parameters such as vital mineralized tissue (VMT), nonvital mineralized tissue (NVMT), nonmineralized tissue (NMT), and vital mineralized tissue in contact with titanium (VMTCT). RESULTS: Clinically, groups 1 and 2 demonstrated bone volume gains of 88.29% ± 25.97% and 98.96% ± 0.00%, respectively. Histomorphometry for groups 1 and 2, respectively, demonstrated the following mean values: VMT, 18.96% ± 8.99% and 28.02% ± 8.76%; NVMT, 28.43% ± 2.44% and 25.57% ± 2.33%; NMT, 52.61% ± 10.80% and 46.42% ± 10.06%; and VMTCT, 4.98% ± 4.30% and 27.29% ± 9.58%. CONCLUSION: The results of this study suggest that the use of the bone marrow mononuclear fraction can improve bone healing and the level of osseointegration.

Role of donor and host cells in muscle-derived stem cell-mediated bone repair: differentiation vs. paracrine effects.

Murine muscle-derived stem cells (MDSCs) have been shown capable of regenerating bone in a critical size calvarial defect model when transduced with BMP 2 or 4; however, the contribution of the donor cells and their interactions with the host cells during the bone healing process have not been fully elucidated. To address this question, C57/BL/6J mice were divided into MDSC/BMP4/GFP, MDSC/GFP, and scaffold groups. After transplanting MDSCs into the critical-size calvarial defects created in normal mice, we found that mice transplanted with BMP4GFP-transduced MDSCs healed the bone defect in 4 wk, while the control groups (MDSC-GFP and scaffold) demonstrated no bone healing. The newly formed trabecular bone displayed similar biomechanical properties as the native bone, and the donor cells directly participated in endochondral bone formation via their differentiation into chondrocytes, osteoblasts, and osteocytes via the BMP4-pSMAD5 and COX-2-PGE2 signaling pathways. In contrast to the scaffold group, the MDSC groups attracted more inflammatory cells initially and incurred faster inflammation resolution, enhanced angiogenesis, and suppressed initial immune responses in the host mice. MDSCs were shown to attract macrophages via the secretion of monocyte chemotactic protein 1 and promote endothelial cell proliferation by secreting multiple growth factors. Our findings indicated that BMP4GFP-transduced MDSCs not only regenerated bone by direct differentiation, but also positively influenced the host cells to coordinate and promote bone tissue repair through paracrine effects.

Cranial bone regeneration after cranioplasty using cryopreserved autogenous bone by a programmed freezer with a magnetic field in rats.

BACKGROUND: The purpose of this study was to develop a bone tissue bank using a programmed freezer with a magnetic field. Parietal bones were removed from rats and used for organ culture examination (non-cryopreserved, cryopreserved with a magnetic field (CAS) and cryopreserved without a magnetic field group). Next, other parietal bones were used for histological examination. The cryopreserved bones by a CAS freezer and dried bones were transplanted respectively. Control bones were replanted without cryopreservation. Animals were sacrificed at 4, 8, 12 and 24 weeks after surgery. After organ culture, the isolated osteoblasts from parietal bones which were cryopreserved by a CAS freezer can survive and proliferate as much as non-cryopreserved group. Histological examinations showed new bone formation in control and CAS group. These results suggest that bone tissue cryopreservation by CAS freezer can be successfully used for bone grafting which may be a novel option for regeneration medicine.

Accelerating bone generation and bone mineralization in the Interparietal sutures of rats using an rhBMP-2/ACS composite after rapid expansion.

This study aims to investigate the effects of rhBMP-2/ACS composite on bone regeneration and mineralization during expansion of the interparietal suture in rats. Forty 10-week-old Sprague-Dawley rats were divided into four groups (n=10). The first group (intact group) did not receive any intervention. The second group (expansion control group) received an expansion force of 60 g. The remaining two groups received an expansion force of 60 g and were implanted with an atelo-type I absorbable collagen sponge and rhBMP-2/ACS composite positioned on the suture beneath the periosteum. The relapse, relapse ratio, relevant bone remodelling, and calcium and osteocalcin contents were evaluated. Bone regeneration in the interparietal suture was estimated by the histological method. The osteocalcin content was measured by radioimmunoassay, and the calcium content was measured by atomic absorption spectrophotometry. Bone regeneration was more active in the suture after application of the expansion force compared with that of the suture without any intervention. Bone bridges formed in the rhBMP-2/collagen composite group. Both osteocalcin and calcium content were higher in the rhBMP-2/collagen composite group than in the other three groups (P<0.01). The relapse ratio in the rhBMP-2/collagen group was much lower than that in the other two expansion groups (P<0.01). RhBMP-2/ACS composite can promote bone regeneration and bone mineralization in the expanded suture and decrease the relapse ratio. Thus, the rhBMP-2/ACS composite may be therapeutically beneficial to the inhibition of relapse and shortening of the retention period during rapid expansion.

Preparation of thermoplastic poly(L-lactic acid) membranes for guided bone regeneration.

