Intervertebral Disc Regeneration Using Stem Cell/Growth Factor-Loaded Porous Particles with a Leaf-Stacked Structure.

Although biological therapies based on growth factors and transplanted cells have demonstrated some positive outcomes for intervertebral disc (IVD) regeneration, repeated injection of growth factors and cell leakage from the injection site remain considerable challenges for human therapeutic use. Herein, we prepare human bone marrow-derived mesenchymal stem cells (hBMSCs) and transforming growth factor-ß3 (TGF-ß3)-loaded porous particles with a unique leaf-stack structural morphology (LSS particles) as a combination bioactive delivery matrix for degenerated IVD. The LSS particles are fabricated with clinically acceptable biomaterials (polycaprolactone and tetraglycol) and procedures (simple heating and cooling). The LSS particles allow sustained release of TGF-ß3 for 18 days and stable cell adhesiveness without additional modifications of the particles. On the basis of in vitro and in vivo studies, it was observed that the hBMSCs/TGF-ß3-loaded LSS particles can provide a suitable milieu for chondrogenic differentiation of hBMSCs and effectively induce IVD regeneration in a beagle dog model. Thus, therapeutically loaded LSS particles offer the promise of an effective bioactive delivery system for regeneration of various tissues including IVD.

Evaluation of treatment with a combination of mycophenolate mofetil and prednisolone in dogs with meningoencephalomyelitis of unknown etiology: a retrospective study of 86 cases (2009-2017).

BACKGROUND: Combination therapy with glucocorticoids and adjunctive immunomodulating drugs has been generally accepted as a standard treatment regimen for meningoencephalomyelitis of unknown etiology (MUE). We hypothesized that treatment with MMF as an adjunctive agent along with glucocorticoids would be effective and well-tolerated protocol in dogs with MUE. Eighty-six dogs with MUE between May 2009 and June 2017 were included (59 females and 27 males; mean age of 5.93 years; mean body weight of 3.83 kg). The medical records of dogs with MUE treated with prednisolone and MMF were retrospectively evaluated to determine the therapeutic response, survival time, and treatment-related adverse effects. RESULTS: A partial or complete response (CR) was recorded for 75 dogs. The overall median survival time from the initiation of treatment was 558 days. Dogs that showed CR with no relapse over the treatment period (from diagnosis to death) had significantly longer median survival times. A significantly higher mortality hazard ratio of 4.546 was recorded in dogs that failed to achieve CR. The interval between the onset of clinical signs and the clinical presentation was not significantly associated with CR, relapse rate, and survival time. Adverse effects included gastrointestinal upsets in 26 dogs (30.23%), sporadic infections in 17 dogs (19.77%), and pancreatitis in seven dogs (8.14%). CONCLUSIONS: The results suggest that adjunctive MMF treatment for MUE is safe and comparable to other immunosuppressive protocols. The treatment should focus on the achievement of CR and preventing relapse for successful management.

Oxygen-Releasing Microparticles for Cell Survival and Differentiation Ability under Hypoxia for Effective Bone Regeneration.

Sufficient oxygen delivery into tissue-engineered three-dimensional (3D) scaffolds to produce clinically applicable tissues/organs remains a challenge for researchers and clinicians. One potential strategy to overcome this limitation is the use of an oxygen releasing scaffold. In the present study, we prepared hollow microparticles (HPs) loaded with an emulsion of the oxygen carrier perfluorooctane (PFO; PFO-HPs) for the timely supply of oxygen to surrounding cells. These PFO-HPs prolonged the survival and preserved the osteogenic differentiation potency of human periosteal-derived cells ( hPDCs) under hypoxia. hPDCs seeded onto PFO-HPs formed new bone at a faster rate and with a higher bone density than hPDCs seeded onto phosphate buffered saline-loaded control HPs. These findings suggest that PFO-HPs provide a suitable environment for the survival and maintenance of differentiation ability of hPDCs at bony defects without vascular networks until new blood vessel ingrowth occurs, thus enhancing bone regeneration. PFO-HPs are a promising system for effective delivery of various functional cells, including stem cells and progenitor cells, to regenerate damaged tissues/organs.

