3D-ink-extruded titanium scaffolds with porous struts and bioactive supramolecular polymers for orthopedic implants.

Porous titanium addresses the longstanding orthopedic challenges of aseptic loosening and stress shielding. This work expands on the evolution of porous Ti with the manufacturing of hierarchically porous, low stiffness, ductile Ti scaffolds via direct-ink write (DIW) extrusion and sintering of inks containing Ti and NaCl particles. Scaffold macrochannels were filled with a subtherapeutic dose of recombinant bone morphogenetic protein-2 (rhBMP-2) alone or co-delivered within a bioactive supramolecular polymer slurry (SPS) composed of peptide amphiphile nanofibrils and collagen, creating four treatment conditions (Ti struts: microporous vs. fully dense; BMP-2 alone or with SPS). The BMP-2-loaded scaffolds were implanted bilaterally across the L4 and L5 transverse processes in a rat posterolateral lumbar fusion model. In-vivo bone growth in these scaffolds is evaluated with synchrotron X-ray computed microtomography (µCT) to study the effects of strut microporosity and added biological signaling agents on the bone formation response. Optical and scanning electron microscopy confirms the ∼100 µm space-holder micropore size, high-curvature morphology, and pore fenestrations within the struts. Uniaxial compression testing shows that the microporous strut scaffolds have low stiffness and high ductility. A significant promotion in bone formation was observed for groups utilizing the SPS, while no significant differences were found for the scaffolds with the incorporation of micropores. STATEMENT OF SIGNIFICANCE: By 2050, the anticipated number of people aged 60 years and older worldwide is anticipated to double to 2.1 billion. This rapid increase in the geriatric population will require a corresponding increase in orthopedic surgeries and more effective materials for longer indwelling times. Titanium alloys have been the gold standard of bone fusion and fixation, but their use has longstanding limitations in bone-implant stiffness mismatch and insufficient osseointegration. We utilize 3D-printing of titanium with NaCl space holders for large- and small-scale porosity and incorporate bioactive supramolecular polymers into the scaffolds to increase bone growth. This work finds no significant change in bone ingrowth via space-holder-induced microporosity but significant increases in bone ingrowth via the bioactive supramolecular polymers in a rat posterolateral fusion model.

A Mechanistic and Preclinical Assessment of BioRestore Bioactive Glass as a Synthetic Bone Graft Extender and Substitute for Osteoinduction and Spine Fusion.

STUDY DESIGN: Preclinical animal study. OBJECTIVE: Evaluate the osteoinductivity and bone regenerative capacity of BioRestore bioactive glass. SUMMARY OF BACKGROUND DATA: BioRestore is a Food and Drug Administration (FDA)-approved bone void filler that has not yet been evaluated as a bone graft extender or substitute for spine fusion. METHODS: In vitro and in vivo methods were used to compare BioRestore with other biomaterials for the capacity to promote osteodifferentiation and spinal fusion. The materials evaluated (1) absorbable collagen sponge (ACS), (2) allograft, (3) BioRestore, (4) Human Demineralized Bone Matrix (DBM), and (5) MasterGraft. For in vitro studies, rat bone marrow-derived stem cells (BMSC) were cultured on the materials in either standard or osteogenic media (SM, OM), followed by quantification of osteogenic marker genes ( Runx2, Osx, Alpl, Bglap, Spp1 ) and alkaline phosphatase (ALP) activity. Sixty female Fischer rats underwent L4-5 posterolateral fusion (PLF) with placement of 1 of 5 implants: (1) ICBG from syngeneic rats; (2) ICBG+BioRestore; (3) BioRestore alone; (4) ICBG+Allograft; or (5) ICBG+MasterGraft. Spines were harvested 8 weeks postoperatively and evaluated for bone formation and fusion via radiography, blinded manual palpation, microCT, and histology. RESULTS: After culture for 1 week, BioRestore promoted similar expression levels of Runx2 and Osx to cells grown on DBM. At the 2-week timepoint, the relative ALP activity for BioRestore-OM was significantly higher ( P <0.001) than that of ACS-OM and DBM-OM ( P <0.01) and statistically equivalent to cells grown on allograft-OM. In vivo, radiographic and microCT evaluation showed some degree of bridging bone formation in all groups tested, with the exception of BioRestore alone, which did not produce successful fusions. CONCLUSIONS: This study demonstrates the capacity of BioRestore to promote osteoinductivity in vitro. In vivo, BioRestore performed similarly to commercially available bone graft extender materials but was incapable of producing fusion as a bone graft substitute. LEVEL OF EVIDENCE: Level V.

