Outer Bone Surface Research Articles

Utility of ultrasound imaging in monitoring fracture healing in rat femur: Comparison with other imaging modalities

ObjectiveFractures are common injuries and various imaging modalities are employed to diagnose and monitor bone union. However, the follow-up of fracture healing using ultrasound imaging (US) remains a topic of debate. In this study, we analyzed of fracture healing process and compared US and radiological analyses with histological analyses to clarify the characteristics and limitations of each modality. MethodsAn osteotomy model was created using the femur of Wistar rats, and US, radiological (radiography and micro-computed tomography (micro-CT)), and histological analyses were performed. Radiological assessments were conducted for the evaluation of calcified tissue. The gap between the bony callus and cartilaginous callus was measured. ResultsUS effectively captured changes on the fracture surface, potentially reflecting the early healing processes. Both US and radiographic findings showed strong correlation in terms of the decrease in the bony callus gap. US was unable to distinguish cartilaginous callus from the surrounding soft tissue. During the remodeling stage, micro-CT offered a detailed assessment of the internal fracture surface, whereas US was limited to evaluating the outer bone surface and lacked accuracy in visualizing the entire fracture site. Radiography provided a general overview of the fractures. The decrease in the bony callus gap measured using US correlated with the reduction in cartilaginous callus observed histologically. ConclusionThis study demonstrated that US could be a valuable tool for evaluating fracture healing. Combining fracture management with US and radiological examinations may provide a more accurate assessment of healing progress.

Effect of hot water maceration, rehydration, and soft tissue presence on 3D geometry of bone.

In forensic medicine, maceration is often essential for examining bone surfaces, serving purposes such as identifying cut marks, making geometric measurements, and determining the victim's age. While hot water maceration removes soft tissue effectively, it is known to cause bone surface shrinkage. This raises the question of whether this effect is permanent or if it can be partially reversed through rehydration, considering the presence of soft tissue. Computed tomography (CT) scans were conducted on the radii of 20 paired human anatomic forearm specimens. Subsequently, the radii were extracted, macerated in 60 °C water, CT-scanned in an air environment, rehydrated, re-implanted into the forearms, and CT-scanned again. Maceration resulted in a mean shrinkage of 0.12mm on the outer bone surface. This shrinkage was nearly fully recoverable for the diaphysis after rehydration and accounting for soft tissue surrounding the bone. In contrast, the epiphysis showed permanent shrinkage, likely due to the loss of small bone fragments. Analysis of the inner bone surface indicated a smaller effect, but with significant standard deviations, especially for the epiphysis, possibly related to the less well-defined nature of the inner bone surface. The epiphyseal surface of hot water-macerated bone will, on average, be approximately 0.15mm deflated and cannot retain the original surface. On the other hand, the diaphyseal surface is less affected and can be nearly completely restored after rehydration and accounting for soft tissue surrounding the bone.

Characterization of the cellular heterogeneity and bone regenerative potential of cultured human periosteal cells

IntroductionCell-based bone regenerative therapy exhibits considerable potential in the treatment of bone defects caused by trauma, disease, and congenital anomalies. The periosteum, a fibrous membrane covering the outer surface of bone, plays a crucial role in bone formation and regeneration by sourcing osteoprogenitor cells. The remarkable osteogenic potential of periosteal cells (PCs) has led to the effective clinical implementation of PC-based regenerative therapies and tissue engineering. The abundance of progenitor cells in cultured PCs is well established; however, the heterogeneity of the cell population and its impact on bone regeneration remain uncertain. In this study, we aimed to characterize the heterogeneity of cultured PCs via single-cell RNA-sequencing (scRNA-seq) and to examine their osteogenic potential in vivo. MethodsHuman PCs cultivated using the tissue explant method were utilized in this study. scRNA-seq and real-time PCR were performed to examine the cellular heterogeneity and osteogenic capacity of the cultured PCs. Experimental bone formation by the cultured PCs was examined using the rat model of subcutaneous implantation. ResultsScRNA-seq analysis showed that the cultured PCs were categorized into three cell types (osteoprogenitor cells, mesenchymal stem cells, and fibroblasts) with specific gene expression patterns. In addition, the cellular population and osteogenic capacity differed between the central and peripheral regions in the culture dish. The PCs in the central region showed higher osteogenic potential than those in the peripheral region. ConclusionsThis study revealed the diversity of the composition of the PCs and their distinct osteogenic capabilities in different regions in the culture dish. The findings may provide promising prospects for the development of more efficacious regenerative therapeutic applications using cultured PCs in the future.

