Blake Eason Hildreth

Comparison of arthroscopy and arthrotomy for diagnosis of medial meniscal pathology: an ex vivo study.

OBJECTIVE To evaluate the sensitivity and specificity of arthroscopy and arthrotomy for diagnosis of medial meniscal pathology and to evaluate the diagnostic value of medial meniscal probing. STUDY DESIGN Ex vivo study. ANIMALS Cadaveric canine stifle joints (n=30). METHODS Stifle joints were assigned to either a cranial cruciate ligament (CrCL) deficient or intact group. Within each stifle joint, no medial meniscal tear, a peripheral detachment, or 1 of 3 variants of vertical longitudinal tears of the medial meniscus were created. Each stifle joint had arthroscopy, craniomedial (CrMed), and caudomedial (CdMed) arthrotomy. Diagnoses were made by both observation and probing. Sensitivity, specificity, and correct classification rate (CCR) for diagnosing the state of the medial meniscus using both observation and probing with all diagnostic methods were calculated. Odds ratios were calculated to determine if probing increased diagnostic accuracy. RESULTS Arthroscopy with probing was the most sensitive and specific diagnostic method and had the highest CCR. For arthrotomy, CrMed was the most sensitive in CrCL-deficient and CdMed the most sensitive in stable, CrCL-intact stifle joints. For all methods, probing increased their diagnostic accuracy. CONCLUSIONS Arthroscopy is the most accurate diagnostic method; however, probing the medial meniscus enhances the diagnostic accuracy of all methods. CLINICAL RELEVANCE Accurate diagnosis of medial meniscal pathology is ideally achieved by means of arthroscopy; however, if arthrotomy is chosen, CrMed should be selected in unstable and CdMed in stable stifle joints. Regardless, medial meniscal probing should be performed to increase diagnostic accuracy.

The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice.

The functions of parathyroid hormone‐related protein (PTHrP) on morphogenesis, cell proliferation, apoptosis, and calcium homeostasis have been attributed to its N terminus. Evidence suggests that many of these effects are not mediated by the N terminus but by the midregion, a nuclear localization sequence (NLS), and C terminus of the protein. A knock‐in mouse lacking the midregion, NLS, and C terminus of PTHrP (Pthrp△/△) was developed. Pthrp△/△ mice had craniofacial dysplasia, chondrodysplasia, and kyphosis, with most mice dying by d 5 of age. In bone, there were fewer chondrocytes and osteoblasts per area, bone mass was decreased, and the marrow was less cellular, with erythroid hypoplasia. Cellular proliferation was impaired, and apoptosis was increased. Runx2, Ocn, Sox9, Crtl1, ß‐catenin, Runx1, ephrin B2, cyclin D1, and Gata1 were underexpressed while P16/ Ink4a, P21, GSK‐3ß, Il‐6, Ffg3, and Ihh were overexpressed. Mammary gland development was aberrant, and energy metabolism was deregulated. These results establish that the midregion, NLS, and C terminus of PTHrP are crucial for the commitment of osteogenic and hematopoietic precursors to their lineages, and for survival, and many of the effects of PTHrP on development are not mediated by its N terminus. The down‐regulation of Runx1, Runx2, and Sox9 indicates that PTHrP is a modulator of transcriptional activation during stem cell commitment. Toribio, R E., Brown, H. A., Novince, C. M., Marlow, B. Hernon, K., Lanigan, L. G., Hildreth III, B. E., Werbeck, J. L., Shu, S. T., Lorch, G., Carlton, M., Foley, J., Boyaka, P., McCauley, L. K., Rosol, T. J. The midregion, nuclear localization sequence, and C terminus of PTHrP regulate skeletal development, hematopoiesis, and survival in mice. FASEB J. 24, 1947–1957 (2010). www.fasebj.org

Animal Models of Bone Metastasis

Bone is one of the most common sites of cancer metastasis in humans and is a significant source of morbidity and mortality. Bone metastases are considered incurable and result in pain, pathologic fracture, and decreased quality of life. Animal models of skeletal metastases are essential to improve the understanding of the molecular pathways of cancer metastasis and growth in bone and to develop new therapies to inhibit and prevent bone metastases. The ideal animal model should be clinically relevant, reproducible, and representative of human disease. Currently, an ideal model does not exist; however, understanding the strengths and weaknesses of the available models will lead to proper study design and successful cancer research. This review provides an overview of the current in vivo animal models used in the study of skeletal metastases or local tumor invasion into bone and focuses on mammary and prostate cancer, lymphoma, multiple myeloma, head and neck squamous cell carcinoma, and miscellaneous tumors that metastasize to bone.