A supramolecular polymer-collagen microparticle slurry for bone regeneration with minimal growth factor.

Recombinant bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive growth factor that can promote bone regeneration for challenging skeletal repair and even for ectopic bone formation in spinal fusion procedures. However, serious clinical side effects related to supraphysiological dosing highlight the need for advances in novel biomaterials that can significantly reduce the amount of this biologic. Novel biomaterials could not only reduce clinical side effects but also expand the indications for use of BMP-2, while at the same time lowering the cost of such procedures. To achieve this objective, we have developed a slurry containing a known supramolecular polymer that potentiates BMP-2 signaling and porous collagen microparticles. This slurry exhibits a paste-like consistency that stiffens into an elastic gel upon implantation making it ideal for minimally invasive procedures. We carried out in vivo evaluation of the novel biomaterial in the rabbit posterolateral spine fusion model, and discovered efficacy at unprecedented ultra-low BMP-2 doses (5 µg/implant). This dose reduces the growth factor requirement by more than 100-fold relative to current clinical products. This observation is significant given that spinal fusion involves ectopic bone formation and the rabbit model is known to be predictive of human efficacy. We expect the novel biomaterial can expand BMP-2 indications for difficult cases requiring large volumes of bone formation or involving patients with underlying conditions that compromise bone regeneration.

Bone resorption and formation in the pedicles of European roe deer (Capreolus capreolus) in relation to the antler cycle-A morphological and microanalytical study.

We analyzed pedicle bone from roe bucks that had died around antler casting or shortly before or during the rutting period. Pedicles obtained around antler casting were highly porous and showed signs of intense osteoclastic activity that had caused the formation of an abscission line. Following the detachment of the antler plus a portion of pedicle bone, osteoclastic activity in the pedicles continued for some time, and new bone was deposited onto the separation plane of the pedicle stump, leading to partial pedicle restoration. Pedicles obtained around the rutting period were compact structures. The newly formed, often very large secondary osteons, which had filled the resorption cavities, exhibited a lower mineral density than the persisting older bone. The middle zones of the lamellar infilling frequently showed hypomineralized lamellae and enlarged osteocyte lacunae. This indicates a deficiency in mineral elements during the formation of these zones that occurred along with peak antler mineralization. We suggest that growing antlers and compacting pedicles compete for mineral elements, with the rapidly growing antlers being the more effective sinks. The competition between the two simultaneously mineralizing structures is probably more severe in Capreolus capreolus than in other cervids. This is because roe bucks regrow their antlers during late autumn and winter, a period of limited food and associated mineral supply. The pedicle is a heavily remodeled bone structure with distinct seasonal variation in porosity. Pedicle remodeling differs in several aspects from the normal bone remodeling process in the mammalian skeleton.

On `Flexible design in the stomatopod dactyl club'.

This commentary discusses loose versus tight control of biomineralization products and how this evolved flexibility. Concomitant improved functionality may be more widespread than commonly thought.

Mesoscale structural gradients in human tooth enamel.