Bone microstructure of bony-toothed birds (Odontopterygiformes) from the Eocene of Ikove, Ukraine: preliminary paleobiological implications

ABSTRACT Histological structure of long bones from bony-toothed birds (Odontopterygiformes) from Early Lutetian locality Ikove (Ukraine) is examined for the first time. Six limb bones from Pelagornithidae cf. Dasornis sp. and Lutetodontopteryx tethyensis are described. The bones, although usually being fragmented, show good preservation at the microscopic level. Their microstructure (e.g. orientation and diameter of primary osteons) significantly varies, but the basic pattern and, therefore, the way of growth matches that of recent Neognathae (as well as a number of extinct taxa). In young birds, the periosteum quickly deposited well-vascularised primary bone, which at the same time was resorbed from inside, then the animal reached adult size and fast bone growth gave way to much slower deposition of circumferential layers of lamellar or parallel-fibred tissue on the outer and inner bone surfaces. The bones also underwent Haversian remodelling of various extent (from nearly absent to nearly full), generally stronger in larger bones and in the inner compacta. Bone microstructure is consistent with high metabolic rates, expected in Odontopterygiformes, and implies that their growth rates were comparable to those of recent Neognathae.

BONE MODIFICATION FEATURES RESULTING FROM BARNACLE ATTACHMENT ON THE BONES OF LOGGERHEAD SEA TURTLES (Caretta caretta), CUMBERLAND ISLAND, GEORGIA, USA: IMPLICATIONS FOR THE PALEOECOLOGICAL, AND TAPHONOMIC ANALYSES OF FOSSIL SEA TURTLES

Abstract Sea turtles are characterized by a wide variety of invertebrate ectoparasites. Few of these ectoparasites leave a permanent indication of their presence on the skeletal remains of their host taxa and thus represent ecological information doomed to be lost in the paleontological record. Some barnacle taxa provide an exception to this, in that they cause the formation of small, subcircular to circular divots, pits, and holes on the skull, mandible, carapace or plastron of sea turtles. Loggerhead Sea Turtle (Caretta caretta) skeletons from Cumberland Island, Georgia, USA were examined to assess the presence, frequency, and loci of occurrence of barnacle pits, and to establish which taxa are involved in pit development. Six types of divot and pit attributed to barnacles are identified in this study. Type I traces are shallow, oval/semi-circular in outline, with smooth, gently sloped bases. Type II traces are deep, hemispherical pits with smooth bases. Type III traces are deep, circular to subcircular pits with flat bases. Type IV traces are deep, circular to subcircular pits with multiple (4–6) small sub-pits on their bases. Type V traces are cylindrical, penetrative holes. Type VI traces comprise shallow ring-shaped grooves on the surface of the bone. Type I through III traces are identical to the ichnotaxon Karethraichnus lakkos. Type IV traces have not, as yet, been described in the rock record. Type V traces are identical to K. fiale. Type VI traces are identical to Thatchtelithichnus holmani. Barnacle taxa identified as emplacing non-penetrative divots and pits on C. caretta skulls, mandibles, and shell bones include Chelonibia caretta (Type I), Platylepas hexastylos (Types I–IV), Calyptolepas bjorndalae (Types I and II), and Stomatolepas elegans (Types I and II). Type V traces were most likely emplaced by either Stephanolepas muricata or Chelolepas cheloniae. Type VI traces reflect the former attachment of balanid or lepadid barnacles. Embedded barnacles were observed in epidermal material associated with Types I through IV traces but not for Type V and VI traces and thus the relationship is inferred for these latter traces. Barnacle-related pits, divots, and holes are believed to result from barnacle mediated chemical corrosion into the outer surface of sea turtle bone. The occurrence of these traces provides one of the few preservable lines of evidence of barnacle interactions with sea turtle hosts. Identification of definitive barnacle borings in fossil material will provide evidence of the evolution of platylepadid barnacles and the development of their commensal relationship with chelonid turtles.

