Adaptation of a Clinical High-Frequency Transrectal Ultrasound System for Prostate Photoacoustic Imaging: Implementation and Pre-clinical Demonstration.

OBJECTIVE: High-frequency, high-resolution transrectal micro-ultrasound (micro-US: ≥15 MHz) imaging of the prostate is emerging as a beneficial tool for scoring disease risk and accurately targeting biopsies. Adding photoacoustic (PA) imaging to visualize abnormal vascularization and accumulation of contrast agents in tumors has potential for guiding focal therapies. In this work, we describe a new imaging platform that combines a transrectal micro-US system with transurethral light delivery for PA imaging. METHODS: A clinical transrectal micro-US system was adapted to acquire PA images synchronous to a tunable laser pulse. A transurethral side-firing optical fiber was developed for light delivery. A polyvinyl chloride (PVC)-plastisol phantom was developed and characterized to image PA contrast agents in wall-less channels. After resolution measurement in water, PA imaging was demonstrated in phantom channels with dyes and biodegradable nanoparticle contrast agents called porphysomes. In vivo imaging of a tumor model was performed, with porphysomes administered intravenously. RESULTS: Photoacoustic imaging data were acquired at 5 Hz, and image reconstruction was performed offline. PA image resolution at a 14-mm depth was 74 and 261 µm in the axial and lateral directions, respectively. The speed of sound in PVC-plastisol was 1383 m/s, and the attenuation was 4 dB/mm at 20 MHz. PA signal from porphysomes was spectrally unmixed from blood signals in the tumor, and a signal increase was observed 3 h after porphysome injection. CONCLUSION: A combined transrectal micro-US and PA imaging system was developed and characterized, and in vivo imaging demonstrated. High-resolution PA imaging may provide valuable additional information for diagnostic and therapeutic applications in the prostate.

Real-time visualisation of peripheral nerve trauma during subepineural injection in pig brachial plexus using micro-ultrasound.

BACKGROUND: Nerve damage is consistently demonstrated after subepineural injection in animal studies, but not after purposeful injection in patients participating in clinical studies. There is a need to better visualise nerves in order to understand the structural changes that occur during subepineural injection. METHODS: We scanned the brachial plexuses of three anaesthetised pigs using micro-ultrasound imaging (55-22 MHz probe), inserted 21 gauge block needles into the radial, median, and axillary nerves, and injected two 0.5 ml boluses of saline into nerves at a rate of 12 ml min-1. Our objectives were to measure the area and diameter of nerves and fascicles, and to describe changes in nerve anatomy, comparing our findings with histology. RESULTS: Images were acquired at 42 sites across 18 nerves in three pigs and compared dimensions (geometric ratio; 95% confidence interval; P value). As expected, the nerve cross-sectional area was greater in the proximal brachial plexus compared with the mid-plexus (2.10; 1.07-4.11; P<0.001) and the distal plexus (2.64; 1.42-4.87; P<0.001). Nerve area expanded after 0.5 ml injection (2.13; 1.48-3.08; P<0.001). Using microultrasound, subepineural injection was characterised by nerve and fascicle rotation, uniform, or localised swelling and epineural rupture. Micro-ultrasound revealed a unique pattern suggestive of subperineural injection after a median nerve injection, and good face validity with histology. Histology showed epineural trauma and inflammation to the perineurium. CONCLUSION: We accurately identified fascicles and real-time structural changes to peripheral nerves using micro-ultrasound. This is the first study to visualise in vivo and in real-time the motion of nerves and fascicles in response to anaesthetic needle insertion and fluid injection.

Tetrazine-Derived Near-Infrared Dye as a Facile Reagent for Developing Targeted Photoacoustic Imaging Agents.

A new photoacoustic (PA) dye was developed as a simple-to-use reagent for creating targeted PA imaging agents. The lead molecule was prepared via an efficient two-step synthesis from an inexpensive commercially available starting material. With the dye's innate albumin-binding properties, the resulting tetrazine-derived dye is capable of localizing to tumor and exhibits a biological half-life of a few hours, allowing for an optimized distribution profile. The presence of tetrazine in turn makes it possible to link the albumin-binding optoacoustic signaling agent to a wide range of targeting molecules. To demonstrate the utility and ease of use of the platform, a novel PA probe for imaging calcium accretion was generated using a single-step bioorthogonal coupling reaction where high-resolution PA images of the knee joint in mice were obtained as early as 1 h post injection. Whole-body distribution was subsequently determined by labeling the probe with 99mTc and performing tissue counting following necropsy. These studies, along with tumor imaging and in vitro albumin binding studies, revealed that the core PA contrast agent can be imaged in vivo and can be easily linked to targeting molecules for organ-specific uptake.

Minor Review: An Overview of a Synthetic Nanophase Bone Substitute.