The outstanding mechanical and chemical properties of dental enamel emerge from its complex hierarchical architecture. An accurate, detailed multiscale model of the structure and composition of enamel is important for understanding lesion formation in tooth decay (dental caries), enamel development (amelogenesis) and associated pathologies (e.g., amelogenesis imperfecta or molar hypomineralization), and minimally invasive dentistry. Although features at length scales smaller than 100 nm (individual crystallites) and greater than 50 µm (multiple rods) are well understood, competing field of view and sampling considerations have hindered exploration of mesoscale features, i.e., at the level of single enamel rods and the interrod enamel (1 to 10 µm). Here, we combine synchrotron X-ray diffraction at submicrometer resolution, analysis of crystallite orientation distribution, and unsupervised machine learning to show that crystallographic parameters differ between rod head and rod tail/interrod enamel. This variation strongly suggests that crystallites in different microarchitectural domains also differ in their composition. Thus, we use a dilute linear model to predict the concentrations of minority ions in hydroxylapatite (Mg2+ and CO32-/Na+) that plausibly explain the observed lattice parameter variations. While differences within samples are highly significant and of similar magnitude, absolute values and the sign of the effect for some crystallographic parameters show interindividual variation that warrants further investigation. By revealing additional complexity at the rod/interrod level of human enamel and leaving open the possibility of modulation across larger length scales, these results inform future investigations into mechanisms governing amelogenesis and introduce another feature to consider when modeling the mechanical and chemical performance of enamel.

Sex-based Difference in Response to Recombinant Human Bone Morphogenetic Protein-2 in a Rat Posterolateral Fusion Model.

STUDY DESIGN: This was a preclinical study. OBJECTIVE: Evaluate sex-dependent differences in the bone healing response to recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rat posterolateral spinal fusion model. SUMMARY OF BACKGROUND DATA: Minimal and conflicting data exist concerning potential sex-dependent differences in rhBMP-2-mediated bone regeneration in the context of spinal fusion. MATERIALS AND METHODS: Forty-eight female and male Sprague-Dawley rats (N=24/group), underwent L4-L5 posterolateral fusion with bilateral placement of an absorbable collagen sponge, each loaded with 5 µg of bone morphogenetic protein-2 (10 µg/animal). At eight weeks postoperative, 10 specimens of each sex were tested in flexion-extension with quantification of range of motion and stiffness. The remaining specimens were evaluated for new bone growth and successful fusion via radiography, blinded manual palpation and microcomputed tomography (microCT). Laboratory microCT quantified bone microarchitecture, and synchrotron microCT examined bone microstructure at the 1 µm level. RESULTS: Manual palpation scores differed significantly between sexes, with mean fusion scores of 2.4±0.4 in females versus 3.1±0.6 in males ( P <0.001). Biomechanical stiffness did not differ between sexes, but range of motion was significantly greater and more variable for females versus males (3.7±5.6° vs. 0.27±0.15°, P <0.005, respectively). Laboratory microCT showed significantly smaller volumes of fusion masses in females versus males (262±87 vs. 732±238 mm 3 , respectively, P <0.001) but significantly higher bone volume fraction (0.27±0.08 vs. 0.12±0.05, respectively, P <0.001). Mean trabecular thickness was not different, but trabecular number was significantly greater in females (3.1±0.5 vs. 1.5±0.4 mm -1 , respectively, P <0.001). Synchrotron microCT showed fine bone structures developing in both sexes at the eight-week time point. CONCLUSIONS: This study demonstrates sex-dependent differences in bone regeneration induced by rhBMP-2. Further investigation is needed to uncover the extent of and mechanisms underlying these sex differences, particularly at different doses of rhBMP-2.

Special Section Guest Editorial: Hard X-Ray Tomography with Micrometer Resolution.

JMI guest editors introduce articles collected in the JMI Special Section on Hard X-Ray Tomography with Micrometer Resolution, a snapshot of this important niche area featured by hard x-ray tomography at the micrometer level.

Distribution, structure, and mineralization of calcified cartilage remnants in hard antlers.

