A preliminary investigation of the subcutaneous tissue reaction to a 3D printed polydioxanone device in horses.

BACKGROUND: A 3D printed self-locking device made of polydioxanone (PDO) was developed to facilitate a standardized ligation technique. The subcutaneous tissue reaction to the device was evaluated after implantation in ten horses of mixed age, sex and breed and compared to loops of poly(lactic-co-glycolic acid) (PLGA). In two of the horses, the implants were removed before closing the skin. The appearance of the implants and surrounding tissue was followed over time using ultrasonography. Implants were removed after 10 and 27 (± 1) days for histologic examination. RESULTS: On macroscopic inspection at day 10, the PDO-device was fragmented and the surrounding tissue was oedematous. On ultrasonographic examination, the device was seen as a hyperechoic structure with strong acoustic shadowing that could be detected 4 months post-implantation, but not at 7 months. Histology revealed a transient granulomatous inflammation, i.e., a foreign body reaction, which surrounded both PDO and PLGA implants. The type and intensity of the inflammation varied between individuals and tissue category. CONCLUSIONS: The 3D printed PDO-device caused a transient inflammatory reaction in the subcutaneous tissue and complete resorption occurred between 4 and 7 months. Considering the intended use as a ligation device the early fragmentation warrants further adjustments of both material and the 3D printing process before the device can be used in a clinical setting.

Pathology of natural Francisella tularensis subsp. holarctica infection in two yellow-necked mice (Apodemus flavicollis).

BACKGROUND: Tularemia is a zoonosis caused by the bacterium Francisella tularensis. It has a wide host range, which includes mammals, birds and invertebrates. F. tularensis has often been isolated from various species of small rodents, but the pathology in naturally infected wild rodent species has rarely been reported. CASE PRESENTATION: Herein, we describe the pathology of tularemia in two naturally infected wild yellow-necked mice (Apodemus flavicollis). To visualize F. tularensis subsp. holarctica, indirect immunofluorescence and immunohistochemistry were applied on tissue sections. Real time polymerase chain reaction detected the bacterium in samples from liver and spleen in both mice. The only finding at necropsy was splenomegaly in one of the mice. Histological examination revealed necrotic foci in the liver associated with mild inflammation in both mice. Immunohistochemistry and indirect immunofluorescence showed bacteria disseminated in many organs, in the cytoplasm of macrophages, and intravascularly. CONCLUSIONS: The two yellow-necked mice died of an acute disease caused by tularemic infection disseminated to many organs. Further investigations of naturally infected small rodents are important to better understand the variability in pathological presentation caused by infection by F. tularensis subsp. holarctica, as well to elucidate the importance of small rodents as transmitters and/or reservoirs.

Online dosimetry for temoporfin-mediated interstitial photodynamic therapy using the canine prostate as model.

Online light dosimetry with real-time feedback was applied for temoporfin-mediated interstitial photodynamic therapy (PDT) of dog prostate. The aim was to investigate the performance of online dosimetry by studying the correlation between light dose plans and the tissue response, i.e., extent of induced tissue necrosis and damage to surrounding organs at risk. Light-dose planning software provided dose plans, including light source positions and light doses, based on ultrasound images. A laser instrument provided therapeutic light and dosimetric measurements. The procedure was designed to closely emulate the procedure for whole-prostate PDT in humans with prostate cancer. Nine healthy dogs were subjected to the procedure according to a light-dose escalation plan. About 0.15 mg/kg temoporfin was administered 72 h before the procedure. The results of the procedure were assessed by magnetic resonance imaging, and gross pathology and histopathology of excised tissue. Light dose planning and online dosimetry clearly resulted in more focused effect and less damage to surrounding tissue than interstitial PDT without dosimetry. A light energy dose-response relationship was established where the threshold dose to induce prostate gland necrosis was estimated from 20 to 30 J/cm2.

Multiplex cytokine analyses in dogs with pyometra suggest involvement of KC-like chemokine in canine bacterial sepsis.

Clinical diagnostic criteria for sepsis (systemic inflammatory response syndrome caused by infection) are unspecific and, therefore, biomarkers for sepsis diagnosis are needed for appropriate treatment and patient survival. Pyometra, a common disease caused by bacterial infection of the uterus, results in sepsis in nearly 60% of cases in dogs. We used dogs with pyometra as a natural model for sepsis and collected serum samples from 39 dogs, of which 22 with pyometra and 17 healthy controls. Dogs with pyometra were further grouped into dogs with sepsis (n=18) and without sepsis (n=4). Serum concentrations of a panel of cytokines, including keratinocyte-derived chemokine (KC)-like, granulocyte-macrophages colony stimulating factor (GM-CSF), interleukin (IL)-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, chemokine C-X-C motif ligand (CXCL)10 and tumor necrosis factor (TNF)-α, were measured using multiplex analyses. Serum C-reactive protein (CRP) levels were determined using an automated immunoturbidimetric assay. In addition to physical examination hematological and serum biochemical analyses were performed to evaluate the overall status of the dogs. Significantly higher concentrations of KC-like (757 vs 304 pg/ml) were detected in dogs with pyometra as compared to healthy dogs. Within the pyometra group, dogs with sepsis compared to dogs without sepsis had a higher KC-like concentration (873 vs 300 pg/ml). Hemoglobin levels were significantly lower in dogs with pyometra compared to healthy dogs, regardless of the presence or absence of sepsis, and correlated negatively with KC-like. KC-like concentrations correlated positively with CRP, number of hospitalization days, number of monocytes, concentrations of IL-8, and percentage band neutrophils. Our data suggest that bacterial infection triggers the expression of KC-like and further studies are warranted of KC-like as a possible biomarker for diagnosing sepsis and uterine bacterial infection in dogs.