Development of Porous Beads to Provide Regulated BMP-2 Stimulation for Varying Durations: In Vitro and In Vivo Studies for Bone Regeneration.

It is commonly accepted that the sustained release of bone morphogenetic protein-2 (BMP-2) can enhance bone regeneration and minimize its safety issues. However, little is known regarding the appropriate duration of BMP-2 stimulation for sufficient osteogenic differentiation and new bone formation because of the short half-life of BMP-2 in the physiological environment and the lack of a well-defined delivery matrix that can regulate the release period of BMP-2. In this study, we prepared porous poly(lactic-co-glycolic acid) (PLGA) beads with different surface pore sizes that can regulate the release period of BMP-2 (i.e., 7, 17, and 30 days) while providing the BMP-2 concentration required for bone regeneration. Our findings in both in vitro cell culture and in vivo animal studies using these BMP-2-loaded beads demonstrate that release of BMP-2 within 7 days affects only the initial differentiation of human periosteum-derived cells (hPDCs) and does not significantly enhance their subsequent differentiation into mature functional cells. However, extending the duration of BMP-2 stimulation over 17 days can provide a suitable environment for osteogenic differentiation of hPDCs and new bone formation.

Immunomodulatory properties and in vivo osteogenesis of human dental stem cells from fresh and cryopreserved dental follicles.

In our previous study, dental follicle tissues from extracted wisdom teeth were successfully cryopreserved for use as a source of stem cells. The goals of the present study were to investigate the immunomodulatory properties of stem cells from fresh and cryopreserved dental follicles (fDFCs and cDFCs, respectively) and to analyze in vivo osteogenesis after transplantation of these DFCs into experimental animals. Third passage fDFCs and cDFCs showed similar expression levels of interferon-γ receptor (CD119) and major histocompatibility complex class I and II (MHC I and MHC II, respectively), with high levels of CD119 and MHC I and nearly no expression of MHC II. Both fresh and cryopreserved human DFCs (hDFCs) were in vivo transplanted along with a demineralized bone matrix scaffold into mandibular defects in miniature pigs and subcutaneous tissues of mice. Radiological and histological evaluations of in vivo osteogenesis in hDFC-transplanted sites revealed significantly enhanced new bone formation activities compared with those in scaffold-only implanted control sites. Interestingly, at 8 weeks post-hDFC transplantation, the newly generated bones were overgrown compared to the original size of the mandibular defects, and strong expression of osteocalcin and vascular endothelial growth factor were detected in the hDFCs-transplanted tissues of both animals. Immunohistochemical analysis of CD3, CD4, and CD8 in the ectopic bone formation sites of mice showed significantly decreased CD4 expression in DFCs-implanted tissues compared with those in control sites. These findings indicate that hDFCs possess immunomodulatory properties that involved inhibition of the adaptive immune response mediated by CD4 and MHC II, which highlights the usefulness of hDFCs in tissue engineering. In particular, long-term preserved dental follicles could serve as an excellent autologous or allogenic stem cell source for bone tissue regeneration as well as a valuable therapeutic agent for immune diseases.

Operative Techniques and Preliminary Outcomes Following Percutaneous Endoscopic Thoracolumbar Pediculectomy in Dogs.