Antlers are paired deciduous bony cranial appendages of deer that undergo a regular cycle of growth, death and casting, and constitute the most rapidly growing bones in mammals. Antler growth occurs in an appositional mode and involves a modified form of endochondral ossification. In endochondral bones, calcified cartilage is typically a transient tissue that is eventually completely replaced by bone tissue. We studied the distribution and characteristics of calcified cartilage in hard antlers from three deer species (Capreolus capreolus, Cervus elaphus, Dama dama), i.e., in antlers from which the skin (velvet) had been shed. Remnants of calcified cartilage were regularly present as part of the trabecular framework in the late formed, distal antler portions in all three species, whereas this tissue was largely or completely missing in the more proximal antler portions. The presence of calcified cartilage remnants in the distal antler portions is attributed to the limited antler lifespan of only a few months, which is also the reason for the virtual lack of bone remodeling in antlers. The calcified cartilage matrix was more highly mineralized than the antler bone matrix. Mineralized deposits were observed in some chondrocyte lacunae and occasionally also in osteocyte lacunae, a phenomenon that has not previously been reported in antlers. Using synchrotron radiation-induced X-ray fluorescence (SR-XRF) mapping, we further demonstrated increased zinc concentrations in cement lines, along the inner borders of incompletely formed primary osteons, along the walls of partly or completely mineral-occluded chondrocyte and osteocyte lacunae, and in intralacunar mineralized deposits. The present study demonstrates that antlers are a promising model for studying the mineralization of cartilage and bone matrices and the formation of mineralized deposits in chondrocyte and osteocyte lacunae.

Microstructure and energy dispersive diffraction reconstruction of 3D patterns of crystallographic texture in a shark centrum.

Purpose: Tomography using diffracted x-rays produces reconstructions mapping quantities such as crystal lattice parameter(s), crystallite size, and crystallographic texture, information quite different from that obtained with absorption or phase contrast. Diffraction tomography is used to map an entire blue shark centrum with its double cone structure (corpora calcerea) and intermedialia (four wedges). Approach: Energy dispersive diffraction (EDD) and polychromatic synchrotron x-radiation at 6-BM-B, the Advanced Photon Source, were used. Different, properly oriented Bragg planes diffract different x-ray energies; these intensities are measured by one of ten energy-sensitive detectors. A pencil beam defines the irradiated volume, and a collimator before each energy-sensitive detector selects which portion of the irradiated column is sampled at any one time. Translating the specimen along X , Y , and Z axes produces a 3D map. Results: We report 3D maps of the integrated intensity of several bioapatite reflections from the mineralized cartilage centrum of a blue shark. The c axis reflection's integrated intensities and those of a reflection with no c axis component reveal that the cone wall's bioapatite is oriented with its c axes lateral, i.e., perpendicular to the backbone's axis, and that the wedges' bioapatite is oriented with its c axes axial. Absorption microcomputed tomography (laboratory and synchrotron) and x-ray excited x-ray fluorescence maps provide higher resolution views. Conclusion: The bioapatite in the cone walls and wedges is oriented to resist lateral and axial deflections, respectively. Mineralized tissue samples can be mapped in 3D with EDD tomography and subsequently studied by destructive methods.

Shark centra microanatomy and mineral density variation studied with laboratory microComputed Tomography.

Centra of shark vertebrae from three species of Lamniformes (Alopias vulpinus, Carcharodon carcharias and Isurus oxyrinchus) and three species of Carcharhiniformes (Carcharhinus plumbeus, Carcharhinus obscurus and Prionace glauca) were imaged with laboratory microcomputed Tomography (microCT) using volume element (voxel) sizes between 16 and 24 µm. Linear attenuation coefficients were the same in the corpus calcarea (hour-glass-shaped cone) and intermedialia of the lamniforms but were smaller in the intermedialia than in the corpus calcarea of the carcharhiniforms. All centra contained growth bands which were visible as small changes in linear attenuation coefficient. In all six cases, the cross-sections of the cones were close to circular, and the cone angles matched those reported in the literature. Cartilage canals were a prominent structure in the intermedialia of all species, 3D renderings of centra of C. obscurus and I. oxyrinchus diameters showed these canals ran radially outward from the cone walls, and canal diameters were consistent with the limited numerical values in the literature. Somewhat higher calcification levels around the periphery of cartilage canals and of outer surfaces of the intermedialia and corpus calcerea suggest microstructural variation exists at scale below that which can be resolved in the present data sets.

Osteoinductivity and biomechanical assessment of a 3D printed demineralized bone matrix-ceramic composite in a rat spine fusion model.