Image Findings of Juxtacortical Lesions

Juxtacortical lesions appear as neoplastic formations on the outer cortical surface of bone. They can arise from nonneoplastic and neoplastic origin, the latter of which might be malignant. Juxtacortical lesions of neoplastic origin can be categorized into bone-forming lesions, cartilaginous lesions, fibrous lesions, lesions of local vascular or neurogenic origin, or metastatic lesions. Forming a differential diagnosis can be challenging for any physician assistant. We selected a series of cases from our tertiary care clinic that have been subject to discussion for their similarity but vastly different prognosis. Our aim is to provide a rubric to compare juxtacortical lesions with guide orthopaedic and sport medicine providers in determining the immediacy of referral and follow-up.

Extramedullary Osseointegration-A Novel Design of Percutaneous Osseointegration Prosthesis for Amputees.

The percutaneous osseointegrated (OI) prostheses have greatly improved the overall quality of life for amputees. However, the long-term maintenance of the OI prostheses is still challenging. A major problem is bone resorption around the bone-implant-skin interface, which might cause implant loosening or osteomyelitis. Another problem is the breakage of connecting components between the intramedullary implant and external prosthesis due to excessive stress. We designed a novel osseointegration implant by changing the bone-implant contact from the inner cortex to the outer surface of cortical bone. In the current study, we compared the extramedullary cap-shaped implants with the intramedullary screw-type implants in rabbits. Osteointegration was confirmed at the interface of bone to implant contact (BIC) in both implant types. The external implant induced intramedullary bone regeneration in the medullary canal and increased the cortical bone density at the end of the stump. This study provides a new perspective on the design of osseointegration implants which might prevent the currently reported complications of the intramedullary OI systems.

Cortical bone microdamage produced by micro-osteoperforation screws versus orthodontic miniscrews: an in vitro study

Abstract Background/objective The alternative use of Orthodontic Miniscrew Implants (OMIs), traditionally used for skeletal anchorage, to facilitate micro-osteoperforations (MOPs) for accelerating orthodontic tooth movement has been reported in previous studies. The objective of the present in vitro study was to compare the microdamage generated by OMIs and MOP-purposed screws of similar dimensions in porcine cortical bone. Materials and methods Forty rectangular porcine cortical bone specimens of 1.5 mm thickness were produced and divided into two equal groups. According to group allocation, either a single MOP screw or OMI was inserted and later removed. A sequential staining protocol was carried out to distinguish true microdamage created upon screw insertion and removal from iatrogenic damage. The bone specimens were imaged by a confocal laser scanning microscope, and five histomorphometric measurements described and quantified the generated microdamage. Results On the entry (outer) bone surface, the OMI screws produced greater microdamage which reached statistical significance across all of the histomorphometric parameters. In contrast, a statistically significant increase in microdamage was created following MOP screw insertion on the exit (inner) bone surface, but only in three assessment parameters, recorded as total damage area, as well as diffuse damage area and radius. Conclusions Overall, the present study showed that 1.5 mm OMIs produced slightly greater microcrack-type and diffuse damage-type microdamage than the 1.6 mm diameter MOP screws. However, these differences were small and considered clinically insignificant.

Mouse Periosteal Cell Culture, in vitro Differentiation, and in vivo Transplantationin Tibial Fractures.

The periosteum covering the outer surface of bone contains skeletal stem/progenitor cells that can efficiently form cartilage and bone during bone repair. Several methods have been described to isolate periosteal cells based on bone scraping and/or enzymatic digestion. Here, we describe an explant culture method to isolate periosteum-derived stem/progenitor cells for subsequent in vitro and in vivo analyses. Periosteal cells (PCs) isolated using this protocol express mesenchymal markers, can be expanded in vitro, and exhibit high regenerative potential after in vivo transplantation at a fracture site, suggesting that this protocol can be employed for PC production to use in new cell-based therapies.

