Murine Skull Research Articles

Wnt3a loaded deformable hydrogel acts as a 3D culture platform for in situ recruitment of stem cells to efficiently repair bone defects via the asymmetric division

Biomaterial-based tissue engineering has emerged as a hotspot in the field of osteanagenesis. Due to invasive operation in the transplantation process always bring irreparable damage to patients, approaches that enable innate repair mechanisms hold considerable promise for bone repair. To this end, a rational design based on in situ recruitment of stem cells is proposed to circumvent the troublesome problem. In this study, an interpenetration network hydrogel is developed utilizing chitosan (CS), in which the enriched bone marrow mesenchymal stem cells (BMSCs) can undergo a tridimensional and freely proliferation. Moreover, the encapsulation of biological growth factor Wnt3a promotes the differentiation of osteoprogenitor cells with an asymmetric cell divisions (ACD) manner, thereby accelerating bone formation. With this “smart biomaterials”, a robust instant stem cell ingrowth within the deformable hydrogel and a high efficiency of bone regeneration on a murine skull are observed, both of which are vital for clinical applications. Such an osteoinductive system represents the advanced design concept to repair bone defects effectively, and offers a promising strategy for the development of future bone tissue engineering.

Micro-CT-Based Bone Microarchitecture Analysis of the Murine Skull.

X-ray micro-computed tomography (micro-CT) imaging has important applications in microarchitecture analysis of cortical and trabecular bone structure. While standardized protocols exist for micro-CT-based microarchitecture assessment of long bones, specific protocols need to be developed for different types of skull bones taking into account differences in embryogenesis, organization, development, and growth compared to the rest of the body. This chapter describes the general principles of bone microarchitecture analysis of murine craniofacial skeleton to accommodate for morphological variations in different regions of interest.

Vitamin D receptor activated by vitamin D administration alleviates Mycobacterium tuberculosis-induced bone destruction by inhibiting NFκB-mediated aberrant osteoclastogenesis.

Clinically, bone destruction caused by Mycobacterium tuberculosis was serious especially in patients with vitamin D (VD) deficiency. However, the role of VD in M. tuberculosis-induced bone destruction remains clear. In this context, we investigate the role of VD and vitamin D receptor (VDR) in the M. tuberculosis-induced bone destruction. First, we infected RAW264.7 and bone marrow-derived macrophages (BMMs) with Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) in vitro. Then, we activated VDR through VD administration. TRAP and FAK staining, bone resorption assays, immunofluorescence staining, qPCR, and western blot were carried out. In vivo, the M. tuberculosis-induced osteolytic model on the murine skull was established and the μCT and histological analyses were performed. We found that VDR and TRAP were upregulated in bone tuberculosis tissue and proved that M. tuberculosis infection promoted osteoclastogenesis in RAW264.7 and BMMs. VD could inhibit osteoclasts differentiation, fusion, and bone resorption dose-dependently. However, when VDR was knocked down, the inhibitory effect of VD on osteoclasts disappeared. In mechanism, activation of VDR inhibits the phosphorylation of IκB α, thereby inhibiting NFκB signaling pathway and alleviating osteoclastogenesis. Furthermore, in the skull osteolysis model, VD administration reduced osteolysis, but not in VDR-/- mice. Our study, for the first time, demonstrates that activation of VDR by VD administration inhibits M. tuberculosis-induced bone destruction. Our results reveal that VD and VDR are potential therapeutic targets for M. tuberculosis-induced bone destruction, and are of great clinical significance for the development of new therapeutic strategies.

Inside Cover

AbstractWavefront shaping technology can correct for wavefront distortion in turbid media and restore the diffraction‐limited imaging. However, the extent of each correction's validity, or the isoplanatic patch, in bone is unknown. In this paper, we present 1) direct measurement and 2) an in‐situ modeling method of the isoplanatic patch in the murine skull. (The authors thank Ms. Rachel Carlyle for her help with the design of the cover illustration.)Further details can be found in the article by Kayvan Forouhesh Tehrani, Nektarios Koukourakis, Jürgen Czarske, and Luke J. Mortensen (e202000160). image

In situ measurement of the isoplanatic patch for imaging through intact bone.

