Selective nanosecond laser removal of retinal pigment epithelium for cell therapy.

Retinal pigment epithelial (RPE) cells play a crucial role in the health of the retina, and their dysfunction is associated with various ocular diseases. The transplantation of RPE cells has been proposed as a potential treatment for numerous degenerative diseases, including geographic atrophy from macular degeneration. However, current models to induce RPE damage in animal models prior to transplantation involve mechanical scraping, chemical administration, or laser photocoagulation techniques, which can damage the overlying neurosensory retina. This study aims to investigate the feasibility and efficacy of nanosecond duration laser treatment to safely remove large areas of RPE cells without causing damage to the adjacent tissue or affecting the retinal architecture. Twelve pigmented rabbits were treated with a nanosecond laser on each eye at a laser energy ranging from 200 to 800 nJ with a treated area of 5 × 5 mm2. Human induced pluripotent stem cells-differentiated to RPE (hiPSC-RPE) cells labeled with indocyanine green (ICG), an FDA approved dye, were transplanted subretinally into the damaged RPE areas at day 14 post-laser treatment. The RPE atrophy and hiPSC-RPE cell survival was evaluated and monitored over a period of 14 days using color photography, fluorescein angiography (FA), photoacoustic microscopy (PAM), and optical coherence tomography (OCT) imaging. All treated eyes demonstrated focal RPE loss with a success rate of 100%. The injured RPE layers and the transplanted hiPSC-RPE cells were visualized in three dimensions using PAM and OCT. By performing PAM at an optical wavelength of 700 nm, the location of hiPSC-RPE cells were identified and distinguished from the surrounding RPE cells, and the induced PA signal increased up to 18 times. Immunohistochemistry results confirmed the grafted hiPSC-RPE replaced regions of RPE damage. This novel technique has the potential to serve as an animal model of RPE degeneration, to improve models of RPE transplantation, and may help accelerate translation of this therapeutic strategy for clinical use.

Ultrasound-assisted laser therapy for selective removal of melanoma cells.

The current study explores the potential of ultrasound-assisted laser therapy (USaLT) to selectively destroy melanoma cells. The technology was tested on an ex vivo melanoma model, which was established by growing melanoma cells in chicken breast tissue. Ultrasound-only and laser-only treatments were used as control groups. USaLT was able to effectively destroy melanoma cells and selectively remove 66.41% of melanoma cells in the ex vivo tumor model when an ultrasound peak negative pressure of 2 MPa was concurrently applied with a laser fluence of 28 mJ/cm2 at 532 nm optical wavelength for 5 min. The therapeutic efficiency was further improved with the use of a higher laser fluence, and the treatment depth was improved to 3.5 mm with the use of 1,064 nm laser light at a fluence of 150 mJ/cm2. None of the laser-only and ultrasound-only treatments were able to remove any melanoma cells. The treatment outcome was validated with histological analyses and photoacoustic imaging. This study opens the possibility of USaLT for melanoma that is currently treated by laser therapy, but at a much lower laser fluence level, hence improving the safety potential of laser therapy.

High Q-factor Polymer Microring Resonators Realized by Versatile Damascene Soft Nanoimprinting Lithography.

High-quality-factor microring resonators are highly desirable in many applications. Fabricating a microring resonator typically requires delicate instruments to ensure a smooth side wall of waveguides and 100-nm critical feature size in the coupling region. In this work, we demonstrate a new method "damascene soft nanoimprinting lithography" that can create high-fidelity waveguide by simply backfill an imprinted cladding template with a high refractive index polymer core. This method can easily realize high Q-factor polymer microring resonators (e.g., ~5 x 105 around 770 nm wavelength) without the use of any expensive instruments and can be conducted in a normal lab environment. The high Q-factors can be attributed to the residual layer-free feature and controllable meniscus cross-section profile of the filled polymer core. Furthermore, the new method is compatible with different polymers, yields low fabrication defects, enables new functionalities, and allows flexible substrate. These benefits can broaden the applicability of the fabricated microring resonator.

Photoacoustic lifetime oxygen imaging of radiotherapy-induced tumor reoxygenation In Vivo.

