Role of α2ß1 integrins in mediating cell shape on microtextured titanium surfaces.

Surface microroughness plays an important role in determining osteoblast behavior on titanium. Previous studies have shown that osteoblast differentiation on microtextured titanium substrates is dependent on alpha-2 beta-1 (α2ß1) integrin signaling. This study used focused ion beam milling and scanning electron microscopy, combined with three-dimensional image reconstruction, to investigate early interactions of individual cells with their substrate and the role of integrin α2ß1 in determining cell shape. MG63 osteoblast-like cells on sand blasted/acid etched (SLA) Ti surfaces after 3 days of culturing indicated decreased cell number, increased cell differentiation, and increased expression of mRNA levels for α1, α2, αV, and ß1 integrin subunits compared to cells on smooth Ti (PT) surfaces. α2 or ß1 silenced cells exhibited increased cell number and decreased differentiation on SLA compared to wild-type cells. Wild-type cells on SLA possessed an elongated morphology with reduced cell area, increased cell thickness, and more apparent contact points. Cells on PT exhibited greater spreading and were relatively flat. Silenced cells possessed a morphology and phenotype similar to wild-type cells grown on PT. These observations indicate that surface microroughness affects cell response via α2ß1 integrin signaling, resulting in a cell shape that promotes osteoblastic differentiation.

Rapidly polymerizing injectable click hydrogel therapy to delay bone growth in a murine re-synostosis model.

Craniosynostosis is the premature fusion of cranial sutures, which can result in progressive cranial deformations, increased intracranial pressure, and restricted brain growth. Most cases of craniosynostosis require surgical reconstruction of the cranial vault with the goal of increasing the intracranial volume and correcting the craniofacial deformities. However, patients often experience rapid post-operative bone regrowth, known as re-synostosis, which necessitates additional surgical intervention. Bone morphogenetic protein (BMP) inhibitors have tremendous potential to treat re-synostosis, but the realization of a clinically viable inhibitor-based therapeutic requires the development of a delivery vehicle that can localize the release to the site of administration. Here, we present an in situ rapidly crosslinking injectable hydrogel that has the properties necessary to encapsulate co-administered proteins and demonstrate that the delivery of rmGremlin1 via our hydrogel system delays bone regrowth in a weanling mouse model of re-synostosis. Our hydrogel is composed of two mutually reactive poly(ethylene glycol) macromolecules, which when mixed crosslink via a bio-orthogonal Cu free click reaction. Hydrogels containing Gremlin caused a dose dependent inhibition of bone regrowth. In addition to craniofacial applications, our injectable click hydrogel has the potential to provide customizable protein, small molecule, and cell delivery to any site accessible via needle or catheter.

Biphasic fusion of the murine posterior frontal suture.

BACKGROUND: Craniosynostosis is the premature fusion of cranial sutures early in development. Mice are commonly used to study the mechanisms driving both normal and pathologic cranial suture development. Despite their frequency of use as a model, the time course of bone formation and mineralization during fusion of mouse posterior frontal suture is not well defined. METHODS: To address this, C57Bl/6J mice were euthanized at ages ranging from 6 to 107 days, and the posterior frontal sutures were imaged using micro-computed tomography. Scans were analyzed with an image-processing algorithm that was previously validated with serial histology to quantify both suture fusion and mineral content. The expression profile of genes associated with key developmental time points was examined using real-time polymerase chain reaction in both the bone and the dura. RESULTS: Results demonstrate that the bones of the posterior frontal suture come together during days 10 to 20 and then increase in mineral content and volume between days 21 and 45. The onset of posterior frontal suture fusion was associated with an increase in cartilage-associated genes on day 12. Later mineralization of the suture was associated with an increase in mRNAs for osteoblast differentiation markers, bone morphogenetic proteins, and bone morphogenetic protein inhibitors. CONCLUSIONS: Complete analysis fusion posterior frontal suture shows that it occurs in a discontinuous biphasic manner. The first phase is from days 10 to 20 and involves production of cartilage. A second mineralization phase from days 21 to 45 was seen with both the imaging algorithm and changes in gene expression.

Rapid re-synostosis following suturectomy in pediatric mice is age and location dependent.