Wavefront-shaping (WS) enables imaging through scattering tissues like bone, which is important for neuroscience and bone-regeneration research. WS corrects for the optical aberrations at a given depth and field-of-view (FOV) within the sample; the extent of the validity of which is limited to a region known as the isoplanatic patch (IP). Knowing this parameter helps to estimate the number of corrections needed for WS imaging over a given FOV. In this paper, we first present direct transmissive measurement of murine skull IP using digital optical phase conjugation based focusing. Second, we extend our previously reported phase accumulation ray tracing (PART) method to provide in-situ in-silico estimation of IP, called correlative PART (cPART). Our results show an IP range of 1 to 3 μm for mice within an age range of 8 to 14 days old and 1.00 ± 0.25 μm in a 12-week old adult skull. Consistency between the two measurement approaches indicates that cPART can be used to approximate the IP before a WS experiment, which can be used to calculate the number of corrections required within a given field of view.

Intravital optoacoustic and ultrasound bio-microscopy reveal radiation-inhibited skull angiogenesis.

Angiogenesis is critical in bone development and growth. Dense, large-scale, and multi-layered vascular networks formed by thin-walled sinusoidal vessels perfuse the plate bones and play an important role in bone repair. Yet, the intricate functional morphology of skull microvasculature remains poorly understood as it is difficult to visualize using existing intravital microscopy techniques. Here we introduced an intravital, fully-transcranial imaging approach based on hybrid optoacoustic and ultrasound bio-microscopy for large-scale observations and quantitative analysis of the vascular morphology, angiogenesis, vessel remodeling, and subsurface roughness in murine skulls. Our approach revealed radiation-inhibited angiogenesis in the skull bone. We also observed previously undocumented sinusoidal vascular networks spanning the entire skullcap, thus opening new vistas for studying the complex interactions between calvarial, pial, and cortical vascular systems.

When the air hits your brain: decreased arterial pulsatility after craniectomy leading to impaired glymphatic flow.

Cranial neurosurgical procedures can cause changes in brain function. There are many potential explanations, but the effect of simply opening the skull has not been addressed, except for research into syndrome of the trephined. The glymphatic circulation, by which CSF and interstitial fluid circulate through periarterial spaces, brain parenchyma, and perivenous spaces, depends on arterial pulsations to provide the driving force for bulk flow; opening the cranial cavity could dampen this force. The authors hypothesized that a craniectomy, without any other pathological insult, is sufficient to alter brain function due to reduced arterial pulsatility and decreased glymphatic flow. Furthermore, they postulated that glymphatic impairment would produce activation of astrocytes and microglia; with the reestablishment of a closed cranial compartment, the glymphatic impairment, astrocytic/microglial activation, and neurobehavioral decline caused by opening the cranial compartment might be reversed. Using two-photon in vivo microscopy, the pulsatility index of cortical vessels was quantified through a thinned murine skull and then again after craniectomy. Glymphatic influx was determined with ex vivo fluorescence microscopy of mice 0, 14, 28, and 56 days following craniectomy or cranioplasty; brain sections were immunohistochemically labeled for GFAP and CD68. Motor and cognitive performance was quantified with rotarod and novel object recognition tests at baseline and 14, 21, and 28 days following craniectomy or cranioplasty. Penetrating arterial pulsatility decreased significantly and bilaterally following unilateral craniectomy, producing immediate and chronic impairment of glymphatic CSF influx in the ipsilateral and contralateral brain parenchyma. Craniectomy-related glymphatic dysfunction was associated with an astrocytic and microglial inflammatory response, as well as with the development of motor and cognitive deficits. Recovery of glymphatic flow preceded reduced gliosis and return of normal neurological function, and cranioplasty accelerated this recovery. Craniectomy causes glymphatic dysfunction, gliosis, and changes in neurological function in this murine model of syndrome of the trephined.

Impacts of the murine skull on high-frequency transcranial photoacoustic brain imaging.