We recently developed a recombinant growth factor-free bone regenerative scaffold composed of stoichiometric hydroxyapatite (HA) ceramic particles and human demineralized bone matrix (DBM) particles (HA-DBM). Here, we performed the first pre-clinical comparative evaluation of HA-DBM relative to the industry standard and established positive control, recombinant human bone morphogenetic protein-2 (rhBMP-2), using a rat posterolateral spinal fusion model (PLF). Female Sprague-Dawley rats underwent bilateral L4-L5 PLF with implantation of the HA-DBM scaffold or rhBMP-2. Fusion was evaluated using radiography and blinded manual palpation, while biomechanical testing quantified the segmental flexion-extension range-of-motion (ROM) and stiffness of the fused segments at 8-weeks postoperatively. For mechanistic studies, pro-osteogenic gene and protein expression at 2-days and 1-, 2-, and 8-weeks postoperatively was assessed with another cohort. Unilateral fusion rates did not differ between the HA-DBM (93%) and rhBMP-2 (100%) groups; however, fusion scores were higher with rhBMP-2 (p = 0.008). Both treatments resulted in significantly reduced segmental ROM (p < 0.001) and greater stiffness (p = 0.009) when compared with non-operated controls; however, the degree of stabilization was significantly higher with rhBMP-2 treatment relative to the HA-DBM scaffold. In the mechanistic studies, PLGA and HA scaffolds were used as negative controls. Both rhBMP-2 and HA-DBM treatments resulted in significant elevations of several osteogenesis-associated genes, including Runx2, Osx, and Alp. The rhBMP-2 treatment led to significantly greater early, mid, and late osteogenic markers, which may be the mechanism in which early clinical complications are seen. The HA-DBM scaffold also induced osteogenic gene expression, but primarily at the 2-week postoperative timepoint. Overall, our findings show promise for this 3D-printed composite as a recombinant growth factor-free bone graft substitute for spinal fusion. STATEMENT OF SIGNIFICANCE: Despite current developments in bone graft technology, there remains a significant void in adequate materials for bone regeneration in clinical applications. Two of the most efficacious bone graft options are the gold-standard iliac crest bone graft and recombinant human-derived bone morphogenetic protein-2 (rhBMP-2), available commercially as Infuse™. Although efficacious, autologous graft is associated with donor-site morbidity, and Infuse™ has known side effects related to its substantial host inflammatory response, possibly associated with a immediate, robust osteoinductive response. Hence, there is a need for a bone graft substitute that provides adequate osteogenesis without associated adverse events. This study represents a significant step in the design of off-the-shelf growth factor-free devices for spine fusion.

Effect of Postoperative Analgesic Exposure to the Cannabinoid Receptor Agonist WIN55 on Osteogenic Differentiation and Spinal Fusion in Rats.

BACKGROUND: After spinal surgery and other orthopaedic procedures, most patients receive opioids for pain, leading to potential complications such as pseudarthrosis and opioid abuse associated with long-term use. As an alternative, the endocannabinoid system has been shown to have antinociceptive activity, while contributing to bone homeostasis via the CB1 and CB2 cannabinoid receptors. This study evaluates the impact of the cannabinoid receptor agonist WIN55,212-2 (WIN55) on osteogenic differentiation in vitro as well as bone regeneration and spinal fusion in a preclinical rat model. METHODS: Primary rat bone marrow stromal cells were cultured in standard or osteogenic media and exposed to vehicle alone or WIN55. Runx2 and Alkaline phosphatase (Alpl) were quantified via qPCR (quantitative real-time polymerase chain reaction), followed by assessment of ALP activity and matrix mineralization. For in vivo evaluation, 45 female Sprague Dawley rats (n = 15 per group) underwent L4-L5 posterolateral spinal fusion with bilateral placement of collagen scaffolds preloaded with low-dose rhBMP-2 (recombinant human bone morphogenetic protein-2; 0.5 µg/implant). Postoperatively, rats received the vehicle alone or 0.5 or 2.5 mg/kg WIN55 via daily intraperitoneal injections for 5 days. Bone regeneration and spinal fusion were assessed using radiography, manual palpation-based fusion scoring, microcomputed tomography imaging, and histology. RESULTS: mRNA expression levels of Runx2 and Alp were similar among cells treated with vehicle alone and WIN55. Likewise, exposure to WIN55 did not inhibit ALP activity or bone matrix mineralization. In this animal model, no significant differences were found among groups with regard to mean fusion score, fusion rate, or new bone volume. CONCLUSIONS: WIN55 showed no adverse impact on osteogenic differentiation, bone regeneration, and spinal fusion. This supports that cannabinoid receptor agonists should be further investigated as a potential alternative approach for postoperative analgesia following spinal fusion and other orthopaedic procedures requiring bone-healing. CLINICAL RELEVANCE: The identification of alternative treatments for postoperative pain following orthopaedic surgical procedures is crucial in combating the ongoing opioid abuse crisis. The endocannabinoid system may represent a viable alternative target for addressing orthopaedic postoperative pain.

