<p>Figure legends to accompany Supplemental Figure S1 and Supplemental Figure S2</p>

<p>ROI analysis of ex vivo and in situ tumor and normal tissues.</p>

<p>Imaging and ROI analysis of ex vivo tumor and normal tissues.</p>

3D Printing (3DP) or additive manufacturing (AM) enables parts with complex shapes, design flexibility, and customization opportunities for defect specific patient-matched implants. 3DP or AM also offers a design platform that can be used to innovate novel alloys for application-specific compositional modifications. In medical applications, the biological response from a host tissue depends on a biomaterial's structural and compositional properties in the physiological environment. Application of 3DP can pave the way towards manufacturing innovative metallic implants, combining structural variations at different length scales and tailored compositions designed for specific biological responses. This study shows how 3DP can be used to design metallic alloys for orthopedic and dental applications with improved biocompatibility using in vitro and in vivo studies. Titanium (Ti) and its alloys are used extensively in biomedical devices due to excellent fatigue and corrosion resistance and good strength to weight ratio. However, Ti alloys' in vivo biological response is poor due to its bioinert surface. Different coatings and surface modification techniques are currently being used to improve the biocompatibility of Ti implants. We focused our efforts on improving Ti's biocompatibility via a combination of tantalum (Ta) chemistry in Ti, the addition of designed micro-porosity, and nanoscale surface modification to enhance both in vitro cytocompatibility and early stage in vivo osseointegration, which was studied in rat and rabbit distal femur models.

Cobalt-chromium-molybdenum (CoCrMo) alloys are widely used in load-bearing implants; specifically, in hip, knee, and spinal applications due to their excellent wear resistance. However, due to in vivo corrosion and mechanically assisted corrosion, metal ion release occurs and accounts for poor biocompatibility. Therefore, a significant interest to find an alternative to CoCrMo alloy exists. In the present work we hypothesize that calcium phosphate (CaP) will behave as a solid lubricant in CoCrMo alloy under tribological testing, thereby minimizing wear and metal ion release concerns associated with CoCrMo alloy. CoCrMo-CaP composite coatings were processed using laser engineered net shaping (LENS™) system. After LENS™ processing, CoCrMo alloy was subjected to laser surface melting (LSM) using the same LENS™ set-up. Samples were investigated for microstructural features, phase identification, and biocompatibility. It was found that LSM treated CoCrMo improved wear resistance by 5 times. CoCrMo-CaP composites displayed the formation of a phosphorus-based tribofilm. In vitro cell-material interactions study showed no cytotoxic effect. Sprague-Dawley rat and rabbit in vivo study displayed increased osteoid formation for CoCrMo-CaP composites, up to 2 wt.% CaP. Our results show that careful surface modification treatments can simultaneously improve wear resistance and in vivo biocompatibility of CoCrMo alloy, which can correlate to a reduction of metal ion release in vivo.

Osteosarcoma is by far the most common primary bone tumour in dogs, accounting for up to 85% of malignancies originating in the skeleton. This chapter covers osteosarcoma in dogs; other primary bone tumours of dogs; metastatic tumours of bone; primary bone tumours of cats.

BLZ-100 is a single intravenous use, fluorescent imaging agent that labels tumor tissue to enable more complete and precise surgical resection. It is composed of a chlorotoxin peptide covalently bound to the near-infrared fluorophore indocyanine green. BLZ-100 is in clinical development for intraoperative visualization of human tumors. The nonclinical safety and pharmacokinetic (PK) profile of BLZ-100 was evaluated in mice, rats, canines, and nonhuman primates (NHP). Single bolus intravenous administration of BLZ-100 was well tolerated, and no adverse changes were observed in cardiovascular safety pharmacology, PK, and toxicology studies in rats and NHP. The single-dose no-observed-adverse-effect-levels (NOAELs) were 7 mg (28 mg/kg) in rats and 60 mg (20 mg/kg) in NHP, corresponding to peak concentration values of 89 400 and 436 000 ng/mL and area-under-the-curve exposure values of 130 000 and 1 240 000 h·ng/mL, respectively. Based on a human imaging dose of 3 mg, dose safety margins are >100 for rat and monkey. BLZ-100 produced hypersensitivity reactions in canine imaging studies (lethargy, pruritus, swollen muzzle, etc). The severity of the reactions was not dose related. In a follow-up study in dogs, plasma histamine concentrations were increased 5 to 60 minutes after BLZ-100 injection; this coincided with signs of hypersensitivity, supporting the conclusion that the reactions were histamine based. Hypersensitivity reactions were not observed in other species or in BLZ-100 human clinical studies conducted to date. The combined imaging, safety pharmacology, PK, and toxicology studies contributed to an extensive initial nonclinical profile for BLZ-100, supporting first-in-human clinical trials.