Purpose: Early detection and diagnosis of cancer is critical for achieving positive therapeutic outcomes. Biomarkers that can provide clinicians with clues to the outcome of a given therapeutic course are highly desired. Oxygen is a small molecule that is nearly universally present in biological tissues and plays a critical role in the effectiveness of radiotherapies by reacting with DNA radicals and subsequently impairing cellular repair of double strand breaks.Techniques for measuring oxygen in biological tissues often use blood oxygen saturation to approximate the oxygen partial pressure in surrounding tissues despite the complex, nonlinear, and dynamic relationship between these two separate oxygen populations. Methods and materials: We combined a directly oxygen-sensitive, tumor-targeted, chemical contrast nanoelement with the photoacoustic lifetime-based (PALT) oxygen imaging technique to obtain image maps of oxygen in breast cancer tumors in vivo. The oxygen levels of patient-derived xenografts in a mouse model were characterized before and after a course of radiotherapy. Results: We show that, independent of tumor size, radiotherapy induced an increase in the overall oxygenation levels of the tumor. Further, this increase in the oxygenation of the tumor significantly correlated with a positive response to radiotherapy, as demonstrated by a reduction in tumor volume over the twenty-day monitoring period following therapy and histological staining. Conclusion: Our PALT imaging presented here is simple, fast, and non-invasive. Facilized by the PALT approach, imaging of tumor reoxygenation may be utilized as a simple, early indicator for evaluating cancer response to radiotherapy. Further characterization of the reoxygenation degree, temporal onset, and possible theragnostic implications are warranted.

Label-free photoacoustic computed tomography of visually evoked responses in the primary visual cortex and four subcortical retinorecipient nuclei of anesthetized mice.

Significance: Many techniques exist for screening retinal phenotypes in mouse models in vision research, but significant challenges remain for efficiently probing higher visual centers of the brain. Photoacoustic computed tomography (PACT), with optical sensitivity to hemodynamic response (HR) in brain and ultrasound resolution, provides unique advantages in comprehensively assessing higher visual function in the mouse brain. Aim: We aim to examine the reliability of PACT in the functional phenotyping of mouse models for vision research. Approach: A PACT-ultrasound (US) parallel imaging system was established with a one-dimensional (1D) US transducer array and a tunable laser. Imaging was performed at three coronal planes of the brain, covering the primary visual cortex and the four subcortical nuclei, including the superior colliculus, the dorsal lateral geniculate nucleus, the suprachiasmatic nucleus, and the olivary pretectal nucleus. The visual-evoked HR was isolated from background signals using an impulse-based data processing protocol. rd1 mice with rod/cone degeneration, melanopsin-knockout (mel-KO) mice with photoreceptive ganglion cells that lack intrinsic photosensitivity, and wild-type mice as controls were imaged. The quantitative characteristics of the visual-evoked HR were compared. Results: Quantitative analysis of the HRs shows significant differences among the three mouse strains: (1) rd1 mice showed both smaller and slower responses compared with wild type ( n = 10,10 , p < 0.01 ) and (2) mel-KO mice had lower amplitude but not significantly delayed photoresponses than wild-type mice ( n = 10,10 , p < 0.01 ). These results agree with the known visual deficits of the mouse strains. Conclusions: PACT demonstrated sufficient sensitivity to detecting post-retinal functional deficits.

Introducing the Special Issue Honoring Lihong V. Wang, Pioneer in Biomedical Optics.

The editorial concludes the JBO Special Issue Honoring Lihong V. Wang, outlining Prof. Wang's salient contributions to advancing the field of biomedical optics.

Physiologically-based pharmacokinetic modelling to investigate the effect of CYP3A4/3A5 maturation on tacrolimus pharmacokinetics in paediatric HSCT patients.