Ossifying Fibroma of the Mandible – Benign Tumor with Aggressive Presentation

Abstract Introduction/Objective Ossifying fibromas are rare benign fibro-osseous lesions of mesenchymal origin affecting the craniofacial bones. Less than 60 cases have been reported in the literature. These tumors are more commonly seen in women and are usually slow growing. Most lesions are incidental, small and treated with curettage or enucleation. Rarely, ossifying fibromas can show a rapidly growing aggressive course that require radical resection. Methods We present a case of a 63-year-old man who presented with an enlarging left mandible lesion that was first noticed two years prior. We discuss the clinical presentation, prognostic factors and management of this rare disease based on the current literature. Results The patient presented with a left mandible mass that had recently grown, caused significant pain and affected chewing. The computed tomography showed a heterogeneous sclerotic lesion with periosteal reaction of the left mandible, but no evident mass. Initial biopsy showed a bland spindle cell proliferation admixed with fragments of woven bone. Due to clinical presentation the patient underwent left hemimandibulectomy with left neck dissection. The gross examination revealed 3 x 1.5 x 0.8 cm tan-pink tumor located at the junction of the mandible body and ramus, abutting the outer bone surface. Microscopic examination showed various shaped spicules of woven bone lined by osteoblasts in a vascularized, cellular, fibroblastic and spindled cell stroma consistent with aggressive ossifying fibroma. The tumor extended into the anterior and posterior bone resection margins. All the examined lymph nodes were negative for malignancy. No adjuvant radiation or other treatment was administered. The patient is alive without evidence of disease after 7 months of follow up. Conclusion The ossifying fibroma is a rare benign fibro-osseous tumor that can rarely have an aggressive behavior. Clinical, imaging and histopathological correlation are crucial to establish the correct diagnosis. Aggressive ossifying fibromas may require radical resection to prevent recurrence.

An Early Myeloma Bone Disease Model in Skeletally Mature Mice as a Platform for Biomaterial Characterization of the Extracellular Matrix.

Multiple myeloma (MM) bone disease is characterized by osteolytic bone tissue destruction resulting in bone pain, fractures, vertebral collapse, and spinal cord compression in patients. Upon initial diagnosis of MM, almost 80% of patients suffer from bone disease. Earlier diagnosis and intervention in MM bone disease would potentially improve treatment outcome and patient survival. New preclinical models are needed for developing novel diagnostic markers of bone structural changes as early as possible in the disease course. Here, we report a proof-of-concept, syngeneic, intrafemoral MOPC315.BM MM murine model in skeletally mature BALB/c mice for detection and characterization of very early changes in the extracellular matrix (ECM) of MM-injected animals. Bioluminescence imaging (BLI) in vivo confirmed myeloma engraftment in 100% of the animals with high osteoclast activity within 21 days after tumor cell inoculation. Early signs of aggressive bone turnover were observed on the outer bone surfaces by high-resolution microcomputed tomography (microCT). Synchrotron phase contrast-enhanced microcomputer tomography (PCE-CT) revealed very local microarchitecture differences highlighting numerous active sites of erosion and new bone at the micrometer scale. Correlative backscattered electron imaging (BSE) and confocal laser scanning microscopy allowed direct comparison of mineralized and nonmineralized matrix changes in the cortical bone. The osteocyte lacunar-canalicular network (OLCN) architecture was disorganized, and irregular-shaped osteocyte lacunae were observed in MM-injected bones after 21 days. Our model provides a potential platform to further evaluate pathological MM bone lesion development at the micro- and ultrastructural levels. These promising results make it possible to combine material science and pharmacological investigations that may improve early detection and treatment of MM bone disease.

The heritability of alveolar bone thickness in siblings seeking orthodontic treatment

The purpose of this study was to calculate heritability estimates of alveolar bone thickness around maxillary and mandibular incisors. The sample consisted of cone-beam computed tomography scans of 69 groups of siblings. The scans were analyzed, and Invivo5 software was used to measure the labial and lingual bone thickness. The central point of the root was identified as the point of rotation in order to construct the point of intersection with the outer bone surface. The linear distance was measured from this constructed point to the root apex. The general linear method was then used to calculate the heritability of alveolar bone thickness. Heritability of alveolar bone thickness was, on average, 58.0% for maxillary incisors and 50.3% for mandibular incisors. These estimates indicate a moderate to high heritability of alveolar bone thickness with an overall average heritability of 54.1%. Genetic factors were found to play significant roles in determining bone thickness around these teeth. The genetic effect on alveolar bone thickness around incisors is moderate to high.