Influence of Geometry and Architecture on the In Vivo Success of 3D-Printed Scaffolds for Spinal Fusion.

We previously developed a recombinant growth factor-free, three-dimensional (3D)-printed material comprising hydroxyapatite (HA) and demineralized bone matrix (DBM) for bone regeneration. This material has demonstrated the capacity to promote re-mineralization of the DBM particles within the scaffold struts and shows potential to promote successful spine fusion. Here, we investigate the role of geometry and architecture in osteointegration, vascularization, and facilitation of spine fusion in a preclinical model. Inks containing HA and DBM particles in a poly(lactide-co-glycolide) elastomer were 3D-printed into scaffolds with varying relative strut angles (90° vs. 45° advancing angle), macropore size (0 µm vs. 500 µm vs. 1000 µm), and strut alignment (aligned vs. offset). The following configurations were compared with scaffolds containing no macropores: 90°/500 µm/aligned, 45°/500 µm/aligned, 90°/1000 µm/aligned, 45°/1000 µm/aligned, 90°/1000 µm/offset, and 45°/1000 µm/offset. Eighty-four female Sprague-Dawley rats underwent spine fusion with bilateral placement of the various scaffold configurations (n = 12/configuration). Osteointegration and vascularization were assessed by using microComputed Tomography and histology, and spine fusion was assessed via blinded manual palpation. The 45°/1000 µm scaffolds with aligned struts achieved the highest average fusion score (1.61/2) as well as the highest osteointegration score. Both the 45°/1000 µm/aligned and 90°/1000 µm/aligned scaffolds elicited fusion rates of 100%, which was significantly greater than the 45°/500 µm/aligned iteration (p < 0.05). All porous scaffolds were fully vascularized, with blood vessels present in every macropore. Vessels were also observed extending from the native transverse process bone, through the protrusions of new bone, and into the macropores of the scaffolds. When viewed independently, scaffolds printed with relative strut angles of 45° and 90° each allowed for osteointegration sufficient to stabilize the spine at L4-L5. Within those parameters, a pore size of 500 µm or greater was generally sufficient to achieve unilateral fusion. However, our results suggest that scaffolds printed with the larger pore size and with aligned struts at an advancing angle of 45° may represent the optimal configuration to maximize osteointegration and fusion capacity. Overall, this work suggests that the HA/DBM composite scaffolds provide a conducive environment for bone regeneration as well as vascular infiltration. This technology, therefore, represents a novel, growth-factor-free biomaterial with significant potential as a bone graft substitute for use in spinal surgery. Impact statement We previously developed a recombinant growth factor-free, three-dimensional (3D)-printed composite material comprising hydroxyapatite and demineralized bone matrix for bone regeneration. Here, we identify a range of 3D geometric and architectural parameters that support the preclinical success of the scaffold, including efficient vascularization, osteointegration, and, ultimately, spinal fusion. Our results suggest that this material holds great promise as a clinically translatable biomaterial for use as a bone graft substitute in orthopedic procedures requiring bone regeneration.

Growth Factors, Carrier Materials, and Bone Repair.