Craniosynostosis is the premature fusion of the cranial sutures early in development. If left untreated, craniosynostosis can lead to complications resulting from cranial deformities or increased intracranial pressure. The standard treatment involves calvarial reconstruction, which in many cases undergoes rapid re-synostosis. This requires additional surgical intervention that is associated with a high incidence of life threatening complications. To better understand this rapid healing, a pediatric mouse model of re-synostosis was developed and characterized. Defects (1.5mm by 2.5mm) over the posterior frontal suture were created surgically in weanling (21 days post-natal) and adolescent (50 days post-natal) C57Bl/6J mice. In addition, defects were created in the frontal bone lateral to the posterior frontal suture. The regeneration of bone in the defect was assessed using advanced image processing algorithms on micro-computed tomography scans. The genes associated with defect healing were assessed by real-time PCR of mRNA isolated from the tissue present in the defect. The results showed that the weanling mouse healed in a biphasic process with bone bridging the defect by post-operative (post-op) day 3 followed by an increase in the bone volume on day 14. In adolescent mice, there was a delay in bone bridging across the defect, and no subsequent increase in bone volume. No bridging of the defect by 14 days post-op was seen in identically sized defects placed lateral to the suture in both weanling and adolescent animals. This study demonstrates that bone regeneration in the cranium is both age and location dependent. Rapid and robust bone regeneration only occurred when the defect was created over the posterior frontal suture in immature weanling mice.

Interrelationship of cranial suture fusion, basicranial development, and resynostosis following suturectomy in twist1(+/-) mice, a murine model of Saethre-Chotzen syndrome.

The interrelationships among suture fusion, basicranial development, and subsequent resynostosis in syndromic craniosynostosis have yet to be examined. The objectives of this study were to determine the potential relationship between suture fusion and cranial base development in a model of syndromic craniosynostosis and to assess the effects of the syndrome on resynostosis following suturectomy. To do this, posterior frontal and coronal suture fusion, postnatal development of sphenooccipital synchondrosis, and resynostosis in Twist1(+/+) (WT) and Twist1(+/-) litter-matched mice (a model for Saethre-Chotzen syndrome) were quantified by evaluating µCT images with advanced image-processing algorithms. The coronal suture in Twist(+/-) mice developed, fused, and mineralized at a faster rate than that in normal littermates at postnatal days 6-30. Moreover, premature fusion of the coronal suture in Twist1(+/-) mice preceded alterations in cranial base development. Analysis of synchondrosis showed faster mineralization in Twist(+/-) mice at postnatal days 25-30. In a rapid resynostosis model, there was an inability to fuse both the midline posterior frontal suture and craniotomy defects in 21-day-old Twist(+/-) mice, despite having accelerated mineralization in the posterior frontal suture and defects. This study showed that dissimilarities between Twist1(+/+) and Twist1(+/-) mice are not limited to a fused coronal suture but include differences in fusion of other sutures, the regenerative capacity of the cranial vault, and the development of the cranial base.

Algorithm to assess cranial suture fusion with varying and discontinuous mineral density.

One of the most difficult challenges in medical imaging is the accurate segmentation of mineralized tissues. This process is complicated when studying developmental or regenerative processes due to the changes in mineral density that these tissues undergo over time. To address these limitations an algorithm was developed to enable the use of computed tomography (CT) to study tissues of varying and heterogeneous mineralization. To examine and validate this algorithm a study was performed on the development of murine cranial sutures. C57Bl/6J mice ranging in age from 6 to 25 days were imaged using micro-CT (µCT). The algorithm was developed to segment the bones of both the posterior frontal (PF) and coronal (COR) sutures. For the curved COR suture, an addition to the algorithm was developed to reconstruct images that were perpendicular to the suture about all three axes. The algorithm showed excellent linear correlation (R (2) > 0.96) with serial histomorphometry and nearly a 1:1 relationship between all measures. The algorithm was validated with serial histology. The algorithm showed that the PF suture fused between days 12 and 20 but then showed a significant increase in bone volume after day 20. The algorithm developed provides an accurate method to segment the irregular sutures of the mouse calvaria.

Use of molecular beacons to image effects of titanium surface microstructure on beta1 integrin expression in live osteoblast-like cells.

This study used molecular beacon technology to examine substrate-dependent changes in integrin subunit expression in living cells. Molecular beacons are oligonucleotide probes that can be delivered into live cells to allow for real-time imaging of mRNA. They have a stem-loop hairpin structure with a fluorophore-quencher pair, which opens when bound to the target mRNA sequence, resulting in a fluorescent signal upon excitation. A novel molecular beacon that is specific to the beta1 integrin subunit mRNA was developed and used to image osteoblast-like MG63 cells in vitro on both glass and titanium surfaces of varying roughness. Specificity was verified by comparing the molecular beacon signal intensities to real-time PCR results in both wild-type cells and cells with shRNA knockdown of beta1 integrin mRNA. The molecular beacon was able to detect changes due to both surface microtopography and silencing of the mRNA target. The results showed that effects of the substrate on beta1 mRNA noted previously in confluent cultures were evident in pre-confluent cells as well, supporting the hypothesis that beta1 integrin pairs are important in proliferation as well as differentiation of osteoblasts. This technique overcomes the limitations of traditional gene assays (PCR, immunofluorescence) by allowing for the real-time measurement and tracking of specific mRNAs in individual live cells prior to confluence.