OBJECTIVE: To examine the technical feasibility of percutaneous endoscopic pediculectomy using a lateral approach and to evaluate its use for decompression and examination of the thoracic and lumbar spinal canals in small dogs. STUDY DESIGN: Experimental study. ANIMALS: Clinically normal adult dogs (n=10). METHODS: After optimizing the technique in cadavers, percutaneous endoscopic pediculectomy was performed using a lateral approach to the thoracic (T12) or lumbar (L2) vertebrae in 5 dogs each. Using fluoroscopic guidance, a K-wire, dilator, and (cannula) working sleeve were placed. A window for visualizing the spinal cord and floor of the spinal canal was created using a specialized drill, rongeurs, trephine, and elevator. Gait and neurologic status were monitored postoperatively, and computed tomography (CT) and magnetic resonance imaging (MRI) performed. RESULTS: All procedures were completed successfully (T12, 45 ± 13 minutes; L2, 59 ± 14 minutes) with clear observation of the spinal cord and floor of the spinal canal. Normal ambulation was maintained in 9 dogs. One dog in the L2 group had ipsilateral hind limb weakness that resolved within 4 days. One dog in the L2 group suffered a fracture of the accessory process. One dog each in the T12 and L2 groups had hyperintense lesions on T2-weighted MRI images, suspicious of focal edema, which reduced at 4 weeks after initial examination, possibly reflecting gliosis. CONCLUSION: Pediculectomy using a percutaneous endoscopic thoracolumbar lateral approach is feasible, provides a good view of the spinal canal, and may be an alternative for treatment of disk disease in dogs.

BMP-2-immobilized PCL 3D printing scaffold with a leaf-stacked structure as a physically and biologically activated bone graft.

Although three-dimensional (3D) printing techniques are used to mimic macro- and micro-structures as well as multi-structural human tissues in tissue engineering, efficient target tissue regeneration requires bioactive 3D printing scaffolds. In this study, we developed a bone morphogenetic protein-2 (BMP-2)-immobilized polycaprolactone (PCL) 3D printing scaffold with leaf-stacked structure (LSS) (3D-PLSS-BMP) as a bioactive patient-tailored bone graft. The unique LSS was introduced on the strand surface of the scaffold via heating/cooling in tetraglycol without significant deterioration in physical properties. The BMP-2 adsorbed on3D-PLSS-BMPwas continuously released from LSS over a period of 32 d. The LSS can be a microtopographical cue for improved focal cell adhesion, proliferation, and osteogenic differentiation.In vitrocell culture andin vivoanimal studies demonstrated the biological (bioactive BMP-2) and physical (microrough structure) mechanisms of3D-PLSS-BMPfor accelerated bone regeneration. Thus, bioactive molecule-immobilized 3D printing scaffold with LSS represents a promising physically and biologically activated bone graft as well as an advanced tool for widespread application in clinical and research fields.

Successful treatment of canine infective endocarditis caused by Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens is a gram-positive bacterial species that is utilised as a probiotic in humans and animals. There are no reports of infective endocarditis (IE) in dogs. An 8-year-old, spayed, female Maltese presented with a 1-month history of fever, depression, weight loss, and hindlimb lameness. Laboratory test results indicated non-regenerative anaemia, neutrophilia, hyperglobulinemia, and proteinuria. Echocardiography revealed vegetation on the septal leaflet of the mitral valve and thromboemboli in the left atrium. Consecutive blood culture results revealed that the blood samples were consistently positive for Bacillus amyloliquefaciens, which is generally considered a probiotic bacterial species for animals. Broad-spectrum antibiotics (amoxicillin-clavulanic acid and cefotaxime) and anticoagulants (clopidogrel and rivaroxaban) were administered for 4 months. The clinical signs were responsive to antibiotic treatment. After 4 months, the dog was no longer febrile and the size of the thromboemboli in the left atrium had decreased. Bacteria were no longer isolated in blood cultures after antibiotic therapy. To the best of our knowledge, this is the first case report of canine IE caused by bactaeremic infection with Bacillus amyloliquefaciens.

Bioactive Porous Particles as Biological and Physical Stimuli for Bone Regeneration.