Tacrolimus (FK506) is a cornerstone of GVHD-prophylaxis treatment in paediatrics undergoing haematopoietic stem cell transplantation (HSCT). However, due to concerns about highly inter/intra-individual variability, precision dosing of FK506 is crucial. Cytochrome P450 (CYP) 3A4 and 3A5 are considered important sources of FK506 pharmacokinetic variability. Nevertheless, the impact of age-related maturation in hepatic and intestinal CYP3A4/3A5 enzymes remains unknown in paediatric HSCT patients. Physiologically-based pharmacokinetic (PBPK) models were developed and verified in adult volunteers and adult HSCT patients using GastroPlus™ (version 9.0), and then extrapolated to paediatric HSCT patients, taking into account the maturation of CYP3A4 and CYP3A5. Default CYP3A4 and CYP3A5 ontogeny profiles were updated based on the latest reports. The paediatric PBPK model was evaluated with independent data collected from Sun Yat-sen Memorial Hospital (86 paediatric HSCT patients, 1 to 16 -year-old). Simulations were performed to evaluate a reported FK506 dosing regimen in infants and children with different CYP3A5 genotypes. Extensive PBPK model validation indicated good predictability, with the predicted/observed (P/O) ratios within the range of 0.80-fold to 1.25-fold. Blood tacrolimus concentration-time curves were comparable between the real and virtual patients. Simulations showed that the higher levels of tacrolimus in 9-month-old to 3-year-old infants were mainly attributed to the CYP3A4/3A5 ontogeny profiles, which resulted in lower clearance and higher exposure relative to dose. The oral dosage of 0.1 mg/kg/day (q12 h) is considered appropriate for paediatric HSCT patients 9 months to 15 years of age with CYP3A5 *1/*1 genotypes. Lower doses were required for paediatric HSCT patients with CYP3A5 *1/*3 (0.08 mg/kg/day, q12h) or CYP3A5 *3/*3 genotypes (0.07 mg/kg/day, q12h), and analyses demonstrated 12.5-20 % decreases in ≤3-year-old patients. The study highlights the feasibility of PBPK modelling to explore age-related enzyme maturation in infants and children (≤3-year-old) undergoing HSCT and emphasizes the need to include hepatic and gut CYP3A4/3A5 maturation parameters.

Development of a Dual-Factor Activatable Covalent Targeted Photoacoustic Imaging Probe for Tumor Imaging.

Photoacoustic imaging (PAI) is an emerging modality in biomedical imaging with superior imaging depth and specificity. However, PAI still has significant limitations, such as the background noise from endogenous chromophores. To overcome these limitations, we developed a covalent activity-based PAI probe, NOx-JS013, targeting NCEH1. NCEH1, a highly expressed and activated serine hydrolase in aggressive cancers, has the potential to be employed for the diagnosis of cancers. We show that NOx-JS013 labels active NCEH1 in live cells with high selectivity relative to other serine hydrolases. NOx-JS013 also presents its efficacy as a hypoxia-responsive imaging probe in live cells. Finally, NOx-JS013 successfully visualizes aggressive prostate cancer tumors in mouse models of PC3, while being negligibly detected in tumors of non-aggressive LNCaP mouse models. These findings show that NOx-JS013 has the potential to be used to develop precision PAI reagents for detecting metastatic progression in various cancers.

Multimodal Imaging-Guided Stem Cell Ocular Treatment.

Stem cell therapies are gaining traction as promising treatments for a variety of degenerative conditions. Both clinical and preclinical studies of regenerative medicine are hampered by the lack of technologies that can evaluate the migration and behavior of stem cells post-transplantation. This study proposes an innovative method to longitudinally image in vivo human-induced pluripotent stem cells differentiated to retinal pigment epithelium (hiPSC-RPE) cells by multimodal photoacoustic microscopy, optical coherence tomography, and fluorescence imaging powered by ultraminiature chain-like gold nanoparticle cluster (GNC) nanosensors. The GNC exhibits an optical absorption peak in the near-infrared regime, and the 7-8 nm size in diameter after disassembly enables renal excretion and improved safety as well as biocompatibility. In a clinically relevant rabbit model, GNC-labeled hiPSC-RPE cells migrated to RPE degeneration areas and regenerated damaged tissues. The hiPSC-RPE cells' distribution and migration were noninvasively, longitudinally monitored for 6 months with exceptional sensitivity and spatial resolution. This advanced platform for cellular imaging has the potential to enhance regenerative cell-based therapies.

Early proactive monitoring of DNA-thioguanine in patients with Crohn's disease predicts thiopurine-induced late leucopenia in NUDT15/TPMT normal metabolizers.

