Calvarial Versus Long Bone: Implications for Tailoring Skeletal Tissue Engineering.

Tissue-engineered graft substitutes have shown great potential to treat large bone defects. While we usually assume that therapeutic approaches developed for appendicular bone healing could be similarly translated for application in craniofacial reconstruction and vice versa, this is not necessarily accurate. In addition to those more well-known healing-associated factors, such as age, lifestyle (e.g., nutrition and smoking), preexisting disease (e.g., diabetes), medication, and poor blood supply, the developmental origins and surrounding tissue of the wound sites can largely affect the fracture healing outcome as well as designed treatments. Therefore, the strategies developed for long bone fracture repair might not be suitable or directly applicable to skull bone repair. In this review, we discuss aspects of development, healing process, structure, and tissue engineering considerations between calvarial and long bones to assist in designing the tailored bone repair strategies. Impact Statement We summarized, in this review, the existing body of knowledge between long bone and calvarial bone with regard to their development and healing, tissue structure, and consideration of current tissue engineering strategies. By highlighting their similarities and differences, we propose that tailored tissue engineering strategies, such as scaffold features, growth factor usage, and the source of cells for tissue or region-specific bone repair, are necessary to ensure an optimized healing outcome.

Molecular Analyses in a Rabbit Model of Craniosynostosis: Likely Exclusion of Known Candidate Genes as the Loci of Origin.

OBJECTIVE: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. We work with a naturally occurring rabbit model of CS with an undefined etiology. Known causes of coronal CS were evaluated to identify potential associations with CS in the rabbit. DESIGN: Candidate genes were sequenced in control New Zealand White (NZW) rabbits (n = 4) and synostotic NZW rabbits (n = 4). Variants were identified by alignment using Clustal Omega. OUTCOME MEASURES: Single nucleotide variants (SNVs) were classified according to phenotypic associations and predicted impact on protein structure. Human correlates were identified in the database of single nucleotide polymorphisms (dbSNP). RESULTS: A total of 21 SNVs were identified in the 10 genes examined. Variant classification and inheritance patterns are inconsistent with causality. CONCLUSIONS: The genetic basis for disease in the CS rabbit likely involves novel loci and is not associated with known causes of coronal synostosis.

Reconstruction of a Calvarial Wound Complicated by Infection: Comparing the Effects of Biopatterned Bone Morphogenetic Protein 2 and Vascular Endothelial Growth Factor.

Bone morphogenetic protein 2 (BMP2) bioprinted on biological matrix induces osseous regeneration in large calvarial defects in rabbits, both uncomplicated and scarred. Healing in unfavorable defects scarred from previous infection is decreased due in part to the lack of vascularity. This impedes the access of mesenchymal stem cells, key to osseous regeneration and the efficacy of BMP2, to the wound bed. The authors hypothesized that bioprinted vascular endothelial growth factor (VEGF) would augment the osseous regeneration achieved with low dose biopatterned BMP2 alone. Thirteen New Zealand white rabbits underwent subtotal calvariectomy using a dental cutting burr. Care was taken to preserve the underlying dura. A 15 mm × 15 mm flap of bone was cut away and incubated in a 1 × 108 cfu/mL planktonic solution of S aureus before reimplantation. After 2 weeks of subsequent infection the flap was removed and the surgical wound debrided followed by 10 days of antibiotic treatment. On postoperative day 42 the calvarial defects were treated with acellular dermal matrix bioprinted with nothing (control), VEGF, BMP2, BMP2/VEGF combined. Bone growth was analyzed with serial CT and postmortem histology. Defects treated with BMP2 (BMP2 alone and BMP2/VEGF combination) showed significantly greater healing than control and VEGF treated defect (P < 0.5). Vascular endothelial growth factor treated defect demonstrated less healing than control and VEGF/BMP2 combination treatments achieved less healing than BMP2 alone though these differences were nonsignificant. Low dose BMP2-patterned acellular dermal matrix improves healing of scarred calvarial defects. Vascular endothelial growth factor at the doses applied in this study failed to increase healing.

Genetic associations and phenotypic heterogeneity in the craniosynostotic rabbit.