Even though bony defects can be recovered to their original condition with full functionality, critical-sized bone injuries continue to be a challenge in clinical fields due to deficiencies in the scaffolding matrix and growth factors at the injury region. In this study, we prepared bone morphogenetic protein-2 (BMP-2)-loaded porous particles as a bioactive bone graft for accelerated bone regeneration. The porous particles with unique leaf-stacked morphology (LSS particles) were fabricated by a simple cooling procedure of hot polycaprolactone (PCL) solution. The unique leaf-stacked structure in the LSS particles provided a large surface area and complex release path for the sufficient immobilization of BMP-2 and sustained release of BMP-2 for 26 days. The LSS was also recognized as a topographical cue for cell adhesion and differentiation. In in vitro cell culture and in vivo animal study using a canine mandible defect model, BMP-2-immobilized LSS particles provided a favorable environment for osteogenic differentiation of stem cells and bone regeneration. In vitro study suggests a dual stimulus of bone mineral-like (leaf-stacked) structure (a physical cue) and continuously supplied BMP-2 (a biological cue) to be the cause of this improved healing outcome. Thus, LSS particles containing BMP-2 can be a promising bioactive grafting material for effective new bone formation.

Undifferentiated pleomorphic sarcoma (malignant fibrous histiocytoma) of the head in a dog.

A six year-old intact female miniature poodle was presented with a soft mass of the forehead region. Computed tomography identified generalized frontal bone loss and a large extracranial mass, which had a low attenuation area of hemorrhagic necrosis with septation and enhancement of solid components. In magnetic resonance imaging, the mass was isointense in T1-weighted images except its fluid parts and hyperintense in T2-weighted images with lobulated by low-signal septa. Surgery was performed to remove the mass, and histopathologic examination revealed that the mass was consistent with undifferentiated pleomorphic sarcoma (malignant fibrous histiocytoma). The dog died from the rapidly recurrent mass and severe pulmonary metastasis.

Evaluation of the effects of thiopental, propofol, and etomidate on glomerular filtration rate measured by the use of dynamic computed tomography in dogs.

OBJECTIVE: To evaluate the effects of thiopental, propofol, and etomidate on glomerular filtration rate (GFR) measured by the use of dynamic computed tomography in dogs. ANIMALS: 17 healthy Beagles. PROCEDURES: Dogs were randomly assigned to receive 2 mg of etomidate/kg (n = 5), 6 mg of propofol/kg (7), or 15 mg of thiopental/kg (5) during induction of anesthesia; anesthesia was subsequently maintained by isoflurane evaporated in 100% oxygen. A 1 mL/kg dosage of a 300 mg/mL solution of iohexol was administered at a rate of 3 mL/s during GFR measurement. Regions of interest of the right kidney were manually drawn to exclude vessels and fatty tissues and highlight the abdominal portion of the aorta. Iohexol clearance per unit volume of the kidney was calculated by use of Patlak plot analysis. RESULTS: Mean ± SD weight-adjusted GFR of the right kidney after induction of anesthesia with thiopental, propofol, and etomidate was 2.04 ± 0.36 mL/min/kg, 2.06 ± 0.29 mL/min/kg, and 2.14 ± 0.43 mL/min/kg, respectively. However, no significant differences in weight-adjusted GFR were detected among the treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Results obtained for the measurement of GFR in anesthetized dogs after anesthetic induction with etomidate, propofol, or thiopental and maintenance with isoflurane did not differ significantly. Therefore, etomidate, propofol, or thiopental can be used in anesthesia-induction protocols that involve the use of isoflurane for maintenance of anesthesia without adversely affecting GFR measurements obtained by the use of dynamic computed tomography in dogs.

Multicystic dysplastic kidney disease in a dog.

A 1-year-old female mongrel dog was evaluated for anorexia and vomiting of 4 days duration. Abdominal ultrasonographic findings revealed small kidneys with multiple anechoic cysts. The dog was euthanized due to poor prognosis. A full necropsy was performed, and the histopathologic findings were consistent with multicystic dysplastic kidney disease.

Metallic foreign body in the liver of a dog.

A 4-year-old beagle had intermittent vomiting and weight loss for 1 month. On plain radiography, an approximately 3.5-cm, radiopaque, linear foreign body was identified in the region of the liver. On ultrasonography, a hyperechoic linear structure with reverberation was identified in the left lobe or quadrate lobe. On computed tomography(CT), a thin hyperattenuating material consistent with a metallic foreign body was identified just medial to the gall bladder. Removal of the foreign body was performed without complications. We present a rare case in which a metallic foreign body was embedded in the liver asymptomatically and found incidentally during a clinical examination performed to ascertain the cause of clinical signs.