Plasma-sprayed hydroxyapatite (HA)-coated titanium implants have been widely used in orthopedic applications due to their inheritance of an excellent mechanical property from titanium and great osteoconductivity from HA. However, the lack of osteoinductivity limits their further applications. In this study, 1 wt % MgO and 0.5 wt % SiO2 were mixed with HA for making plasma-sprayed coatings on titanium implants. Plasma-sprayed HA- and MgO/SiO2-HA-coated titanium implants showed adhesive bond strengths of 25.73 ± 1.92 and 23.44 ± 2.89 MPa, respectively. The presence of MgO and SiO2 significantly increased the osteogenesis, osseointegration, and bone mineralization of HA-coated titanium implants by the evaluation of their histomorphology after 6, 10, and 14 weeks of implantation in rat distal femoral defects. Implant pushout tests also showed a shear modulus of 149.83 ± 3.69 MPa for MgO/SiO2-HA-coated implants after 14 weeks of implantation, compared to 52.68 ± 10.41 MPa for uncoated implants and 83.92 ± 3.68 MPa for pure HA-coated implants; These are differences in the shear modulus of 96% and 56.4%, respectively. This study assesses for the first time the quality of the bone-implant interface of induction plasma-sprayed MgO and SiO2 binary-doped HA coatings on load-bearing implants compared to bare titanium and pure HA coatings in a quantitative manner. Relating the osseointegration and interface shear modulus to the quality of implant fixation is critical to the advancement and implementation of HA-coated orthopedic implants.

Over 230,000 new breast cancer cases occur in the US annually. Complete surgical removal of tumor tissue is critical to reduce the risk of local recurrence. About 24% of patients who undergo breast conservation surgery require a second surgery due to positive margins. Second surgeries can be psychologically and financially costly to patients. BLZ-100, a peptide-fluorophore conjugate, is a Tumor Paint product candidate being developed to provide real-time, high resolution, intraoperative optical contrast between tumor and normal tissue, potentially helping surgeons accomplish more complete and accurate tumor resections. Intraoperative imaging with BLZ-100 may potentially improve the percentage of breast cancer patients achieving a complete resection and negative margins at their first surgical procedure. To better define the relationship between tumor pathology and BLZ-100 fluorescence, data from mouse models and canine mammary cancer cases were evaluated. Human breast cancer was modeled in mice using cell lines and xenograft models using patient-derived tumors. Review of histopathology was also conducted for each dog case. Animals received a single IV dose of BLZ-100, and tumors were excised one day after dosing. Excised tumor and normal tissues were imaged using the Odyssey Clx (LI-COR) to determine the ratio of signal in tumor compared to normal (background) tissue (TBR). Detailed histopathology reports for the dog cases were also analyzed to determine factors potentially associated with fluorescence intensity and TBR. A significant BLZ-100 TBR was observed. Histopathologic features potentially associated with TBR in dogs included cellularity and the degree of infiltration and demarcation. The studies supported the concept that BLZ-100 might potentially serve as an intraoperative optical imaging agent for breast cancer surgery. Citation Format: Stacey Hansen, Janean Fidel, Katie Kennedy, William Dernell, Boel Fransson, Josh Ramsay, Valorie Wiss, Mark Stroud, Julia E. Parrish-Novak. BLZ-100 provides optical contrast in animal models of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4245.

Abstract There is a need in surgical oncology for contrast agents that can enable real-time intraoperative visualization of solid tumors that can enable complete resections while sparing normal surrounding tissues. The Tumor Paint agent BLZ-100 is a peptide–fluorophore conjugate that can specifically bind solid tumors and fluoresce in the near-infrared range, minimizing light scatter and signal attenuation. In this study, we provide a preclinical proof of concept for use of this imaging contrast agent as administered before surgery to dogs with a variety of naturally occurring spontaneous tumors. Imaging was performed on excised tissues as well as intraoperatively in a subset of cases. Actionable contrast was achieved between tumor tissue and surrounding normal tissues in adenocarcinomas, squamous cell carcinomas, mast cell tumors, and soft tissue sarcomas. Subcutaneous soft tissue sarcomas were labeled with the highest fluorescence intensity and greatest tumor-to-background signal ratio. Our results establish a foundation that rationalizes clinical studies in humans with soft tissue sarcoma, an indication with a notably high unmet need. Cancer Res; 75(20); 4283–91. ©2015 AACR.