Material is reviewed that consists of reconstituted collagen fibril gel mineralized in a manner that produces biomimetically sized nanoapatites intimately associated with the fibrils. This gel is formed into usable shapes with a modulus and strength that allow it to be surgically press fitted into bony defects. The design paradigm for the material is that the nanoapatites will dissolve into soluble Ca2+ as the collagen is degraded into RGD-containing peptide fragments due to osteoclastic action. This is intended to signal to the osteoclasts to continue removing the material in a biomimetic fashion similar to bony remodeling. Preliminary experiments in a subcutaneous rat model show that the material is biocompatible with respect to inflammatory and immunogenic responses, and that it supports cellular invasion. Preliminary experiments in a critical-sized mandibular defect in rats show that the material is resorbable and functions well as a bone morphogenetic 2 (BMP-2) carrier. We have produced a range of mechanical and biological responses by varying mechanical and chemical processing of the material.

Nanophase bone substitute for craniofacial load bearing application: Pilot study in the rodent.

An exploratory pilot study shows that a rodent mandibular defect model is useful in determining the biological response to a nanophase collagen/apatite composite designed as a biomimetic load-bearing bone substitute. Using a critical size defect, eight groups of rats (n = 3) were implanted with four renditions of the nanophase bone substitute (NBS) biomaterial. Each rendition was tested with and without recombinant human bone morphogenetic protein 2 (BMP2). NBS biomaterial renditions were: baseline, hyper-densified, d-ribose crosslinked, and d-ribose crosslinked and hyper-densified. Biological outcomes were assessed surgically, radiologically, and histologically. With the limited power available due to the small N's involved, some interesting hypotheses were generated that will be more fully investigated in future studies. BMP2 loaded NBS, when uncrosslinked, resulted in robust bone formation in the entire defect volume (regardless of porosity). Unloaded NBS were well tolerated but did not cause significant new bone formation in the defect volume. Densification alone had little effect on in vivo performance. Crosslinking thwarted implant uptake of BMP2 and resulted in fibrous encapsulation. It is concluded that the nanophase bone substitute is well tolerated in this bone defect model. When loaded with BMP2, implantation resulted in complete bony healing and defect closure with implant density (porosity) having little effect on bone healing or remodeling. Without BMP2 the biomaterial did not result in defect closure. Crosslinking, necessary to increase mechanical properties in an aqueous environment, disrupts osteointegration and BMP2 uptake. Alternate implant fabrication strategies will be necessary to achieve an improved balance between material strength and osteointegration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 520-532, 2018.

The role of bone sialoprotein in the tendon-bone insertion.

Tendons/ligaments insert into bone via a transitional structure, the enthesis, which is susceptible to injury and difficult to repair. Fibrocartilaginous entheses contain fibrocartilage in their transitional zone, part of which is mineralized. Mineral-associated proteins within this zone have not been adequately characterized. Members of the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family are acidic phosphoproteins expressed in mineralized tissues. Here we show that two SIBLING proteins, bone sialoprotein (BSP) and osteopontin (OPN), are present in the mouse enthesis. Histological analyses indicate that the calcified zone of the quadriceps tendon enthesis is longer in Bsp(-/-) mice, however no difference is apparent in the supraspinatus tendon enthesis. In an analysis of mineral content within the calcified zone, micro-CT and Raman spectroscopy reveal that the mineral content in the calcified fibrocartilage of the quadriceps tendon enthesis are similar between wild type and Bsp(-/-) mice. Mechanical testing of the patellar tendon shows that while the tendons fail under similar loads, the Bsp(-/-) patellar tendon is 7.5% larger in cross sectional area than wild type tendons, resulting in a 16.5% reduction in failure stress. However, Picrosirius Red staining shows no difference in collagen organization. Data collected here indicate that BSP is present in the calcified fibrocartilage of murine entheses and suggest that BSP plays a regulatory role in this structure, influencing the growth of the calcified fibrocartilage in addition to the weakening of the tendon mechanical properties. Based on the phenotype of the Bsp(-/-) mouse enthesis, and the known in vitro functional properties of the protein, BSP may be a useful therapeutic molecule in the reattachment of tendons and ligaments to bone.

Nanophase bone substitute in vivo response to subcutaneous implantation.

A collagen-apatite composite designed as a load-bearing bone substitute implant is used to characterize the relationship between implant morphology and in vivo behavior. This nanophase bone substitute (NBS) is studied morphologically using a nondestructive imaging technique and biologically using the rodent subcutaneous model. Porosity and pore interconnectivity are correlated with histological outcomes showing cellular invasion occurs with average pore sizes below 100 µm. Crosslinking with D-ribose is shown to affect cellular infiltration in a dose-response manner. These data suggest that collagen-apatite bone substitutes can support cellular infiltration with pore size significantly smaller than 100 µm, an encouraging result regarding development of the NBS into a platform of biomaterials with enhanced mechanical properties. The data also indicate that increasing crosslinking density decreases cellular infiltration of NBS. Thus, modulating mechanical properties of the material by altering crosslink density is likely to produce decreased biological response within the material.