Non-invasive photoacoustic tomography (PAT) of mouse brains with intact skulls has been a challenge due to the skull's strong acoustic attenuation, aberration, and reverberation, especially in the high-frequency range (>15 MHz). In this paper, we systematically investigated the impacts of the murine skull on the photoacoustic wave propagation and on the PAT image reconstruction. We studied the photoacoustic acoustic wave aberration due to the acoustic impedance mismatch at the skull boundaries and the mode conversion between the longitudinal wave and shear wave. The wave's reverberation within the skull was investigated for both longitudinal and shear modes. In the inverse process, we reconstructed the transcranial photoacoustic computed tomography (PACT) and photoacoustic microscopy (PAM) images of a point target enclosed by the mouse skull, showing the skull's different impacts on both modalities. Finally, we experimentally validated the simulations by imaging an in vitro mouse skull phantom using representative transcranial PAM and PACT systems. The experimental results agreed well with the simulations and confirmed the accuracy of our forward and inverse models. We expect that our results will provide better understanding of the impacts of the murine skull on transcranial photoacoustic brain imaging and pave the ways for future technical improvements.

Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration.

Innate immune cells recruited to inflammatory sites have short life spans and originate from the marrow, which is distributed throughout the long and flat bones. While bone marrow production and release of leukocyte increases after stroke, it is currently unknown if its activity rises homogeneously throughout the entire hematopoietic system. To address this question, we employed spectrally resolved in vivo cell labeling in the murine skull and tibia. We show that in murine models of stroke and aseptic meningitis, skull bone marrow derived neutrophils are more likely to migrate to the adjacent brain tissue than cells that reside in the tibia. Confocal microscopy of the skull-dura interface revealed myeloid cell migration through microscopic vascular channels crossing the inner skull cortex. These observations point to a direct local interaction between the brain and the skull bone marrow through the meninges.

Prediction and near-field observation of skull-guided acoustic waves

Ultrasound waves propagating in water or soft biological tissue are strongly reflected when encountering the skull, which limits the use of ultrasound-based techniques in transcranial imaging and therapeutic applications. Current knowledge on the acoustic properties of the cranial bone is restricted to far-field observations, leaving its near-field unexplored. We report on the existence of skull-guided acoustic waves, which was herein confirmed by near-field measurements of optoacoustically-induced responses in ex-vivo murine skulls immersed in water. Dispersion of the guided waves was found to reasonably agree with the prediction of a multilayered flat plate model. We observed a skull-guided wave propagation over a lateral distance of at least 3 mm, with a half-decay length in the direction perpendicular to the skull ranging from 35 to 300 μm at 6 and 0.5 MHz, respectively. Propagation losses are mostly attributed to the heterogenous acoustic properties of the skull. It is generally anticipated that our findings may facilitate and broaden the application of ultrasound-mediated techniques in brain diagnostics and therapy.

Syringe needle skull penetration reduces brain injuries and secondary inflammation following intracerebral neural stem cell transplantation.

Intracerebral neural stem cell (NSC) transplantation is beneficial for delivering stem cell grafts effectively, however, this approach may subsequently result in brain injury and secondary inflammation. To reduce the risk of promoting brain injury and secondary inflammation, two methods were compared in the present study. Murine skulls were penetrated using a drill on the left side and a syringe needle on the right. Mice were randomly divided into three groups (n=84/group): Group A, receiving NSCs in the left hemisphere and PBS in the right; group B, receiving NSCs in the right hemisphere and PBS in the left; and group C, receiving equal NSCs in both hemispheres. Murine brains were stained for morphological analysis and subsequent evaluation of infiltrated immune cells. ELISA was performed to detect neurotrophic and immunomodulatory factors in the brain. The findings indicated that brain injury and secondary inflammation in the left hemisphere were more severe than those in the right hemisphere, following NSC transplantation. In contrast to the left hemisphere, more neurotrophic factors but less pro-inflammatory cytokines were detected in the right hemisphere. In addition, increased levels of neurotrophic factors and interleukin (IL)-10 were observed in the NSC transplantation side when compared with the PBS-treated hemispheres, although lower levels of IL-6 and tumor necrosis factor-α were detected. In conclusion, the present study indicated that syringe needle skull penetration vs. drill penetration is an improved method that reduces the risk of brain injury and secondary inflammation following intracerebral NSC transplantation. Furthermore, NSCs have the potential to modulate inflammation secondary to brain injuries.