What about limb long bone nutrient canal(s)? – a 3D investigation in mammals

The nutrient arteries, located in the long bone diaphysis, are the major blood supply to long bones, especially during the early phases of growth and ossification. Their intersection with the central axis of the medullary area corresponds to the ossification center, and their opening on the outer bone surface to the nutrient foramen. Nutrient arteries/foramen have essentially been analyzed in humans, and only to a much lesser extent in a few mammals. Some studies have taken measurements of the nutrient foramen; others have investigated the shape and orientation of the nutrient canals, although only partially. No studies have analyzed the nutrient canal in three dimensions inside the bone and the relationships between nutrient foramen, nutrient canal, growth, and physiology require further investigation. The current study proposes to investigate in three dimensions the shape of the nutrient canal in stylopod bones of various mammals. Qualitative and quantitative parameters are defined to discuss the diversity in, for example, morphology, orientation, and diameter encountered, resorting to two different datasets to maximize differences within mammals and then analyze variation within morphologically and phylogenetically closer taxa. This study highlights a strong intraspecific variation for various parameters, with limited biological signal, but also shows trends. It notably provides evidence that canals are generally more numerous and relatively thinner in less elongated bones. Moreover, it shows that the growth center is located distally in the humerus and proximally in the femur, and that the canals are essentially oriented towards the faster growing end, so that the nutrient foramen does not indicate the location of the growth center. This result seems general in mammals but cannot be generalized outside of Mammalia. Further analyses of the features of nutrient arteries in reptiles are required to make comparisons with the trends observed in mammals.

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the invivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible forlifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration invivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.

Caught in the act: A case study on microscopic scale physicochemical effects of fossilization on stable isotopic composition of bone

Geochemical zoning in a partially fossilized Pleistocene artiodactyl metapodial was contrasted with a fully fossilized Cretaceous ceratopsian femur as distinct stages along the fossilization process to investigate the physicochemical effects of fossilization and fossilization mechanisms. Bone fragments were analyzed via laser ablation ICP-MS, FT-IR, and stable isotope microanalysis to determine zoning patterns in trace elements, relative carbonate content and collagen contents, and stable isotopes of both the CO3 and PO4 components (δ13CCO3, δ18OCO3, and δ18OPO4). The Pleistocene sample shows a pronounced step in U concentration from high values (up to 90 ppm) within 1 mm of the outer bone surface and inner medullary cavity to ppb-level concentrations typical of in-vivo bone in the interior (inner ∼6 mm). High U broadly coincides with reduced collagen, higher δ13CCO3, and lower δ18OCO3. Rare earth elements (REE), however, show typical exponential decreases from bone edges inward, and δ18OPO4 shows no clear trend. The Cretaceous bone shows high REE and U contents throughout, a complete absence of collagen, and relatively uniform isotope compositions. The Pleistocene data, especially U zoning, implicate a diffusion-reaction (DR) fossilization mechanism, in which collagen degradation facilitates a recrystallization front that propagates as a front towards the bone interior, reducing OH-site CO3 content, and shifting δ13CCO3, δ18OCO3, and δ18OPO4. Disequilibrium O-isotope partitioning between the CO3 and PO4 components in the interior of the Pleistocene bone suggests that both the CO3 and PO4 components have exchanged with diagenetic waters, likely through abiotic and microbially-induced exchange, respectively. Strong partitioning between apatite and soil water, coupled with uptake of REE in apatite from surrounding sediment matrix during fossilization, leads to exponential decreases in concentrations, which are passively recorded in apatite in the bone interior. The Cretaceous data do not implicate a unique fossilization mechanism but instead reflect the consequences of protracted fossilization and the final state that the Pleistocene bone might eventually have reached had the bone not been excavated. That is, the Cretaceous bone fossilization history resulted in no collagen, reduced or no OH-site CO3, and more uniform isotopic compositions. The multi-analytical methods employed in this study may benefit studies of diagenetic alteration of other apatitic tissues, e.g., tooth enamel.

Periostin in Bone Regeneration.

Bone regeneration is an efficient regenerative process depending on the recruitment and activation of skeletal stem cells that allowcartilage and bone formation leading to fracture consolidation. Periosteum, the tissue located at the outer surface of bone is now recognized as an essential player in the bone repair process and contains skeletal stem cells with high regenerative potential. The matrix composition of the periosteum defines its roles in bone growth, in cortical bone modeling and remodeling in response to mechanical strain, and in bone repair. Periostinis a key extracellular matrix component of the periosteum involved in periosteum functions. In this chapter, we summarize the current knowledge on the bone regeneration process, the role of the periosteum and skeletal stem cells, and Periostin functions in this context. The matricellular protein Periostinhas several roles through all stages of bone repair: in the early days of repair during the initial activation of stem cells within periosteum, in the active phase of cartilage and bone deposition in the facture callus, and in the final phase of bone bridging and reconstitution of the stem cell pool within periosteum.