Ligation of the spermatic cord in dogs with a self-locking device of a resorbable polyglycolic based co-polymer--feasibility and long-term follow-up study.

BACKGROUND: New surgical techniques are developed to enable a quicker, easier and safer surgery with reduced risk of complications and shortened time needed for recovery. A resorbable device, a self-locking loop, was designed for surgical ligation. The objective of this pilot study was to investigate the feasibility of ligating the spermatic cord with the device, its biocompatibility and long-term resorption in dogs. RESULTS: The device was made of a block co-polymer (glycolide and trimethylene carbonate), manufactured by injection moulding and consisted of a flexible band running through a case with a locking mechanism. Ten devices were tested for ligation of the spermatic cords in five dogs admitted for routine neutering. The dogs were monitored by physical examination and ultrasonography of the site of ligation, area of spermatic cord and medial iliac lymph nodes regularly until no hyperechoic remnants of the device or acoustic shadowing or local tissue reactions were observed. Haemostasis of the spermatic cords was achieved with the devices. On ultrasonography the devices were seen as hyperechoic structures for 2 months after neutering causing acoustic shadowing for 1 month. The dogs were monitored for 3 - 5 months after surgery. Gradual decrease in echogenicity and final disappearance of the hyperechoic structures suggested resorption. Macroscopic and histological post mortem examinations were performed in one dog at 3 months after surgery. Post mortem examination showed a tissue reaction of a suture granuloma that was restricted in extent at site of the device. CONCLUSIONS: The results of this pilot study suggest biocompatibility and indicate that ligation of the spermatic cord is feasible with the device.

Serum insulin-like growth factor-I, iron, C-reactive protein, and serum amyloid A for prediction of outcome in dogs with pyometra.

Pyometra, accumulation of pus in the uterus, is a bacterial infection that frequently initiates systemic inflammation. The disease may have lethal consequences when the systemic effects are severe or complications occur. Markers for identifying high-risk patients and predicting outcome are therefore in high demand. The objective of this study was to measure serum concentrations of insulin-like growth factor-I (IGF-I), iron, C-reactive protein (CRP), and serum amyloid A (SAA) in bitches with pyometra and to explore the possible value of these variables for detection of increased morbidity. In total, 31 bitches were diagnosed with pyometra and destined for surgical treatment (ovariohysterectomy) and 17 healthy bitches were included in the study. Concentrations of IGF-I and iron were lower in the pyometra group (mean concentration 221.2 ± 22.5 ng/mL and 16.9 ± 1.6 µmol/L, respectively) compared with the healthy control group (mean concentration 366.7 ± 46.2 ng/mL and 38.1 ± 2.7 µmol/L, respectively). In contrast, concentrations of CRP and SAA were significantly higher in bitches with pyometra (mean concentrations 212.9 ± 17.3 mg/L and 119.9 ± 8.5 mg/L, respectively) compared with the control group (<5 mg/L and <10 mg/L, respectively). None of the explored variables were associated with morbidity as measured by duration of postoperative hospitalization. In conclusion, IGF-I and iron concentrations were decreased in pyometra, whereas SAA and CRP concentrations were increased in the disease. Although unspecific, measurement of these variables may be valuable as adjunctive markers for prognosis in cases of pyometra.

Immunolocalization of collagens (I and III) and cartilage oligomeric matrix protein in the normal and injured equine superficial digital flexor tendon.

This is a descriptive study of tendon pathology with different structural appearances of repair tissue correlated to immunolocalization of cartilage oligomeric matrix protein (COMP) and type I and III collagens and expression of COMP mRNA. The material consists of nine tendons from seven horses (5-25 years old; mean age of 10 years) with clinical tendinopathy and three normal tendons from horses (3, 3, and 13 years old) euthanized for non-orthopedic reasons. The injured tendons displayed different repair-tissue appearances with organized and disorganized fibroblastic regions as well as areas of necrosis. The normal tendons presented distinct immunoreactivity for COMP and expression of COMP mRNA and type I collagen in the normal aligned fiber structures, but no immunolabeling of type III collagen. However, immunoreactivity for type III collagen was present in the endotenon surrounding the fiber bundles, where no expression of COMP could be seen. Immunostaining for type I and III collagens was present in all of the pathologic regions indicating repair tissue. Interestingly, the granulation tissues showed immunostaining for COMP and expression of COMP mRNA, indicating a role for COMP in repair and remodeling of the tendon after fiber degeneration and rupture. The present results suggest that not only type III collagen but also COMP is involved in the repair and remodeling processes of the tendon.