BACKGROUND: Thiopurine-induced leucopenia significantly hinders the wide application of thiopurines. Dose optimization guided by nudix hydrolase 15 (NUDT15) has significantly reduced the early leucopenia rate, but there are no definitive biomarkers for late risk leucopenia prediction. AIM: To determine the predictive value of early monitoring of DNA-thioguanine (DNATG) or 6-thioguanine nucleotides (6TGN) for late leucopenia under a NUDT15-guided thiopurine dosing strategy in patients with Crohn's disease (CD). METHODS: Blood samples were collected within two months after thiopurine initiation for detection of metabolite concentrations. Late leucopenia was defined as a leukocyte count < 3.5 × 109/L over two months. RESULTS: Of 148 patients studied, late leucopenia was observed in 15.6% (17/109) of NUDT15/thiopurine methyltransferase (TPMT) normal and 64.1% (25/39) of intermediate metabolizers. In patients suffering late leucopenia, early DNATG levels were significantly higher than in those who did not develop late leucopenia (P = 4.9 × 10-13). The DNATG threshold of 319.43 fmol/µg DNA could predict late leucopenia in the entire sample with an area under the curve (AUC) of 0.855 (sensitivity 83%, specificity 81%), and in NUDT15/TPMT normal metabolizers, the predictive performance of a threshold of 315.72 fmol/µg DNA was much more remarkable with an AUC of 0.902 (sensitivity 88%, specificity 85%). 6TGN had a relatively poor correlation with late leucopenia whether in the entire sample (P = 0.021) or NUDT15/TPMT normal or intermediate metabolizers (P = 0.018, P = 0.55, respectively). CONCLUSION: Proactive therapeutic drug monitoring of DNATG could be an effective strategy to prevent late leucopenia in both NUDT15/TPMT normal and intermediate metabolizers with CD, especially the former.

Real-Time Cavitation Monitoring During Optical Coherence Tomography Guided Photo-Mediated Ultrasound Therapy of the Retina.

OBJECTIVE: Photo-mediated ultrasound therapy (PUT) is a novel antivascular therapeutic modality based on cavitation-induced bioeffects. During PUT, synergistic combinations of laser pulses and ultrasound bursts are used to remove the targeted microvessels selectively and precisely without harming nearby tissue. In the current study, an integrated system combining PUT and spectral domain optical coherence tomography (SD-OCT) was developed, where the SD-OCT system was used to guide PUT by detecting cavitation in real time in the retina of the eye. METHOD: We first examined the capability of SD-OCT in detecting cavitation on a vascular-mimicking phantom and compared the results with those from a passive cavitation detector. The performance of the integrated system in treatment of choroidal microvessels was then evaluated in rabbit eyes in vivo. RESULTS: During the in vivo PUT experiments, several biomarkers at the subretinal layer in the rabbit eye were identified on OCT images. The findings indicate that, by evaluating biomarkers of treatment effect, real-time SD-OCT monitoring could help to avoid micro-hemorrhage, which is a potential major side effect. CONCLUSION: Real-time OCT monitoring can thus improve the safety and efficiency of PUT in removing the retinal and choroidal microvasculature.

Biomechanical properties of articular cartilage in different regions and sites of the knee joint: acquisition of osteochondral allografts.

Osteochondral allograft (OCA) transplantation involves grafting of natural hyaline cartilage and supporting subchondral bone into the cartilage defect area to restore its biomechanical and tissue structure. However, differences in biomechanical properties and donor-host matching may impair the integration of articular cartilage (AC). This study analyzed the biomechanical properties of the AC in different regions of different sites of the knee joint and provided a novel approach to OCA transplantation. Intact stifle joints from skeletally mature pigs were collected from a local abattoir less than 8 h after slaughter. OCAs were collected from different regions of the joints. The patella and the tibial plateau were divided into medial and lateral regions, while the trochlea and femoral condyle were divided into six regions. The OCAs were analyzed and compared for Young's modulus, the compressive modulus, and cartilage thickness. Young's modulus, cartilage thickness, and compressive modulus of OCA were significantly different in different regions of the joints. A negative correlation was observed between Young's modulus and the proportion of the subchondral bone (r = - 0.4241, P < 0.0001). Cartilage thickness was positively correlated with Young's modulus (r = 0.4473, P < 0.0001) and the compressive modulus (r = 0.3678, P < 0.0001). During OCA transplantation, OCAs should be transplanted in the same regions, or at the closest possible regions to maintain consistency of the biomechanical properties and cartilage thickness of the donor and recipient, to ensure smooth integration with the surrounding tissue. A 7 mm depth achieved a higher Young's modulus, and may represent the ideal length.

Association of Valproic Acid and Its Main Metabolites' Plasma Concentrations with Clinical Outcomes among Epilepsy Patients: A 10-Year Retrospective Study Based on Therapeutic Drug Monitoring.