Anti-resorptive effect of pamidronate on extraction socket wall in dogs.

The aim of this experimental in vivo investigation was to assess the anti-resorptive effect of low concentration pamidronate on the buccal plate in fresh extraction sockets. The distal roots of the third premolars were extracted bilaterally in six dogs. A collagen matrix loaded with either pamidronate (test group) or saline (control group) was positioned on the outer surface of buccal bone immediately after tooth extraction and subsequently covered with a coronally advanced flap. Histological and histomorphometric outcomes were evaluated 12weeks later. The mean vertical distance between the buccal and lingual bone crest differed significantly between the test and control groups (0.52±0.43 and 2.21±1.15mm, respectively; p=.037). The width of the buccal bone 1mm below the crest was significantly wider in the test group than the control group (4.68±0.68 vs. 3.44±0.60mm, p<.001). Local application of pamidronate onto a collagen matrix may reduce the dimensional changes of the buccal bone plate both vertically and horizontally.

CBCT study of mandibular first molars with a distolingual root in Koreans

ObjectivesThis study aimed to investigate the prevalence of a separate distolingual root and to measure the thickness of the buccal cortical bone in mandibular first molars in Koreans using cone-beam computed tomography (CBCT) images.Materials and MethodsHigh-quality CBCT data from 432 patients were analyzed in this study. The prevalence of a separate distolingual root of the mandibular first molar was investigated. The distance from the distobuccal and distolingual root apices to the outer surface of the buccal cortical bone was measured. We also evaluated the thickness of the buccal cortical bone.ResultsThe prevalence of a separate distolingual root (2 separate distal roots with 1 canal in each root; 2R2C) was 23.26%. In mandibular first molars with 2R2C, the distance from the distobuccal root apex to the outer surface of the buccal cortical bone was 5.51 mm. Furthermore, the distance from the distolingual root apex to the outer surface of the buccal cortical bone was 12.09 mm. In mandibular first molars with 2R2C morphology, the thickness of the buccal cortical bone at the distobuccal root apex of the mandibular first molar was 3.30 mm. The buccal cortical bone at the distobuccal root apex was significantly thicker in the right side (3.38 mm) than the left side (3.09 mm) (p < 0.05).ConclusionsA separate distolingual root is not rare in mandibular first molars in the Korean population. Anatomic and morphologic knowledge of the mandibular first molar can be useful in treatment planning, including surgical endodontic treatment.

Congestion of mastoid mucosa and influence on middle ear pressure – Effect of retroauricular injection of adrenaline

Micro-CT scanning of temporal bones has revealed numerous retroauricular microchannels, which connect the outer bone surface directly to the underlying mastoid air cells. Their structure and dimensions have suggested a separate vascular supply to the mastoid mucosa, which may play a role in middle ear (ME) pressure regulation. This role may be accomplished by changes in the mucosa congestion resulting in volumetric changes, which ultimately affect the pressure of the enclosed ME gas pocket (Boyle's law). Further, such mucosa congestion may be susceptible to α-adrenergic stimulation similar to the mucosa of the nose. The purpose of our study was to investigate these hypotheses by recording the ME pressure in response to adrenergic stimulation administered by retroauricular injections at the surface of the microchannels.In a group of 20 healthy adults we measured the ME pressure by tympanometry initially in the sitting position, and then in the supine position over a 5 min period with 30 s intervals. In each subject, the study included 1) a control reference experiment with no intervention, 2) a control experiment with subcutaneously retroauricular injection of 1 ml isotonic NaCl solution, and 3) a test experiment with subcutaneously retroauricular injection of 1 ml NaCl-adrenaline solution. In both control experiments the ME pressure displayed an immediate increase in response to changing body position; this pressure increase remained stable for the entire period up to five minutes. In the test experiments the ME pressure also showed an initial pressure increase, but it was followed by a distinct significant pressure decrease with a maximum after 90 s. The test group was injected with both a 5 and 10% adrenaline solution, but the responses appeared similar for the two concentrations.Subcutaneous retroauricular injection of adrenaline caused a significant pressure decrease in ME pressure compared with control ears. This may be explained by the microchannels conveying the adrenaline to the underlying mastoid mucosa, where it may result in a vascular constriction and decongestion, ultimately resulting in a ME pressure decrease. These findings suggest that the microchannels contain vascular connections to the mastoid mucosa, and that the mastoid mucosa is susceptible to vasoactive mediators, which may play a role in ME pressure regulation. Further anatomical and physiological experiments should be carried out to confirm these suggestions including pharmacological interactions with the mastoid mucosa.