This chapter provides an overview of the growth factors active in bone regeneration and healing. Both normal and impaired bone healing are discussed, with a focus on the spatiotemporal activity of the various growth factors known to be involved in the healing response. The review highlights the activities of most important growth factors impacting bone regeneration, with a particular emphasis on those being pursued for clinical translation or which have already been marketed as components of bone regenerative materials. Current approaches the use of bone grafts in clinical settings of bone repair (including bone grafts) are summarized, and carrier systems (scaffolds) for bone tissue engineering via localized growth factor delivery are reviewed. The chapter concludes with a consideration of how bone repair might be improved in the future.

X-ray fluorescence microscopy: A method of measuring ion concentrations in the ear.

This technical note describes synchrotron x-ray fluorescence microscopy (XFM) as a method for measuring the concentrations of different elements in cross-sections of the ear at extremely high resolution. This method could be of great importance for addressing many open questions in hearing research. XFM uses synchrotron radiation to evoke emissions from many biologically relevant elements in the tissue. The intensity and wavelength of the emitted radiation provide a fingerprint of the tissue composition that can be used to measure the concentration of the elements in the sampled location. Here, we focus on energies that target biologically-relevant elements of the periodic table between magnesium and zinc. Since a highly focused x-ray beam is used, the spot size is well below 1 µm and the samples can be scanned at a nanometer lateral resolution. This study shows that measurement of the concentrations of different elements is possible in a mid-modiolar cross-section of a mouse cochlea. Images are presented that indicate potassium and chloride "hot spots" in the spiral ligament and the spiral limbus, providing experimental evidence for the potassium recycling pathway and showing the cochlear structures involved. Scans of a section obtained from the incus, one of the middle ear ossicles, in a developing mouse have shown that zinc is not uniformly distributed This supports the hypothesis that zinc plays a special role in the process of ossification. Although limited by sophisticated sample preparation and sectioning, the method provides ample exciting opportunities, to understand the role of genetics and epigenetics on hearing mechanisms in ontogeny and phylogeny.

3D-Printed Ceramic-Demineralized Bone Matrix Hyperelastic Bone Composite Scaffolds for Spinal Fusion.

Although numerous spinal biologics are commercially available, a cost-effective and safe bone graft substitute material for spine fusion has yet to be proven. In this study, "3D-Paints" containing varying volumetric ratios of hydroxyapatite (HA) and human demineralized bone matrix (DBM) in a poly(lactide-co-glycolide) elastomer were three-dimensional (3D) printed into scaffolds to promote osteointegration in rats, with an end goal of spine fusion without the need for recombinant growth factor. Spine fusion was evaluated by manual palpation, and osteointegration and de novo bone formation within scaffold struts were evaluated by laboratory and synchrotron microcomputed tomography and histology. The 3:1 HA:DBM composite achieved the highest mean fusion score and fusion rate (92%), which was significantly greater than the 3D printed DBM-only scaffold (42%). New bone was identified extending from the host transverse processes into the scaffold macropores, and osteointegration scores correlated with successful fusion. Strikingly, the combination of HA and DBM resulted in the growth of bone-like spicules within the DBM particles inside scaffold struts. These spicules were not observed in DBM-only scaffolds, suggesting that de novo spicule formation requires both HA and DBM. Collectively, our work suggests that this recombinant growth factor-free composite shows promise to overcome the limitations of currently used bone graft substitutes for spine fusion. Impact Statement Currently, there exists a no safe, yet highly effective, bone graft substitute that is well accepted for use in spine fusion procedures. With this work, we show that a three-dimensional printed scaffold containing osteoconductive hydroxyapatite and osteoinductive demineralized bone matrix that promotes new bone spicule formation, osteointegration, and successful fusion (stabilization) when implemented in a preclinical model of spine fusion. Our study suggests that this material shows promise as a recombinant growth factor-free bone graft substitute that could safely promote high rates of successful fusion and improve patient care.

Alcohol exposure decreases osteopontin expression during fracture healing and osteopontin-mediated mesenchymal stem cell migration in vitro.