Accumulation of 90-Sr by murine skulls at Chornobyl exclusion zone and variability of their craniometric features

Accumulation of 90-Sr by murine skulls at Chornobyl exclusion zone and variability of their craniometric features

Broadband acoustic properties of a murine skull

It has been well recognized that the presence of a skull imposes harsh restrictions on the use of ultrasound and optoacoustic techniques in the study, treatment and modulation of the brain function. We propose a rigorous modeling and experimental methodology for estimating the insertion loss and the elastic constants of the skull over a wide range of frequencies and incidence angles. A point-source-like excitation of ultrawideband acoustic radiation was induced via the absorption of nanosecond duration laser pulses by a 20 μm diameter microsphere. The acoustic waves transmitted through the skull are recorded by a broadband, spherically focused ultrasound transducer. A coregistered pulse-echo ultrasound scan is subsequently performed to provide accurate skull geometry to be fed into an acoustic transmission model represented in an angular spectrum domain. The modeling predictions were validated by measurements taken from a glass cover-slip and ex vivo adult mouse skulls. The flexible semi-analytical formulation of the model allows for seamless extension to other transducer geometries and diverse experimental scenarios involving broadband acoustic transmission through locally flat solid structures. It is anticipated that accurate quantification and modeling of the skull transmission effects would ultimately allow for skull aberration correction in a broad variety of applications employing transcranial detection or transmission of high frequency ultrasound.

Osteogenic Potential of Mouse Periosteum-Derived Cells Sorted for CD90 In Vitro and In Vivo.

The treatment of bone defects still presents complex problems, although various techniques have been developed. The periosteum is considered a good source of osteogenic precursor cells for new bone formation. It can be collected easily in the clinical setting and is less invasive to the donor site. However, the murine skull periosteum has a poor cellular component, and growth is very slow, making it important to identify a culture method for efficient growth. In the present study, we used three-dimensional cell migration with atelocollagen and gelatin media and found that both were effective for promoting the proliferation of periosteum-derived cells. Moreover, atelocollagen medium is expected to provide an added benefit as a scaffold structure in the ambient temperature of the human body. The selection of a proper surface marker for osteogenesis is imperative for bone regeneration. CD90 is a mesenchymal stem cell marker. Periosteum-derived cells sorted with CD90 showed higher proliferative capacity and osteogenic potential than that of unsorted periosteum-derived cells in vivo and in vitro. Thus, periosteum-derived cells sorted with CD90 are expected to be a good source for bone regeneration. Significance: Periosteum-derived cells showed higher proliferative capacity and osteogenic potential. Periosteum can be collected easily in the clinical setting and is less invasive to the donor site. Thus, periosteum-derived cells can be expected to be a good source for bone regeneration.

MNGO-08MENINGIOMA GROWTH INHIBITION AND RADIOSENSITIZATION BY THE SMALL MOLECULE YAP INHIBITOR VERTEPORFIN

Meningiomas frequently harbor inactivating mutations in the tumor suppressor protein Merlin (NF2), an upstream activator of the Hippo signaling pathway. This aberration drives tumor growth and aggressiveness through inactivation of this pathway and unchecked activation of its downstream effector, Yes-associated protein (YAP). The transcriptional coactivator YAP has been previously implicated in driving meningioma growth and malignant transformation of non-neoplastic arachnoid cells. YAP exerts its effect by direct binding to the TEAD family of transcription factors and transactivation of growth-related genes. However, the efficacy of pharmacologic modulation of this pathway in meningioma has yet to be explored. Verteporfin is a small molecule inhibitor of the porphyrin family, recently implicated as a direct inhibitor of YAP. Here, we report that Verteporfin modulates growth and radiation resistance in NF2 driven meningiomas through inhibition of the YAP-TEAD interaction. In particular, our data demonstrates that Verteporfin treatment in the NF2 mutant KT21MG1 human malignant meningioma cell line, results in a decreased activity of oncogenic YAP, resulting in decreased mRNA expression of YAP downstream targets (CTGF, Mcl-1 and ANKRD1) (p ≤ 0.05). Moreover, Verteporfin inhibits meningioma cell growth and proliferation in a dose dependent manner (MTT reduction and viable cell number) (p ≤ 0.05). Interestingly, pretreatment with Verteporfin results in increased sensitivity to irradiation in vitro (p ≤ 0.05). In addition, we explore the in vivo efficacy of Verteporfin treatment in a murine skull base xenograft model of human meningioma. Taken together, our results show that Verteporfin is a potent inhibitor of meningioma cell growth and radioresistance, making it an attractive drug candidate for treatment of this disease. Hence, this targeted therapy towards the NF2-YAP axis holds promising alternatives to current standard of care.