Self-assembled bioactive membranes for bone regeneration

Event Abstract Back to Event Self-assembled bioactive membranes for bone regeneration Sofia C. Ribeiro1, 2, 3, Daniela S. Ferreira3, 4, Rogério P. Pirraco1, 2, Isabel B. Leonor1, 2, Rui L. Reis1, 2, Álvaro Mata3, 4 and Helena S. Azevedo1, 2, 3, 4 1 University of Minho, 3B’s Research Group – Biomaterials, Biodegradables and Biomimetics, Portugal 2 University of Minho, ICVS/3B’s – PT Government Associate Laboratory, Portugal 3 Queen Mary University of London, School of Engineering and Materials Science, United Kingdom 4 Queen Mary University of London, Institute of Bioengineering, United Kingdom Introduction: Peptide self-assembly offers the possibility of fabricating highly organized biomaterials with the ability of recreating the nano-architecture and functionality of the extracellular matrix of tissues. The self-assembly of peptides with the negatively charged biopolymer hyaluronan (HA) has been explored to fabricate membranes with different biochemical and physical features[1]-[3]. Periosteum consists of microvascularized connective tissue covering the outer surface of cortical bone formed by two distinct layers: an outer fibrous layer that contains fibroblasts and Sharpey’s fibers; an inner layer called cambium, which contains multipotent mesenchymal stem cells and osteoprogenitor cells that contribute to normal bone growth, healing, and regeneration. To mimic the structure and function of periosteum, we designed new multi-domain peptides (MPDs) known to form filamentous nano-structures by self-assembly[4], to fabricate bilayer, periosteum-like, membranes by self-assembly with HA. Materials and Methods: MPDs used in this study are composed of a positively charged block (to interact with HA), a β-sheet forming segment and bioactive domain aiming to promote cell adhesion (RGDS), mineralization (EE) and osteogenic differentiation (YGFGG) (Fig.1-A). The peptides were synthesised by solid phase and purified as described elsewhere[3]. Their secondary structure and charge were analysed by circular dichroism and zeta-potential, respectively. Membranes were obtained by interfacial self-assembly between aqueous solutions of HA (700 kDa, 2%) and the various MPDs (3%) (Fig.1-B). The microstructure of the membranes was analysed by scanning electron microscopy (SEM) and a preliminary cell adhesion assay was performed using periosteum-derived cells (PDCs) to assess their ability to support cell adhesion in the absence of serum. The cells were seeded on top of the membranes and cultured for 24 h. To examine the morphology of adherent cells, cells were first stained with TRITC-conjugated phalloidin and DAPI and then observed under a confocal microscope. For cell proliferation studies, PDCs were cultured with serum for 14 days. The samples were fixed and dehydrated for observation by SEM. Results and Discussion: All designed peptides were successfully synthesised, maintaining the ability to form beta-sheet secondary structures. The HA-peptide membranes show a well-defined stratified organization with a thickness of ∼55 μm (Fig.1-C, cross-section) and a nanofibrillar structure. The presence of RGDS clearly enhances the adhesion of PDCs, when compared with control membranes (Fig.2). After 14 days of culture, a large number of well-spread cells with an elongated morphology are observed adhered to the membrane. Conclusions: We anticipate that these highly organized biomimetic membranes can provide biochemical signals for stem cell growth and differentiation and be used to promote bone regeneration. This work was supported by the Portuguese Foundation for Science and Technology under the scope of the project PTDC/CTM-BIO/0814/2012 and by the European Regional Development Fund (ERDF) through the Operational Competitiveness Programme “COMPETE” (FCOMP-01-0124-FEDER-028491).