Craniosynostosis (CS) is a disorder that involves the premature ossification of one or more cranial sutures. Our research team has described a naturally occurring rabbit model of CS with a variable phenotype and unknown etiology. Restriction-site associated DNA (RAD) sequencing is a genomic sampling method for identifying genetic variants in species with little or no existing sequence data. RAD sequencing data was analyzed using a mixed linear model to identify single nucleotide polymorphisms (SNPs) associated with disease occurrence and onset in the rabbit model of CS. SNPs achieving a genome-wide significance of p ≤ 5 x 10-8 were identified on chromosome 2 in association with disease occurrence and on chromosomes 14 and 19 in association with disease onset. Genotyping identified a coding variant in fibroblast growth factor binding protein 1 (FGFBP-1) on chromosome 2 and a non-coding variant upstream of integrin alpha 3 (ITGA3) on chromosome 19 that associated with disease occurrence and onset, respectively. Retrospective analysis of patient data revealed a significant inverse correlation between FGFBP-1 and ITGA3 transcript levels in patients with coronal CS. FGFBP-1 and ITGA3 are genes with roles in early development that warrant functional study to further understand suture biology.

Molecular Analysis of Gli3, Ihh, Rab23, and Jag1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

OBJECTIVE: Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. The etiology of CS is complex and mutations in more than 50 distinct genes have been causally linked to the disorder. Many of the genes that have been associated with CS in humans play an essential role in tissue patterning and early craniofacial development. Among these genes are members of the Hedgehog (HH) and Notch signal transduction pathways, including the GLI family member Gli3, Indian Hedgehog ( Ihh), the RAS oncogene family member Rab23, and the Notch ligand JAGGED1 ( Jag1). We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if coding errors in Gli3, Ihh, Rab23, or Jag1 could be causally linked to craniosynostosis in this unique animal model. DESIGN: Sequencing of cDNA templates was performed using samples obtained from wild-type and craniosynostotic rabbits. RESULTS: Several nucleotide polymorphisms were identified in Gli3, Ihh, and Rab23, although these variants failed to segregate by phenotype. No nucleotide polymorphisms were identified in Jag1. CONCLUSIONS: These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.

TLR4 Inactivation in Myeloid Cells Accelerates Bone Healing of a Calvarial Defect Model in Mice.

BACKGROUND: Toll-like receptor 4 (TLR4) has been implicated in inflammation-induced bone destruction in various chronic bone diseases; however, its direct influence on bone healing is not well understood. The authors' previous study showed accelerated bone healing with higher osteoclastogenesis gene expression in toll-like receptor 4 knockout mice (TLR4). This study aimed to further elucidate the underlying cellular mechanisms during fracture healing by generating a myeloid cell-specific toll-like receptor 4 knockout model (Lyz-TLR4 mice). METHODS: Calvarial defects, 1.8 mm in diameter, were created in wild-type, TLR4, and Lyz-TLR4 mice. Bone healing was investigated using micro-computed tomography and histologic, histomorphometric, and immunohistochemistry analyses. Primary bone marrow-derived cells were also isolated from wild-type, TLR4, and Lyz-TLR4 mice to measure their osteoclast differentiation and resorption properties. RESULTS: A similar faster bone healing response, with active intramembranous bone formation, intense osteopontin staining, and more osteoblast infiltration, was observed in TLR4 and Lyz-TLR4 mice. Tartrate-resistant acid phosphatase staining showed more osteoclast infiltration in Lyz-TLR4 mice than in wild-type mice at day 7. Primary bone marrow-derived cells isolated from TLR4 and Lyz-TLR4 mice presented enhanced osteoclastogenesis and resorption activity compared with those from wild-type mice. Comparable M0, M1, and M2 macrophage infiltration was found among all groups at days 1, 4, and 7. CONCLUSIONS: This study revealed that inactivation of toll-like receptor 4 in myeloid cells enhanced osteoclastogenesis and accelerated healing response during skull repair. Together with the role of toll-like receptor 4 in inflammation-mediated bone destruction, it suggests that toll-like receptor 4 might regulate inflammation-induced osteoclastogenesis under different clinical settings.

Enhanced Calvarial Bone Healing in CD11c-TLR4-/- and MyD88-/- Mice.