BACKGROUND: Alcohol consumption is a risk factor for impaired fracture healing, though the mechanism(s) by which this occurs are not well understood. Our laboratory has previously shown that episodic alcohol exposure of rodents negatively affects fracture callus development, callus biomechanics, and cellular signaling which regulates stem cell differentiation. Here, we examine whether alcohol alters chemokine expression and/or signaling activity in the mouse fracture callus during early fracture healing. METHODS: A mouse model for alcohol-impaired tibia fracture healing was utilized. Early fracture callus was examined for alcohol-effects on tissue composition, expression of chemokines involved in MSC migration to the fracture site, and biomechanics. The effects of alcohol on MSC migration and cell adhesion receptors were examined in an in vitro system. RESULTS: Mice exposed to alcohol showed decreased evidence of external callus formation, decreased callus-related osteopontin (OPN) expression levels, and decreased biomechanical stiffness. Alcohol exposure decreased rOPN-mediated MSC migration and integrin ß1 receptor expression in vitro. CONCLUSIONS: The effects of alcohol exposure demonstrated here on fracture callus-associated OPN expression, rOPN-mediated MSC migration in vitro, and MSC integrin ß1 receptor expression in vitro have not been previously reported. Understanding the effects of alcohol exposure on the early stages of fracture repair may allow timely initiation of treatment to mitigate the long-term complications of delayed healing and/or fracture non-union.

Fluvastatin protects cochleae from damage by high-level noise.

Exposure to noise and ototoxic drugs are responsible for much of the debilitating hearing loss experienced by about 350 million people worldwide. Beyond hearing aids and cochlear implants, there have been no other FDA approved drug interventions established in the clinic that would either protect or reverse the effects of hearing loss. Using Auditory Brainstem Responses (ABR) in a guinea pig model, we demonstrate that fluvastatin, an inhibitor of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway, protects against loss of cochlear function initiated by high intensity noise. A novel synchrotron radiation based X-ray tomographic method that imaged soft tissues at micrometer resolution in unsectioned cochleae, allowed an efficient, qualitative evaluation of the three-dimensional internal structure of the intact organ. For quantitative measures, plastic embedded cochleae were sectioned followed by hair cell counting. Protection in noise-exposed cochleae is associated with retention of inner and outer hair cells. This study demonstrates the potential of HMG-CoA reductase inhibitors, already vetted in human medicine for other purposes, to protect against noise induced hearing loss.

In vitro effect of amorphous calcium phosphate paste applied for extended periods of time on enamel remineralization.

OBJECTIVES: Dental applications based on the unique characteristics of amorphous calcium phosphate stabilized by casein phosphopeptides (CPP-ACP) have been proposed, as well as the improvement of its properties. The objective of this study was to determine the ability of topically applied CPP-ACP from a commercial product to remineralize subsurface lesions when applied for extended periods of time (3 h and 8 h). MATERIAL AND METHODS: Artificially induced carious lesions were produced in 50 bovine enamel blocks previously selected by surface hardness. After treatments with gel without F and CPP-ACP applied for 1 minute (Placebo); 2% NaF neutral gel applied for 1 minute (Fluoride 1 min); CPP-ACP applied for 3 min (ACP 3 min); and CPP-ACP applied for 3 h (ACP 3 h) and for 8 h (ACP 8 h), the enamel blocks were submitted to the remineralization pH-cycling. Surface hardness and synchrotron micro-tomography were used to determine the percentage of surface hardness recovery (%SHR) and to calculate mineral concentration (gHAp.cm-3), respectively. The data were submitted to ANOVA followed by the Student-Newman-Keuls test (p<0.05). RESULTS: Fluoride gel presented higher %SHR followed by ACP 3 min (p<0.001). No difference (p = 0.148) was found for Placebo, ACP 3 h and ACP 8 h groups for %SHR. Fluoride gel showed greater mineral concentration (p<0.001) when compared with the other groups. ACP 3 min demonstrated a significant difference (p<0.001) from ACP 3 h and ACP 8 h. The ACP 3 h and 8 h presented a subsurface lesion with development of laminations in all blocks. CONCLUSION: In this in vitro study the use of CPP-ACP for extended periods of time did not produce an additive effect in the remineralization process.