Effect of zoledronic acid and amputation on bone invasion and lung metastasis of canine osteosarcoma in nude mice

Osteosarcoma (OSA) is an aggressive, highly metastatic and lytic primary bone neoplasm commonly affecting the appendicular skeleton of dogs and children. Current treatment options include amputation of the afflicted limb, limb-sparing procedures, or palliative radiation with or without adjunct chemotherapy. Therapies that inhibit bone resorption, such as the bisphosphonates, may be an effective palliative therapy by limiting the local progression of OSA in those patients that are not viable candidates for amputation. We have developed a mouse model of canine skeletal OSA following intratibial inoculation of OSCA40 cells that spontaneously metastasized to the lungs. We demonstrated that therapy with a nitrogen-containing bisphosphonate, zoledronic acid (Zol), reduced OSA-induced bone lysis; however, Zol monotherapy or in combination with amputation was not effective at inhibiting pulmonary metastasis. While not reaching statistical significance, amputation of the tumor-bearing limb reduced the average incidence of lung metastases; however, this effect was nullified when Zol was added to the treatment protocol. In untreated mice, the magnitude of proximal tibial lysis was significantly correlated with the incidence of metastasis. The data support amputation alone for the management of appendicular OSA rather than combining amputation with Zol. However, in patients that are not viable candidates for amputation, Zol may be a useful palliative therapy for OSA by reducing the magnitude of lysis and therefore bone pain, despite the risk of increased pulmonary metastasis.

Expression of DGCR8‐Dependent MicroRNAs Is Indispensable for Osteoclastic Development and Bone‐Resorbing Activity

Recently, microRNAs (miRs) have been implicated in bone formation and homeostasis. We previously reported that Dicer generated miRs have pivotal roles in differentiation and activity of osteoclasts. However, recent studies have demonstrated that Dicer is implicated in production of endogenous small interfering RNAs, non‐canonical miRs, and other small RNAs in mammals. Hence, a challenging question is the extent to which expression of canonical miRs is obligatory for osteoclastic control of bone metabolism. DiGeorge syndrome critical region gene 8 (DGCR8) is exclusively related to expression of miRs by a canonical processing pathway together with the nuclear RNase III enzyme Drosha. Osteoclast‐specific deletion of DGCR8 led to impaired osteoclastic development and bone resorption so that bone development was significantly retarded. In culture, the expression levels of osteoclastic phenotype‐related genes and proteins were remarkably inhibited during osteoclastogenesis in DGCR8‐deficiency. Thus, we have identified that DGCR8‐dependent miRs are indispensable for osteoclastic control of bone metabolism. J. Cell. Biochem. 115: 1043–1047, 2014.

PTHrP 1-141 and 1-86 increase in vitro bone formation.

BACKGROUND Parathyroid hormone-related protein (PTHrP) has anabolic effects in bone, which has led to the clinical use of N-terminal fragments of PTHrP and PTH. Since 10% to 20% of fractures demonstrate healing complications and osteoporosis continues to be a debilitating disease, the development of bone-forming agents is of utmost importance. Due to evidence that regions of PTHrP other than the N-terminus may have bone-forming effects, this study was designed to compare the effects of full-length PTHrP 1-141 to N-terminal PTHrP 1-86 on in vitro bone formation. MATERIALS AND METHODS MC3T3-E1 pre-osteoblasts were treated once every 6 d for 36 d with 5, 25, and 50 pM of PTHrP 1-141 or 1-86 for 1 or 24 h. Cells were also treated after blocking the N-terminus, the nuclear localization sequence (NLS), and the C-terminus of PTHrP, individually and in combination. Area of mineralization, alkaline phosphatase (ALP), and osteocalcin (OCN) were measured. RESULTS PTHrP 1-141 and 1-86 increased mineralization after 24-h treatments, but not 1-h. PTHrP 1-141 was more potent than 1-86. Treatment with PTHrP 1-141 for 24-h, but not 1-86, resulted in a concentration-dependent increase in ALP, with no effect after 1-h. Exposure to both peptides for 1- or 24-h induced a concentration-dependent increase in OCN, with 24-h exceeding 1-h. Antibody blocking revealed that the NLS and C-terminus are anabolic. CONCLUSIONS Both PTHrP 1-141 and 1-86 increased in vitro bone formation; however, PTHrP 1-141 was more effective. The NLS and C-terminus have anabolic effects distinct from the N-terminus. This demonstrates the advantage of PTHrP 1-141 as a skeletal anabolic agent.