Thymic Hematoma Found in a Wild Eurasian Otter (Lutra lutra).

Thymic hematomas are uncommon in animals, and the few reported cases are all in dogs. In May 2019, we necropsied a wild Eurasian otter (Lutra lutra) in Junju, Republic of Korea, and found a thymic hematoma without any signs of trauma or hemorrhage in other organs, except for a hemothorax. This study describes the macroscopic and microscopic examinations of a thymic hematoma in an Eurasian otter.

Determination of optimal dosage and delay time for computed tomographic lymphography after percutaneous injection of Iohexol into popliteal lymph nodes in dogs.

The purposes of this study were to determine the optimal dose and delay time for lymphography by injection of Iohexol into popliteal lymph nodes and to assess images of computed tomography by the established protocol. Three different doses (30, 60 and 90 mgI/kg) of water-soluble iodinated contrast medium were injected into 15 popliteal lymph nodes of 10 adult beagles, and fluoroscopy was performed. Filling and duration of contrast media and the number of visible ducts from popliteal lymph nodes to the thoracic duct and its branches were recorded. CT lymphography was performed, and the number of visible thoracic ducts was compared with that found by radiographic lymphography. Radiographs obtained between 130 and 800 seconds after injection of contrast medium provided a detailed view of the thoracic duct. The dose of 60 mgI/kg was determined to enable quality diagnostic imaging without extranodal leakage in radiographic lymphography. There was no significant difference in the number of thoracic ducts between the two modalities at each anatomic location. However, CT lymphography provided images of the thoracic duct with better spatial resolution and without superimposition of surrounding tissue. The present study provides an adequate delay time and injection for identification of the canine thoracic duct, and therefore, this technique could be applied to diagnosis of disease associated with chest lymphatic drainage.

Classification of radiographic lung pattern based on texture analysis and machine learning.

This study evaluated the feasibility of using texture analysis and machine learning to distinguish radiographic lung patterns. A total of 1200 regions of interest (ROIs) including four specific lung patterns (normal, alveolar, bronchial, and unstructured interstitial) were obtained from 512 thoracic radiographs of 252 dogs and 65 cats. Forty-four texture parameters based on eight methods of texture analysis (first-order statistics, spatial gray-level-dependence matrices, gray-level-difference statistics, gray-level run length image statistics, neighborhood gray-tone difference matrices, fractal dimension texture analysis, Fourier power spectrum, and Law's texture energy measures) were used to extract textural features from the ROIs. The texture parameters of each lung pattern were compared and used for training and testing of artificial neural networks. Classification performance was evaluated by calculating accuracy and the area under the receiver operating characteristic curve (AUC). Forty texture parameters showed significant differences between the lung patterns. The accuracy of lung pattern classification was 99.1% in the training dataset and 91.9% in the testing dataset. The AUCs were above 0.98 in the training set and above 0.92 in the testing dataset. Texture analysis and machine learning algorithms may potentially facilitate the evaluation of medical images.

Development of bone regeneration strategies using human periosteum-derived osteoblasts and oxygen-releasing microparticles in mandibular osteomyelitis model of miniature pig.

Hypoxia and limited vascularization inhibit bone growth and recovery after surgical debridement to treat osteomyelitis. Similarly, despite significant efforts to create functional tissue-engineered organs, clinical success is often hindered by insufficient oxygen diffusion and poor vascularization. To overcome these shortcomings, we previously used the oxygen carrier perfluorooctane (PFO) to develop PFO emulsion-loaded hollow microparticles (PFO-HPs). PFO-HPs act as a local oxygen source that increase cell viability and maintains the osteogenic differentiation potency of human periosteum-derived cells (hPDCs) under hypoxic conditions. In the present study, we used a miniature pig model of mandibular osteomyelitis to investigate bone regeneration using hPDCs seeded on PFO-HPs (hPDCs/PFO-HP) or hPDCs seeded on phosphate-buffered saline (PBS)-HPs (hPDCs/PBS-HP). Osteomyelitis is characterized by a series of microbial invasion, vascular disruption, bony necrosis, and sequestrum formation due to impaired host defense response. Sequential plain radiography, computed tomography (CT), and 3D reconstructed CT images revealed new bone formation was more advanced in defects that had been implanted with the hPDCs/PFO-HPs than in defects implanted with the hPDCs/PBS-HP. Thus, PFO-HPs are a promising tissue engineering approach to repair challenging bone defects and regenerate structurally organized bone tissue with 3D architecture.