Ligation of the ovarian pedicles in dogs with a resorbable self-locking device--a long-term follow-up study.

A prospective clinical trial was performed to evaluate applicability, hemostatic efficiency, and long-term tissue reactions of a novel medical device. The device was made of polydioxanone and designed in principle as a cable-tie with a flexible band running through a locking case. It was tested for ligation of the ovarian pedicles during ovariohysterectomy in nine intact female dogs. The dogs were followed-up monthly by physical examination and ultrasonography of the abdomen for 5-12 months after surgery. Macroscopic and histological post mortem examinations were performed in two dogs 7 and 18 months after surgery. Hemostasis was achieved with the devices although 1 of the 18 devices needed further tightening during a reoperation. Ultrasonography revealed a hyperechoic device that caused acoustic shadowing, which decreased over time indicating gradual resorption. Post mortem examinations demonstrated adhesions and a local transient tissue reaction at site of the device. The results show that the device may be a future alternative for ligation of ovarian pedicles in dogs.

Ultrastructural immunolocalization of cartilage oligomeric matrix protein, thrombospondin-4, and collagen fibril size in rodent achilles tendon in relation to exercise.

Fourteen 3-week-old Sprague-Dawley rats were housed in pairs in standard cages (5 controls) and in individual cages with a running wheel. Four of these rats had run 27-36 km/week (low training - LT) and 5 had run 56-92 km/week (high training - HT). After 4 weeks, the rats were euthanized and Achilles tendons were fixed for electron microscopy. The ultrastructural distribution of cartilage oligomeric matrix protein (COMP) and thrombospondin (TSP)-4 and collagen fibril thickness in two different extracellular compartments were studied. The immunolabeling of COMP decreased with longer running distance and was significantly lower in both the pericellular (p = 0.009) and interterritorial (p = 0.03) compartments of the HT rats compared with the controls. TSP-4 immunolabeling was higher in the pericellular compared with the interterritorial compartments in all rats (p = 0.013) but was not correlated with COMP immunolabeling. No alterations in collagen fibril size were found in relation to running; however, the gold markers representing COMP and TSP-4 were mostly found at the dark bands, representing the gap region of the fibril.

Thrombospondin-4 and cartilage oligomeric matrix protein form heterooligomers in equine tendon.

Injuries of the equine superficial digital flexor tendon are common in racing horses. Knowledge of the tendon matrix composition is crucial to understand physiological and pathological processes in the tendon. The aim of this study was to analyze TSP-4 expressed in equine tendon. Equine tendons were extracted with 10 mM EDTA-containing buffer and TSP-4 purified with ion-exchange chromatography followed by heparin affinity chromatography. The purified TSP-4 was analyzed by one- and two-dimensional SDS-PAGE, immunoblotting, and MALDI-TOF mass spectrometry. Purified TSP-4 gave bands reacting with a TSP-4 specific antiserum, but also with an antiserum to COMP, when submitted to SDS-PAGE under nonreducing conditions. Two-dimensional SDS-PAGE (nonreducing followed by reducing conditions) and immunoprecipitation as well as MALDI-TOF mass spectrometry analysis showed that TSP-4 and COMP are both present in equine tendon and cannot be separated under nonreducing conditions despite significant differences in subunit size. This suggests that they are connected via disulfide bridges into heterooligomers.

Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in relation to collagen fibrils in the equine tendon.

The structure and organisation of the extracellular matrix, and in particular the axial alignment of type I collagen fibrils, are essential for the tensile strength of tendons. The resident tenocytes synthesize and maintain the composition of the extracellular matrix, which changes with age and maturation. Other components of the extracellular matrix include less abundant collagen types II, III, V, VI, XII, proteoglycans and glycoproteins. Cartilage oligomeric matrix protein (COMP) is an abundant non-collagenous pentameric glycoprotein in the tendon, which can bind to collagen types I and II. The function of COMP in the tendon is not clear, but it may act as a catalyst in fibrillogenesis. Its concentration changes with age, maturation and load. The present study delineates the ultrastructural distribution of COMP and its correlation to collagen fibril thickness in different compartments in two flexor tendons from horses of different ages (foetus, 8 months, 3 years, 12 years). The immunolabeling for COMP was higher in the superficial digital flexor tendon compared with the deep digital flexor tendon and it increased with the age of the animal, with the highest concentration in the 3-year-olds. Fibril diameter differed between age groups and a more homogenous fibril population was found in the fetal tendons. A positive correlation between high COMP immunolabeling and the percentage of small fibrils (<60 nm) were present in the SDFT. COMP immunolabeling was enriched at the gap region of the collagen fibril. In situ hybridization revealed the strongest expression in tendons from the 3-year-old horses whereas there was no expression in foetal tendon.