The presence of interconnected macro pores allows guided tissue regeneration in tissue engineering scaffolds. However, highly porous scaffolds suffer from having poor mechanical strength. Previously, we showed that microwave sintering could successfully be used to improve mechanical strength of macro porous tricalcium phosphate (TCP) scaffolds. This study reports the presence of SrO and MgO as dopants in TCP scaffolds improves mechanical and in vivo biological performance. We have used direct three dimensional printing (3DP) technology for scaffold fabrication. These 3DP scaffolds possessed multiscale porosity, that is, 3D interconnected designed macro pores along with intrinsic micro pores. A significant increase in mechanical strength, between 37 and 41%, was achieved due to SrO and MgO doping in TCP as compared with pure TCP. Maximum compressive strengths of 9.38 ± 1.86 MPa and 12.01 ± 1.56 MPa were achieved by conventional and microwave sintering, respectively, for SrO-MgO-doped 3DP scaffolds with 500 μm designed pores. Histomorphological and histomorphometric analysis revealed a significantly higher osteoid, bone and haversian canal formation induced by the presence of SrO and MgO dopants in 3DP TCP as compared with pure TCP scaffolds when tested in rabbit femoral condyle defect model. Increased osteon and thus enhanced network of blood vessel formation, and osteocalcin expression were observed in the doped TCP scaffolds. Our results show that these 3DP SrO-MgO-doped TCP scaffolds have the potential for early wound healing through accelerated osteogenesis and vasculogenesis.

Abstract Tumor PaintTM technology is designed to provide real-time visualization of tumors during surgery at improved resolution over existing methods. BLZ-100 is a Tumor Paint product consisting of a chlorotoxin (CTX) peptide conjugated to a near-infrared (NIR) fluorophore that is being advanced toward clinical studies. CTX-based Tumor Paint products have been shown to illuminate a broad range of cancers in mouse models. To facilitate clinical translation, a preclinical feasibility study was conducted in dogs with naturally occurring solid tumors. Many types of canine tumors resemble human disease, including sarcomas, mammary and lung cancers, mucosal squamous cell cancers, and gliomas. The diversity of tumor size and type, surrounding tissue, and patient body mass provides a model that is superior to the mouse in predicting the clinical characteristics of BLZ-100, including tumor penetration, background staining, and effective imaging dose. Twenty-seven dogs were given BLZ-100 intravenously 24 - 48 hours before surgery. Doses, normalized to body surface area (mg/m2), were 0.25 - 0.8 (7 dogs), 0.8 - 1.2 (15 dogs) and 1.2 - 1.6 (5 dogs). Tumor types were sarcoma, adenocarcinoma, mastocytoma, squamous cell carcinoma, and meningioma. Dogs received standard of care including tumor resection with intent to control or cure local disease. Excised tissues were imaged using the IVIS Spectrum (Caliper) and the Odyssey NIR scanner (Li-Cor) to determine overall signal in tumor and gross tumor to normal ratios. Tissues were then embedded in OCT, sectioned on a cryostat, and scanned on the Odyssey. Serial sections were stained with H&E, and comparison with the fluorescence scans was used to validate the specificity of BLZ-100 for tumor tissue. Ex vivo imaging data showed maximal tumor fluorescence at doses above 0.9 mg/m2. In dogs treated with 0.8 mg/m2 or higher (20 dogs in total), ratios of fluorescence in tumour to normal surrounding tissue ranged from <1 (no specific signal, 2 dogs) to >200, with good differentiation in several tumor types including meningioma, carcinomas (lung, thyroid, and mammary), and sarcomas. Highest signals and gross tumor to background ratios were seen in a subset of soft tissue sarcomas, suggesting preferential uptake of the conjugate in these tumor types. Histologic analysis of these cases showed 95% sensitivity and 85% specificity. Intraoperative imaging was conducted in several cases, using a prototype open NIR imaging device. These cases showed good contrast between gross tumors and surrounding structures, detection of tumor through 0.5 cm of normal fatty tissue, and low background in tissues such as fat and muscle. The information obtained in this study provides data that will be valuable in clinical translation of BLZ-100. This project was funded in whole with Federal funds from National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN2612012000054C. Citation Format: William S. Dernell, Janean Fidel, Katie Kennedy, Stacey Hansen, Valorie Wiss, Mark Stroud, Julia E. Parrish-Novak. Tumor PaintTM technology detects naturally occurring solid tumors in dogs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4936. doi:10.1158/1538-7445.AM2014-4936