PURPOSE: The objective of this study was to evaluate the feasibility of application of thermoplastic poly-L lactic acid (PLLA) membranes for guided bone regeneration in rabbit parietal bone. MATERIALS AND METHODS: PLLA membranes with a molecular weight of 100,000 (PLLA-100,000) and a molecular weight of 380,000 (PLLA-380,000) were dissolved in chloroform to prepare concentrations of 8% by weight and 4% by weight, respectively. The compression strength, temperature, and time to prepare each formulation were measured. Moreover, the pH was noted and cytotoxicity of the membrane was determined by monotetrazolium assay. In vivo experiments were performed to measure the volume of newly formed bone tissue in hematoxylin and eosin-stained tissue sections 4 and 12 weeks after implantation. RESULTS: The membrane prepared from PLLA-380,000 showed excellent thermoplasticity at 75°C to 80°C and the compressive strength was equal to that of titanium mesh, in contrast to that of PLLA-100,000 and poly(lactic acid-co-glycolic acid). There was a significant change in the pH of an aqueous solution in which the PLLA-380,000 membrane was placed, but there was no cytotoxic activity. The membrane made of PLLA-380,000 induced new bone formation in a dome shape without any membrane deformation. CONCLUSION: Thermoplastic PLLA membrane shows promise for guided bone regeneration in vertical bone augmentation.

A sensitivity analysis to the role of the fronto-parietal suture in Lacerta bilineata: a preliminary finite element study.

Cranial sutures are sites of bone growth and development but micromovements at these sites may distribute the load across the skull more evenly. Computational studies have incorporated sutures into finite element (FE) models to assess various hypotheses related to their function. However, less attention has been paid to the sensitivity of the FE results to the shape, size, and stiffness of the modeled sutures. Here, we assessed the sensitivity of the strain predictions to the aforementioned parameters in several models of fronto-parietal (FP) suture in Lacerta bilineata. For the purpose of this study, simplifications were made in relation to modeling the bone properties and the skull loading. Results highlighted that modeling the FP as either an interdigitated suture or a simplified butt suture, did not reduce the strain distribution in the FP region. Sensitivity tests showed that similar patterns of strain distribution can be obtained regardless of the size of the suture, or assigned stiffness, yet the exact magnitudes of strains are highly sensitive to these parameters. This study raises the question whether the morphogenesis of epidermic scales in the FP region in the Lacertidae is related to high strain fields in this region, because of micromovement in the FP suture.

Femtosecond plasma mediated laser ablation has advantages over mechanical osteotomy of cranial bone.

BACKGROUND: Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma-mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration. METHODS: Critical-size circular calvarial defects were created with a trephine drill bit or with a Ti:Sapphire femtosecond pulsed laser. Healing was followed using micro-CT scans for 8 weeks. Calvaria were also harvested at various time points for histological analysis. Finally, scanning electron microscopy was used to analyze the microstructure of bone tissue treated with the Ti:Sapphire laser, and compared to that treated with the trephine bur. RESULTS: Laser-created defects healed significantly faster than those created mechanically at 2, 4, and 6 weeks post-surgery. However, at 8 weeks post-surgery, there was no significant difference. In the drill osteotomy treatment group, empty osteocyte lacunae were seen to extend 699 ± 27 µm away from the edge of the defect. In marked contrast, empty osteocyte lacunae were seen to extend only 182 ± 22 µm away from the edge of the laser-created craters. Significantly less ossification and formation of irregular woven bone was noted on histological analysis for drill defects. CONCLUSIONS: We demonstrate accelerated bone healing after femtosecond laser ablation in a calvarial defect model compared to traditional mechanical drilling techniques. Improved rates of early regeneration make plasma-mediated ablation of the craniofacial skeleton advantageous for applications to osteotomy.

Precise control of osteogenesis for craniofacial defect repair: the role of direct osteoprogenitor contact in BMP-2-based bioprinting.

BACKGROUND: Success with bone morphogenetic protein-2 (BMP-2) has been widely reported in the osseous reconstruction of large calvarial defects. These efforts have required enormous doses of BMP-2 and are not sufficiently refined to facilitate the detail-oriented repair required for intricate craniofacial structures. We have previously shown that inkjet-based bioprinting technologies allow for precisely customized low-dose protein patterns to induce spatially regulated osteogenesis. Here, we investigate the importance of direct contact between bioprinted BMP-2 and the dura mater (a source of osteoprogenitors) in mediating calvarial healing. METHODS: Five-millimeter osseous defects were trephinated in mouse parietal bones (N=8). Circular acellular dermal matrix (ADM) implants were prepared such that 1 semicircle of 1 face per implant was printed with BMP-2 bio-ink. These implants were then placed ink-toward (N=3) or ink-away (N=5) from the underlying dura mater. After 4 weeks, osteogenesis was assessed in each of the 4 possible positions (BMP-2-printed area toward dura, BMP-2-printed area away from dura, unprinted area toward dura, and unprinted area away from dura) by faxitron. RESULTS: The BMP-2-printed portion of the ADM generated bone covering an average of 66.5% of its surface area when it was face-down (printed surface directly abutting dura mater). By comparison, the BMP-2-printed portion of the ADM generated bone covering an average of only 21.3% of its surface area when it was face-up (printed surface away from dura). Similarly, the unprinted portion of the ADM generated an average of only 18.6% osseous coverage when face-down and 18.4% when face-up. CONCLUSIONS: We have previously shown that inkjet-based bioprinting has the potential to significantly enhance the role of regenerative therapies in craniofacial surgery. This technology affords the precise control of osteogenesis necessary to reconstruct this region's intricate anatomical architecture. In the present study, we demonstrate that direct apposition of BMP-2-printed ADM to a source of osteoprogenitor cells (in this case dura mater) is necessary for bio-ink-directed osteogenesis to occur. These results have important implications for the design of more complex bioprinted osseous structures.