Combined zoledronic acid and meloxicam reduced bone loss and tumour growth in an orthotopic mouse model of bone-invasive oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is common in cats and humans and invades oral bone. We hypothesized that the cyclooxygenase (COX)-2 inhibitor, meloxicam, with the bisphosphonate, zoledronic acid (ZOL), would inhibit tumour growth, osteolysis and invasion in feline OSCC xenografts in mice. Human and feline OSCC cell lines expressed COX-1 and COX-2 and the SCCF2 cells had increased COX-2 mRNA expression with bone conditioned medium. Luciferase-expressing feline SCCF2Luc cells were injected beneath the perimaxillary gingiva and mice were treated with 0.1 mg kg(-1) ZOL twice weekly, 0.3 mg kg(-1) meloxicam daily, combined ZOL and meloxicam, or vehicle. ZOL inhibited osteoclastic bone resorption at the tumour-bone interface. Meloxicam was more effective than ZOL at reducing xenograft growth but did not affect osteoclastic bone resorption. Although a synergistic effect of combined ZOL and meloxicam was not observed, combination therapy was well-tolerated and may be useful in the clinical management of bone-invasive feline OSCC.

Canine prostate cancer cell line (Probasco) produces osteoblastic metastases in vivo

In 2012, over 240,000 men were diagnosed with prostate cancer and over 28,000 died from the disease. Animal models of prostate cancer are vital to understanding its pathogenesis and developing therapeutics. Canine models in particular are useful due to their similarities to late‐stage, castration‐resistant human disease with osteoblastic bone metastases. This study established and characterized a novel canine prostate cancer cell line that will contribute to the understanding of prostate cancer pathogenesis.

Ulnocarpal arthrodesis for the treatment of radial agenesis in a dog

Forelimb deformity caused by radial agenesis was diagnosed in a one-year-old Shih Tzu dog. In contrast to most of the previously reported cases of radial agenesis, the humeroulnar joint was inherently stable. The deformity was treated by means of a one-stage ulnocarpal arthrodesis with the application of an eight hole dorsolateral arthrodesis bone plate and autogenous corticocancellous bone graft from the ilial wing. Radiographic evaluation at the eighth and sixteenth post-operative week showed evidence of union of the arthrodesis. At sixteen weeks post-operatively, the dog had much improved limb function. In humans afflicted with radial agenesis, ulnocarpal arthrodesis is used to restore forearm function by minimizing pain and decreasing the magnitude of angular deformity and instability at the level of the ulnocarpal joint. However, to our knowledge, this is the first report of treatment of radial agenesis in the dog by means of a one-stage, ulnocarpal arthrodesis.

Failure to Target RANKL Signaling Through p38-MAPK Results in Defective Osteoclastogenesis in the Microphthalmia Cloudy-Eyed Mutant

The Microphthalmia‐associated transcription factor (MITF) is a basic helix‐loop‐helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of Receptor Activator of NFkB Ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C‐terminal end. Here we show that the Mitfce allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitfce/ce mice. The loss of the leucine zipper domain in Mitfce/ce mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitfce. This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site‐directed mutations in the docking site interfered with the interaction between MITF and its co‐factors FUS and BRG1. MITF‐ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/p38 MAPK interactions in osteoclast differentiation. J. Cell. Physiol. 231: 630–640, 2016.