Spermatogenesis is a multistep synchronized process. Diploid spermatogonia differentiate into haploid spermatozoa following mitosis, meiosis and spermiogenesis. Division and differentiation of male germ cells is achieved through the sequential expression of several genes. Numerous mRNAs in the differentiating germ cells undergo post-transcriptional and translational regulation. MiRNAs are powerful negative regulators of mRNA transcription, stability, and translation and recognize their mRNA targets through base-pairing. Retinoic acid (RA) signaling is essential for spermatogenesis and testicular function. Testicular RA level is critical for RA signal transduction. This study investigated the miRNAs modulation in an RA- induced testicular environment following the administration of all-trans RA (2 µM) and CYP26B1- inhibitor (1 µM) compared to control. Eighty four canine mature miRNAs were analyzed and their expression signatures were distinguished using real-time PCR based array technology. Of the miRNAs analyzed, miRNA families such as miR-200 (cfa-miR-200a, cfa-miR-200b and cfa-miR-200c), Mirlet-7 (cfa-let-7a, cfa-let-7b, cfa-let-7c, cfa-let-7g and cfa-let-7f), miR-125 (cfa-miR-125a and cfa-miR-125b), miR-146 (cfa-miR-146a and cfa-miR-146b), miR-34 (cfa-miR-34a, cfa-miR-34b and cfa-miR-34c), miR-23 (cfa-miR-23a and cfa-miR-23b), cfa-miR-184, cfa-miR-214 and cfa-miR-141 were significantly up-regulated with testicular RA intervention via administration of CYP26B1 inhibitor and all-trans-RA and species of miRNA such as cfa-miR-19a, cfa-miR-29b, cfa-miR-29c, cfa-miR-101 and cfa-miR-137 were significantly down-regulated. This study explored information regarding chromosome distribution, human orthologous sequences and the interaction of target genes of miRNA families significantly distinguished in this study using prediction algorithms. This study importantly identified dysregulated miRNA species resulting from RA-induced spermatogenesis. The present contribution serves as a useful resource for further elucidation of the regulatory role of individual miRNA in RA synchronized canine spermatogenesis.

Veterinary SurgeryVolume 39, Issue 2 p. 224-225 Preparation of Canine and Feline Cadavers for Surgical Laboratories KYLE G. MATHEWS DVM, MS, Diplomate ACVS, KYLE G. MATHEWS DVM, MS, Diplomate ACVS Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this authorKRISTEN RILEY BS, CVT, KRISTEN RILEY BS, CVT Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this authorB. D. X. LASCELLES BVSc, PhD, Diplomate ECVS & ACVS, B. D. X. LASCELLES BVSc, PhD, Diplomate ECVS & ACVS Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this authorWILLIAM S. DERNELL DVM, MS, Diplomate ACVS, WILLIAM S. DERNELL DVM, MS, Diplomate ACVS Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this author KYLE G. MATHEWS DVM, MS, Diplomate ACVS, KYLE G. MATHEWS DVM, MS, Diplomate ACVS Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this authorKRISTEN RILEY BS, CVT, KRISTEN RILEY BS, CVT Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this authorB. D. X. LASCELLES BVSc, PhD, Diplomate ECVS & ACVS, B. D. X. LASCELLES BVSc, PhD, Diplomate ECVS & ACVS Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this authorWILLIAM S. DERNELL DVM, MS, Diplomate ACVS, WILLIAM S. DERNELL DVM, MS, Diplomate ACVS Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC; K9 Care, LLC, Washington, DC Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WASearch for more papers by this author First published: 03 February 2010 https://doi.org/10.1111/j.1532-950X.2009.00586.xCitations: 2 Corresponding author: Dr. Kyle G. Mathews, DVM, MS, Diplomate ACVS, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606. E-mail: [email protected]. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume39, Issue2February 2010Pages 224-225 RelatedInformation

Survival following amputation and administration of single-agent carboplatin for treatment of appendicular osteosarcoma (OSA) in dogs was retrospectively examined. Records of 155 dogs with appendicular OSA treated with amputation and single-agent carboplatin were included from 14 centers. Any carboplatin dosage, number of doses, and protocol schedule were eligible for inclusion. The median disease-free interval (DFI) was 256 days. The median overall survival time was 307 days. Similar prognostic survival factors were identified in this study as reported in prior studies of canine appendicular OSA. Median DFI and survival were comparable to those reported in the original Bergman et al publication. Carboplatin treatment improves the survival probability in dogs with appendicular OSA compared to amputation alone and remains an acceptable alternative to adjuvant treatment with cisplatin.

Being the first lymph node or nodes to which many primary tumours reliably drain, the disease status of the sentinel lymph node/s (SLN) is important in the prediction of survival. SLN identification and biopsy are critical in the staging of human cancers. The status of the SLN helps determine prognosis and shape treatment plans. SLN evaluation is currently not routinely performed in veterinary oncology, not even at specialty oncology practices. Given the prognostic importance of lymph node involvement in tumours such as mammary gland carcinoma, osteosarcoma, synovial cell sarcoma and mast cell tumours, SLN evaluation should be incorporated into routine clinical practice so as to improve our clinical assessment of veterinary oncologic patients.