BACKGROUND: Inflammation is integral to the injury response. The inflammatory response is essential to the host defense against infection and also to tissue regeneration and repair. Toll-like receptors (TLRs) are critical activators of the innate immune response and present attractive therapeutic targets for inflammation-modulated tissue regeneration. The authors' previous study showed that depletion of TLR4 resulted in accelerated skull bone healing concurrent with increased expression of osteoclastogenic genes. As such, in the present study, the authors used various knockout mouse models for TLR4 and its associated signaling mediators as tools to further understand the role of Toll-like receptor-mediated inflammation in calvarial bone healing. METHODS: Calvarial defects (1.8-mm diameter) were created in wild-type, TLR4 knockout (TLR4), TLR2, MyD88, TRIF, TLR4 knockout in myeloid cell (Lyz-TLR4), and TLR4 knockout in dendritic-lineage cell (CD11c-TLR4) mice. Bone healing was examined using micro-computed tomographic, histologic, and histomorphometric analyses. RESULTS: Micro-computed tomographic and histomorphometric analyses revealed that TLR4-deficient mice (TLR4, Lyz-TLR4, and CD11c-TLR4) exhibited a faster intramembraneous healing response at postoperative day 7, whereas MyD88 and CD11c-TLR4 mice showed enhanced bone healing at day 28. CONCLUSIONS: The authors' data suggest a detrimental role for TLR4 in CD11c cells, mediated by Myd88 signaling, during calvarial bone healing. The authors have demonstrated that Toll-like receptor signaling components affect calvarial bone healing, establishing a link between the skeletal and immune systems during craniofacial bone healing. Toll-like receptor signaling components might be used to initiate enhanced healing in bone defects to improve clinical outcomes.

Genetic Homozygosity and Phenotypic Variability in Craniosynostotic Rabbits.

BACKGROUND: Craniosynostosis ranges in severity from single suture involvement with prenatal onset to multiple suture involvement with postnatal onset. The present study was designed to test the hypothesis that increasing homozygosity may be responsible for more severe phenotypic expression by examining the relationship between inbreeding and phenotypic expression in synostotic rabbits. METHODS: Data were obtained from 173 litters and 209 rabbits with familial craniosynostosis. Five distinct phenotypes were identified (normal n = 62; unicoronal delayed onset synostosis (DOS) n = 47; bicoronal DOS n = 21; unicoronal early onset synostosis (EOS) n = 26, and bicoronal EOS n= 53). Wright's coefficients of inbreeding (CI) were calculated using CompuPed software. Radiographs were taken at 10, 25, 42, 84, and 126 days of age to assess coronal suture, craniofacial, and skeletal growth. The relationship between CI and growth data was assessed using correlation coefficients. RESULTS: Mean CIs ranged from 15.68 (±2.22) in normal rabbits to 25.89 (±5.03) in bicoronal DOS, to 36.29 (±2.10) in unicoronal EOS to 42.85 (±2.10) in bicoronal EOS rabbits. Significant differences were noted among groups (F = 11.48; P < .001). Significant negative correlations were noted between CI and sutural and craniofacial growth at 25 (r = -.45, P < .001; and r = -.66, P < .001) through 126 (r = -.40, P < .001 and r = -.46, P < .001) days of age. CONCLUSIONS: While the synostotic phenotype is inherited in an autosomal dominant fashion in these rabbits, increasing homozygosity is associated with more severely affected phenotypes. These findings suggest that an accumulation of additional, modifier genes may determine the severity of the synostotic phenotype in rabbits.

Google Haul Out: Earth Observation Imagery and Digital Aerial Surveys in Coastal Wildlife Management and Abundance Estimation.

As the sampling frequency and resolution of Earth observation imagery increase, there are growing opportunities for novel applications in population monitoring. New methods are required to apply established analytical approaches to data collected from new observation platforms (e.g., satellites and unmanned aerial vehicles). Here, we present a method that estimates regional seasonal abundances for an understudied and growing population of gray seals (Halichoerus grypus) in southeastern Massachusetts, using opportunistic observations in Google Earth imagery. Abundance estimates are derived from digital aerial survey counts by adapting established correction-based analyses with telemetry behavioral observation to quantify survey biases. The result is a first regional understanding of gray seal abundance in the northeast US through opportunistic Earth observation imagery and repurposed animal telemetry data. As species observation data from Earth observation imagery become more ubiquitous, such methods provide a robust, adaptable, and cost-effective solution to monitoring animal colonies and understanding species abundances.

Toll-like receptor 4 mediates the regenerative effects of bone grafts for calvarial bone repair.

Craniofacial trauma is difficult to repair and presents a significant burden to the healthcare system. The inflammatory response following bone trauma is critical to initiate healing, serving to recruit inflammatory and progenitor cells and to promote angiogenesis. A role for inflammation in graft-induced bone regeneration has been suggested, but is still not well understood. The current study assessed the impact of Toll-like receptor (TLR4) signaling on calvarial repair in the presence of morselized bone components. Calvarial defects in wild-type and global TLR4(-/-) knockout mouse strains were treated with fractionated bone components in the presence or absence of a TLR4 neutralizing peptide. Defect healing was subsequently evaluated over 28 days by microcomputed tomography and histology. The matrix-enriched fraction of morselized bone stimulated calvarial bone repair comparably with intact bone graft, although the capacity for grafts to induce calvarial bone repair was significantly diminished by inhibition or genetic ablation of TLR4. Overall, our findings suggest that the matrix component of bone graft stimulates calvarial bone repair in a TLR4-dependent manner. These results support the need to better understand the role of inflammation in the design and implementation of strategies to improve bone healing.