Valproic acid (VPA) is a first-line antiepileptic drug with broad efficacy. Due to significant individual differences in its metabolism, therapeutic drug monitoring is commonly used. However, the recommended therapeutic range (50-100 µg/mL) is inadequate for predicting clinical outcomes. Additionally, the relationship between VPA metabolites and clinical outcomes remains unclear. In this retrospective study, 485 Chinese Southern Han epilepsy patients receiving VPA monotherapy were analyzed after reaching steady-state levels. Plasma concentrations of VPA and its five main metabolites were determined by liquid chromatography-mass spectrometry (LC-MS). We assessed the relevance of the recommended therapeutic VPA range for clinical outcomes and explored the association between VPA/metabolites levels and treatment efficacy/adverse effects. Vitro experiments were conducted to assess 4-ene-VPA hepatotoxicity. The therapeutic range of VPA exhibited no significant correlation with clinical outcomes, and plasma concentrations of VPA failed to serve as predictive indicators for treatment response/adverse effects. Treatment responders had higher 2-PGA concentrations (median, 26.39 ng/mL versus 13.68 ng/mL), with a threshold of 36.5 ng/mL for optimal epilepsy treatment. Patients with abnormal liver function had a higher 4-ene-VPA median concentration (6.41 µg/mL versus 4.83 µg/mL), and the ratio of 4-ene-VPA to VPA better predicted VPA-induced hepatotoxicity (area under the curve, 0.718) than 4-ene-VPA concentration. Vitro experiments revealed that 4-ene-VPA was more hepatotoxic than VPA in HepaRG and L02 cell lines. Total plasma VPA concentration does not serve as a predictor of clinical outcomes. 2-PGA concentrations may be associated with efficacy, whereas the ratio of 4-ene-VPA to VPA may be considered a better biomarker (threshold 10.03%) for VPA-induced hepatotoxicity. SIGNIFICANCE STATEMENT: This was the first and largest observational cohort in China to explore the relationship between patients' parent and metabolites concentrations of VPA and clinical outcomes during the maintenance of VPA monotherapy in epileptic patients. This study provided feasible references of VPA for epilepsy clinical treatment with a larger sample of patients compared with previous studies for a more definitive conclusion based on real-world situations. We found two potential biomarkers in predicting efficacy and liver injury, respectively. This breakthrough has the potential to assist in the rational use of VPA.

Germline USP36 Mutation Confers Resistance to EGFR-TKIs by Upregulating MLLT3 Expression in Patients with Non-Small Cell Lung Cancer.

PURPOSE: Although somatic mutations were explored in depth, limited biomarkers were found to predict the resistance of EGFR tyrosine kinase inhibitors (EGFR-TKI). Previous studies reported N6-methyladenosine (m6A) levels regulated response of EGFR-TKIs; whether the germline variants located in m6A sites affected resistance of EGFR-TKIs is still unknown. EXPERIMENTAL DESIGN: Patients with non-small cell lung cancer (NSCLC) with EGFR-activating mutation were enrolled to investigate predictors for response of EGFR-TKIs using a genome-wide-variant-m6A analysis. Bioinformatics analysis and series of molecular biology assays were used to uncover the underlying mechanism. RESULTS: We identified the germline mutation USP36 rs3744797 (C > A, K814N) was associated with survival of patients with NSCLC treated with gefitinib [median progression-free survival (PFS): CC vs. CA, 16.30 vs. 10.50 months, P < 0.0001, HR = 2.45] and erlotinib (median PFS: CC vs. CA, 14.13 vs. 9.47 months, P = 0.041, HR = 2.63). Functionally, the C > A change significantly upregulated USP36 expression by reducing its m6A level. Meanwhile, rs3744797_A (USP36 MUT) was found to facilitate proliferation, migration, and resistance to EGFR-TKIs via upregulating MLLT3 expression in vitro and in vivo. More importantly, MLLT3 and USP36 levels are tightly correlated in patients with NSCLC, which were associated with prognosis of patients. Mechanistically, USP36 MUT stabilized MLLT3 by deubiquitinating MLLT3 in nucleoli and consequently activating its downstream signaling (HIF1α and Snai). Furthermore, inhibition of MLLT3 alleviated USP36 variant-induced EGFR-TKIs resistance in EGFR-mutant NSCLC. CONCLUSIONS: These findings characterized rs3744797 as an oncogenic variant in mediating EGFR-TKI resistance and tumor aggressiveness through deubiquitinating MLLT3, highlighting the variant as a predictive biomarker for EGFR-TKI response in NSCLC.