Characterization of Reversibly Immortalized Calvarial Mesenchymal Progenitor Cells

Bone morphogenetic proteins (BMPs) play a sentinel role in osteoblastic differentiation, and their implementation into clinical practice can revolutionize cranial reconstruction. Preliminary data suggest a therapeutic role of adenoviral gene delivery of BMPs in murine calvarial defect healing. Poor transgene expression inherent in direct adenoviral therapy prompted investigation of cell-based strategies. To isolate and immortalize calvarial cells as a potential progenitor source for osseous tissue engineering. Cells were isolated from murine skulls, cultured, and transduced with a retroviral vector bearing the loxP-flanked SV40 large T antigen. Immortalized calvarial cells (iCALs) were evaluated via light microscopy, immunohistochemistry, and flow cytometry to determine whether the immortalization process altered cell morphology or progenitor cell profile. Immortalized calvarial cells were then infected with adenoviral vectors encoding BMP-2 or GFP and assessed for early and late stages of osteogenic differentiation. Immortalization of calvarial cells did not alter cell morphology as demonstrated by phase contrast microscopy. Mesenchymal progenitor cell markers CD166, CD73, CD44, and CD105 were detected at varying levels in both primary cells and iCALs. Significant elevations in alkaline phosphatase activity, osteocalcin mRNA transcription, and matrix mineralization were detected in BMP-2 treated iCALs compared with GFP-treated cells. Gross and histological analyses revealed ectopic bone production from treated cells compared with controls in an in vivo stem cell implantation assay. We have established an immortalized osteoprogenitor cell line from juvenile calvarial cells that retain a progenitor cell phenotype and can successfully undergo osteogenic differentiation upon BMP-2 stimulation. These cells provide a valuable platform to investigate the molecular mechanisms underlying intramembranous bone formation and to screen for factors/small molecules that can facilitate the healing of osseous defects in the craniofacial skeleton.

Effects of the murine skull in optoacoustic brain microscopy.

Despite the great promise behind the recent introduction of optoacoustic technology into the arsenal of small-animal neuroimaging methods, a variety of acoustic and light-related effects introduced by adult murine skull severely compromise the performance of optoacoustics in transcranial imaging. As a result, high-resolution noninvasive optoacoustic microscopy studies are still limited to a thin layer of pial microvasculature, which can be effectively resolved by tight focusing of the excitation light. We examined a range of distortions introduced by an adult murine skull in transcranial optoacoustic imaging under both acoustically- and optically-determined resolution scenarios. It is shown that strong low-pass filtering characteristics of the skull may significantly deteriorate the achievable spatial resolution in deep brain imaging where no light focusing is possible. While only brain vasculature with a diameter larger than 60 µm was effectively resolved via transcranial measurements with acoustic resolution, significant improvements are seen through cranial windows and thinned skull experiments.

The effect of a Beare‐Stevenson syndrome Fgfr2 Y394C mutation on early craniofacial bone volume and relative bone mineral density in mice

Quantifying the craniofacial skeletal phenotype during development highlights potential effects of known mutations on bone maturation and is an informative first step for the analysis of animal models. We introduce a novel technique to easily and efficiently quantify individual cranial bone volume and relative bone mineral density across the murine skull from high resolution computed tomography images. The approach can be combined with existing quantitative morphometric methods to provide details of bone growth and bone quality, which can be used to make inferences about regulatory effects local to individual bones and identify locations and developmental times for which additional analyses are warranted. Analysis of the Fgfr2(+/Y394C) mouse model of Beare-Stevenson cutis gyrata syndrome, an FGFR-related craniosynostosis syndrome, is used to demonstrate the method. Mutants and unaffected littermates display similar bone volume and relative bone density at birth, followed by significant differences at postnatal day eight. The change in rates of bone volume growth occurs similarly for all bones of the skull, regardless of origin, location or association with craniosynostosis. These results suggest an association between low bone density, low bone volume, and Fgfr craniosynostosis mutations. Our novel technique provides an initial quantitative evaluation of local shifts in bone maturation across the skull of animal models.