Evaluation of partial cranial cruciate ligament rupture with positive contrast computed tomographic arthrography in dogs.

Computed tomographic arthrography (CTA) of four cadaveric canine stifles was performed before and after partial cranial cruciate ligament rupture in order to verify the usefulness of CTA examination for the diagnosis of partial cranial cruciate ligament rupture. To obtain the sequential true transverse image of a cranial cruciate ligament, the computed tomography gantry was angled such that the scanning plane was parallel to the fibula. True transverse images of cranial cruciate ligaments were identified on every sequential image, beginning just proximal to the origin of the cranial cruciate ligament distal to the tibial attachment, after the administration of iodinated contrast medium. A significant decrease in the area of the cranial cruciate ligament was identified on CTA imaging after partial surgical rupture of the cranial cruciate ligament. This finding implies that CTA can be used for assessing partial cranial cruciate ligament ruptures in dogs.

Sustained Release of BMP-2 from Porous Particles with Leaf-Stacked Structure for Bone Regeneration.

Sustained release of bioactive molecules from delivery systems is a common strategy for ensuring their prolonged bioactivity and for minimizing safety issues. However, residual toxic reagents, the use of harsh organic solvents, and complex fabrication procedures in conventional delivery systems are considered enormous impediments toward clinical use. Herein, we describe bone morphogenetic protein-2 (BMP-2)-immobilized porous polycaprolactone particles with unique leaf-stacked structures (LSS particles) prepared using clinically feasible materials and procedures. The BMP-2 immobilized in these LSS particles is continuously released up to 36 days to provide an appropriate environment for osteogenic differentiation of human periosteum-derived cells and new bone formation. Thus, the leaf-stacked structures of these LSS particles provide a simple but clinically applicable platform for effectively delivering a variety of bioactive molecules, such as growth factors, hormones, cytokines, peptides, etc.

Operative techniques of percutaneous endoscopic mini-hemilaminectomy using a uniportal approach in dogs.

The present study aimed to investigate the technical feasibility of percutaneous endoscopic mini-hemilaminectomy via a uniportal approach, and to evaluate the possibility of decompression and endoscopic examination of the thoracic and lumbar spinal canals in small dogs during such procedures. Fresh canine cadavers of mixed-breed dogs (n=7) were used in this study. Following injection of a barium and agarose mixture (BA-gel) to stimulate intervertebral disc herniation, percutaneous endoscopic mini-hemilaminectomy was performed using a lateral approach to the thoracic and lumbar vertebrae. BA-gel was removed to decompress the spinal cord using an elevator and rongeurs after mini-hemilaminectomy. Pre and post-operative computed tomography (CT) scans were obtained to evaluate surgical outcomes. Intra-operative complications, incision length, and procedure time were recorded. All procedures were completed with clear visualization of the spinal cord and floor of the spinal canal. The mean total operating time was 58.00 ± 18.06 min. Lengths of incision were under 1 cm in all dogs. Intra-operative complications included iatrogenic nerve root injuries caused by the micro-rongeur in two dogs. CT imaging revealed that removal of BA-gel resulted in sufficient spinal cord decompression. Our findings indicated that percutaneous endoscopic thoracolumbar mini-hemilaminectomy is feasible for spinal cord decompression and allows for adequate observation of the spinal canal. Thus, this technique may be an alternative surgical option for treatment of thoracolumbar disk disease in dogs.