High-Speed Centrifugation Efficiently Removes Immunogenic Elements in Osteochondral Allografts.

OBJECTIVES: The current clinical pulse lavage technique for flushing fresh osteochondral allografts (OCAs) to remove immunogenic elements from the subchondral bone is ineffective. This study aimed to identify the optimal method for removing immunogenic elements from OCAs. METHODS: We examined five methods for the physical removal of immunogenic elements from OCAs from the femoral condyle of porcine knees. We distributed the OCAs randomly into the following seven groups: (1) control, (2) saline, (3) ultrasound, (4) vortex vibration (VV), (5) low-pulse lavage (LPL), (6) high-pulse lavage (HPL), and (7) high-speed centrifugation (HSC). OCAs were evaluated using weight measurement, micro-computed tomography (micro-CT), macroscopic and histological evaluation, DNA quantification, and chondrocyte activity testing. Additionally, the subchondral bone was zoned to assess the bone marrow and nucleated cell contents. One-way ANOVA and paired two-tailed Student's t-test are used for statistical analysis. RESULTS: Histological evaluation and DNA quantification showed no significant reduction in marrow elements compared to the control group after the OCAs were treated with saline, ultrasound, or VV treatments; however, there was a significant reduction in marrow elements after LPL, HPL, and HSC treatments. Furthermore, HSC more effectively reduced the marrow elements of OCAs in the middle and deep zones compared with LPL (p < 0.0001) and HPL (p < 0.0001). Macroscopic evaluation revealed a significant reduction in blood, lipid, and marrow elements in the subchondral bone after HSC. Micro-CT, histological analyses, and chondrocyte viability results showed that HSC did not damage the subchondral bone and cartilage; however, LPL and HPL may damage the subchondral bone. CONCLUSION: HSC may play an important role in decreasing immunogenicity and therefore potentially increasing the success of OCA transplantation.

Photoacoustic imaging of squirrel monkey cortical responses induced by peripheral mechanical stimulation.

Non-human primates (NHPs) are crucial models for studies of neuronal activity. Emerging photoacoustic imaging modalities offer excellent tools for studying NHP brains with high sensitivity and high spatial resolution. In this research, a photoacoustic microscopy (PAM) device was used to provide a label-free quantitative characterization of cerebral hemodynamic changes due to peripheral mechanical stimulation. A 5 × 5 mm area within the somatosensory cortex region of an adult squirrel monkey was imaged. A deep, fully connected neural network was characterized and applied to the PAM images of the cortex to enhance the vessel structures after mechanical stimulation on the forelimb digits. The quality of the PAM images was improved significantly with a neural network while preserving the hemodynamic responses. The functional responses to the mechanical stimulation were characterized based on the improved PAM images. This study demonstrates capability of PAM combined with machine learning for functional imaging of the NHP brain.

Development of a semi-anthropomorphic photoacoustic calcaneus phantom based on nano computed tomography and stereolithography 3D printing.

Osteoporosis is a major public health threat with significant physical, psychosocial, and financial consequences. The calcaneus bone has been used as a measurement site for risk prediction of osteoporosis by noninvasive quantitative ultrasound (QUS). By adding optical contrast to QUS, our previous studies indicate that a combination of photoacoustic (PA) and QUS, that is, PAQUS, provides a novel opportunity to assess the health of human calcaneus. Calibration of the PAQUS system is crucial to realize quantitative and repeatable measurements of the calcaneus. Therefore, a phantom which simulates the optical, ultrasound, and architectural properties of the human calcaneus, for PAQUS system calibration, is required. Additionally, a controllable phantom offers researchers a versatile framework for developing versatile structures, allowing more controlled assessment of how varying bone structures cause defined alterations in PA and QUS signals. In this work, we present the first semi-anthropomorphic calcaneus phantom for PAQUS. The phantom was developed based on nano computed-tomography (nano-CT) and stereolithography 3D printing, aiming to maximize accuracy in the approximation of both trabecular and cortical bone microstructures. Compared with the original digital input calcaneus model from a human cadaveric donor, the printed model achieved accuracies of 71.15% in total structure and 87.21% in bone volume fraction. Inorganic materials including synthetic blood, mineral oil, intralipid, and agar gel were used to model the substitutes of bone marrow and soft tissue, filling and covering the calcaneus phantom. The ultrasound and optical properties of this phantom were measured, and the results were consistent with those measured by a commercialized device and from previous in vivo studies. In addition, a short-term stability test was conducted for this phantom, demonstrating that the optical and ultrasound properties of the phantom were stable without significant variation over 1 month. This semi-anthropomorphic calcaneus phantom shows structural, ultrasound, and optical properties similar to those from a human calcaneus in vivo and, thereby, can serve as an effective source for equipment calibration and the comprehensive study of human patients.