Identifying the Inertial Cavitation Threshold and Skull Effects in a Vessel Phantom Using Focused Ultrasound and Microbubbles

Focused ultrasound (FUS) in combination with microbubbles has been shown capable of delivering large molecules to the brain parenchyma through opening of the blood-brain barrier (BBB). However, the mechanism behind the opening remains unknown. To investigate the pressure threshold for inertial cavitation of preformed microbubbles during sonication, passive cavitation detection in conjunction with B-mode imaging was used. A cerebral vessel was simulated by generating a cylindrical hole of 610 μm in diameter inside a polyacrylamide gel and saturating its volume with microbubbles. Definity microbubbles (Mean diameter range: 1.1-3.3 μm, Lantheus Medical Imaging, N. Billerica, MA, USA) were injected prior to sonication (frequency: 1.525 MHz; pulse length: 100 cycles; PRF: 10 Hz; sonication duration: 2 s) through an excised mouse skull. The acoustic emissions due to the cavitation response were passively detected using a cylindrically focused hydrophone, confocal with the FUS transducer and a linear-array transducer with the field of view perpendicular to the FUS beam. The broadband spectral response acquired at the passive cavitation detector (PCD) and the B-mode images identified the occurrence and location of the inertial cavitation, respectively. Findings indicated that the peak-rarefactional pressure threshold was approximately equal to 0.45 MPa, with or without the skull present. Mouse skulls did not affect the threshold of inertial cavitation but resulted in a lower inertial cavitation dose. The broadband response could be captured through the murine skull, so the same PCD set-up can be used in future in vivo applications. (E-mail: ek2191@columbia.edu)

Proliferation, Osteogenic Differentiation, and FGF-2 Modulation of Posterofrontal/Sagittal Suture–Derived Mesenchymal Cells In Vitro

Fibroblast growth factor (FGF) signaling is of central importance in premature cranial suture fusion. In the murine skull, the posterofrontal suture normally fuses in early postnatal life, whereas the adjacent sagittal suture remains patent. The authors used a recently developed isolation technique for in vitro culture of suture-derived mesenchymal cells to examine the effects of FGF-2 on proliferation and differentiation of posterofrontal and sagittal suture-derived mesenchymal cells. Skulls were harvested from 40 mice (5-day-old). Posterofrontal and sagittal sutures were dissected, separating sutural mesenchymal tissue from dura mater and pericranium, and cultured. After cell migration from the explant and subculture, differences in proliferation and osteogenic differentiation of these distinct populations were studied. The mitogenic and osteogenic effects of recombinant FGF-2 were then assessed. FGF-2 regulation of gene expression was evaluated. Suture-derived mesenchymal cells isolated from the posterofrontal suture demonstrated significantly higher proliferation rates and a robust mitogenic response to FGF-2 as compared with suture-derived mesenchymal cells isolated from the sagittal suture. Interestingly, posterofrontal suture-derived mesenchymal cells retained a higher in vitro osteogenic potential, as shown by alkaline phosphatase activity and bone nodule formation. FGF-2 significantly diminished osteogenesis in both suture-derived mesenchymal cell populations. Subsequently, Ob-cadherin and Sox9 were found to be differentially expressed in posterofrontal versus sagittal suture-derived mesenchymal cells and dynamically regulated by FGF-2. In vitro osteogenesis of suture-derived mesenchymal cells recapitulates in vivo posterofrontal and sagittal sutural fates. Posterofrontal rather than sagittal suture-derived mesenchymal cells are more responsive to FGF-2 in vitro, in terms of both mitogenesis and osteogenesis.