Accelerated calvarial healing in mice lacking Toll-like receptor 4.

The bone and immune systems are closely interconnected. The immediate inflammatory response after fracture is known to trigger a healing cascade which plays an important role in bone repair. Toll-like receptor 4 (TLR4) is a member of a highly conserved receptor family and is a critical activator of the innate immune response after tissue injury. TLR4 signaling has been shown to regulate the systemic inflammatory response induced by exposed bone components during long-bone fracture. Here we tested the hypothesis that TLR4 activation affects the healing of calvarial defects. A 1.8 mm diameter calvarial defect was created in wild-type (WT) and TLR4 knockout (TLR4(-/-)) mice. Bone healing was tested using radiographic, histologic and gene expression analyses. Radiographic and histomorphometric analyses revealed that calvarial healing was accelerated in TLR4(-/-) mice. More bone was observed in TLR4(-/-) mice compared to WT mice at postoperative days 7 and 14, although comparable healing was achieved in both groups by day 21. Bone remodeling was detected in both groups on postoperative day 28. In TLR4(-/-) mice compared to WT mice, gene expression analysis revealed that higher expression levels of IL-1ß, IL-6, TNF-α,TGF-ß1, TGF-ß3, PDGF and RANKL and lower expression level of RANK were detected at earlier time points (≤ postoperative 4 days); while higher expression levels of IL-1ß and lower expression levels of VEGF, RANK, RANKL and OPG were detected at late time points (> postoperative 4 days). This study provides evidence of accelerated bone healing in TLR4(-/-) mice with earlier and higher expression of inflammatory cytokines and with increased osteoclastic activity. Further work is required to determine if this is due to inflammation driven by TLR4 activation.

A novel short hairpin RNA (shRNA) expression system promotes Sox9-dependent gene silencing.

Cartilage development and function are dependent on a temporally integrated program of gene expression. With the advent of RNA interference (RNAi), artificial control of these complex programs becomes a possibility, limited only by the ability to regulate and express the RNAi. Using existing methods for production of RNAi's, we have constructed a plasmid-based short hairpin RNA (shRNA) expression system under control of the human pol III H1 promoter and supplemented this promoter with DNA binding sites for the cartilage-specific transcription factor Sox9. The resulting shRNA expression system displays robust, Sox9-dependent gene silencing. Dependence on Sox9 expression was confirmed by electrophoretic mobility shift assays. The ability of the system to regulate heterologously expressed Sox9 was demonstrated by Western blot, as a function of both Sox9 to shRNA ratio, as well as time from transfection. This novel expression system supports auto-regulatory gene silencing, providing a tissue-specific feedback mechanism for temporal control of gene expression. Its applications for both basic mechanistic studies and therapeutic purposes should facilitate the design and implementation of innovative tissue engineering strategies.

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation.

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.

Targeting vascular injury using Hantavirus-pseudotyped lentiviral vectors.

Restenosis is a pathological condition involving intimal hyperplasia and negative arterial remodeling. Gene therapy vectors have shown modest therapeutic effects, but the level of infectivity has been relatively poor. In the present study we have designed a modified lentiviral vector (LV) pseudotyped with a strain of Hantavirus (HTNV) to improve the transduction efficiency into vascular smooth muscle and endothelial cells in vitro and in vivo. In vivo studies using adult New Zealand White rabbits demonstrated that local delivery of HTNV-pseudotyped LV (2 x 10(7) TU) into balloon-injured carotid arteries led to highly efficient transduction into endothelial and smooth muscle cells more effectively than VSV-G-pseudotyped LV (2 x 10(7) TU) or replication-defective adenoviral vectors (1-1.5 x 10(9) pfu) as determined by beta-gal immunohistochemistry. Overexpression of extracellular superoxide dismutase in balloon-injured carotid arteries 6 weeks after LV administration resulted in a significant reduction (P = 0.0024) of the intima/media ratio (0.18 +/- 0.09; n = 4) compared to vehicle-infused carotid arteries (0.69 +/- 0.08; n = 7). No beta-gal immunostaining was detected in other systemic organs, including the spleen, liver, heart, lung, kidneys, and brain. Moreover, no changes in plasma alanine aminotransferase or aspartate aminotransferase were detected following LV administration. In all, these data show that LV pseudotyped with Hantaviral glycoproteins can be a useful vector for targeting therapeutic genes to the vasculature in vivo.