Longitudinal Monitoring of Angiogenesis in a Murine Window Chamber Model In Vivo.

Angiogenesis induced by growth factor administration, which can augment the blood supply in regenerative applications, has drawn wide attention in medical research. Longitudinal monitoring of vascular structure and development in vivo is important for understanding and evaluating the dynamics of involved biological processes. In this work, a dual-modality imaging system consisting of photoacoustic microscopy (PAM) and optical coherence tomography (OCT) was applied for noninvasive in vivo imaging of angiogenesis in a murine model. Fibrin scaffolds, with and without basic fibroblast growth factor (bFGF), were implanted in a flexible imaging window and longitudinally observed over 9 days. Imaging was conducted at 3, 5, 7, and 9 days after implantation to monitor vascularization in and around the scaffold. Several morphometric parameters were derived from the PAM images, including vessel area density (VAD), total vessel length (TVL), and vessel mean diameter (VMD). On days 7 and 9, mice receiving bFGF-laden fibrin gels exhibited significantly larger VAD and TVL compared to mice with fibrin-only gels. In addition, VMD significantly decreased in +bFGF mice versus fibrin-only mice on days 7 and 9. Blood vessel density, evaluated using immunohistochemical staining of explanted gels and underlying tissue on day 9, corroborated the findings from the PAM images. Overall, the experimental results highlight the utility of a dual-modality imaging system in longitudinally monitoring of vasculature in vivo with high resolution and sensitivity, thereby providing an effective tool to study angiogenesis.

Cutaneous Hypervascularization Treatment Using Photo-Mediated Ultrasound Therapy.

Photo-mediated ultrasound therapy (PUT) is a cavitation-based, highly selective antivascular technique. In this study, the effectiveness and safety of PUT on cutaneous vascular malformation was examined through in vivo experiments in a clinically relevant chicken wattle model, whose microanatomy is similar to that of port-wine stain and other hypervascular dermal diseases in humans. Assessed by optical coherence tomography angiography, the blood vessel density in the chicken wattle decreased by 73.23% after one session of PUT treatment in which 0.707 J/cm2 fluence laser pulses were applied concurrently with ultrasound bursts (n = 7, P < .01). The effectiveness of removing blood vessels in the skin at depth up to 1 mm was further assessed by H&E-stained histology at multiple time points, which included days 1, 3, 7, 14, and 21 after treatment. Additional immunohistochemical analyses with CD31, caspase-3, and Masson's trichrome stains were performed on day 3 after treatment. The results show that the PUT-induced therapeutic effect was confined and specific to blood vessels only, whereas unwanted collateral damage in other skin tissues such as collagen was avoided. The findings from this study demonstrate that PUT can efficiently and safely remove hypervascular dermal capillaries using laser fluence at a level that is orders of magnitude smaller than that used in conventional laser treatment of vascular lesions, thus offering a safer alternative technique for clinical management of cutaneous vascular malformations.

Photoacoustic Chemical Imaging Sodium Nano-Sensor Utilizing a Solvatochromic Dye Transducer for In Vivo Application.

Sodium has many vital and diverse roles in the human body, including maintaining the cellular pH, generating action potential, and regulating osmotic pressure. In cancer, sodium dysregulation has been correlated with tumor growth, metastasis, and immune cell inhibition. However, most in vivo sodium measurements are performed via Na23 NMR, which is handicapped by slow acquisition times, a low spatial resolution (in mm), and low signal-to-noise ratios. We present here a plasticizer-free, ionophore-based sodium-sensing nanoparticle that utilizes a solvatochromic dye transducer to circumvent the pH cross-sensitivity of most previously reported sodium nano-sensors. We demonstrate that this nano-sensor is non-toxic, boasts a 200 µM detection limit, and is over 1000 times more selective for sodium than potassium. Further, the in vitro photoacoustic calibration curve presented demonstrates the potential of this nano-sensor for performing the in vivo chemical imaging of sodium over the entire physiologically relevant concentration range.