MMP-9 Concentration in Peritoneal Fluid Is a Valuable Biomarker Associated with Endotoxemia in Equine Colic

The purpose of the study was to compare the results of sepsis scoring (clinical examination and clinical pathology) to the concentrations of matrix-metalloproteinases (MMPs) -2, -8, and -9; tissue-inhibitor of metalloproteinases (TIMPs) -1 and -2; and inflammatory chemokines interleukin (IL) 1 beta and tumor-necrosis-factor-alpha (TNF-alpha) in plasma and peritoneal fluid of equine colic patients. A modified sepsis scoring including general condition, heart and respiratory rate, rectal temperature, mucous membranes, white blood cell count (WBC), and ionized calcium was applied in 47 horses presented with clinical signs of colic. Using this scoring system, horses were classified as negative (n=32, = 10/19 points) for sepsis. MMPs, TIMPs, IL-1 beta, and TNF-alpha concentrations were evaluated in plasma and peritoneal fluid using species-specific sandwich ELISA kits. In a linear discriminant analysis, all parameters of sepsis scoring apart from calcium separated well between sepsis severity groups (P113 ng/ml in the peritoneal fluid was found to be the ideal cutoff to identify positive sepsis scoring (>= 10/19 points; sensitivity of 83.3% and specificity of 82.9%). In conclusion, MMP-9 was found to be a biomarker of high diagnostic value for sepsis and endotoxemia in equine colic. The evaluation of peritoneal fluid seems preferable in comparison to plasma. As abdominocentesis is commonly performed in the diagnostic work-up of equine colic, a pen-side assay would be useful and easy-to-perform diagnostic support in the decision for therapeutic intervention

Functional Scaffold‐Free Bone Equivalents Induce Osteogenic and Angiogenic Processes in a Human In Vitro Fracture Hematoma Model

After trauma, the formed fracture hematoma within the fracture gap contains all the important components (immune/stem cells, mediators) to initiate bone regeneration immediately. Thus, it is of great importance but also the most susceptible to negative influences. To study the interaction between bone and immune cells within the fracture gap, up-to-date in vitro systems should be capable of recapitulating cellular and humoral interactions and the physicochemical microenvironment (eg, hypoxia). Here, we first developed and characterized scaffold-free bone-like constructs (SFBCs), which were produced from bone marrow-derived mesenchymal stromal cells (MSCs) using a macroscale mesenchymal condensation approach. SFBCs revealed permeating mineralization characterized by increased bone volume (mu CT, histology) and expression of osteogenic markers (RUNX2, SPP1, RANKL). Fracture hematoma (FH) models, consisting of human peripheral blood (immune cells) mixed with MSCs, were co-cultivated with SFBCs under hypoxic conditions. As a result, FH models revealed an increased expression of osteogenic (RUNX2, SPP1), angiogenic (MMP2, VEGF), HIF-related (LDHA, PGK1), and inflammatory (IL6, IL8) markers after 12 and 48 hours co-cultivation. Osteogenic and angiogenic gene expression of the FH indicate the osteoinductive potential and, thus, the biological functionality of the SFBCs. IL-6, IL-8, GM-CSF, and MIP-1 beta were detectable within the supernatant after 24 and 48 hours of co-cultivation. To confirm the responsiveness of our model to modifying substances (eg, therapeutics), we used deferoxamine (DFO), which is well known to induce a cellular hypoxic adaptation response. Indeed, DFO particularly increased hypoxia-adaptive, osteogenic, and angiogenic processes within the FH models but had little effect on the SFBCs, indicating different response dynamics within the co-cultivation system. Therefore, based on our data, we have successfully modeled processes within the initial fracture healing phase in vitro and concluded that the cross-talk between bone and immune cells in the initial fracture healing phase is of particular importance for preclinical studies. (c) 2021 American Society for Bone and Mineral Research (ASBMR)

Development of a camera-marker system (CMS) for locking nails during intramedullary osteosynthesis in large animals – First results

In large animals, fractures of the upper long tubular bones such as forearm and humerus as well as lower and upper leg, continue to represent a major surgical challenge. An implant and a corresponding surgical procedure for intramedullary osteosynthesis in fractures of long bones were developed at fzmb GmbH. Fixation of the implanted nail to the distal drill holes has proven to be difficult and requires a high level of X-ray monitoring. Therefore, a camera-marker system (CMS) was developed without the need for X-ray monitoring during drilling. The test was performed on 10 equine femora. The results showed that 8 of 10 implants could be securely fixed and no complications occurred in the experimental procedure. For the first two implants, the distal drill holes could not be hit in the first attempt. This is due to the circumstance, that the handling of the CMS needs to be practiced to hit the distal drill holes accurately and appropriately, resulting in a hit rate of 80 %. However, the described method has great potential and does not require X-ray exposure

Investigation of knee joint stability in surgically repaired canine cruciate ligament ruptures by cyclic passive joint motions

The cranial cruciate ligament (CCL) rupture is a prevalent injury in dogs. A consequence of a cruciate ligament rupture is instability in the affected knee joint. A veterinary, mostly surgical treatment of the cruciate ligament rupture is usually unavoidable. The suitability of an arthroscopic surgical method with ligament replacement material was investigated. The stability of the knee joint was determined several times during 1,200 passive robotic motion cycles with movement radius between 90° flexion and 140° extension. The stability condition was measured by triggering the drawer test. After 300 motion cycles, the drawer test could be triggered (positive drawer test). In the following movement cycles up to 1,200 cycles, the drawer test could also be triggered. However, no significant differences occurred between these triggered drawer tests. The ligament replacement material showed no damage and no loosening after the tests. The first results showed that the developed arthroscopic surgical method could be a promising approach for the surgical treatment of cruciate ligament ruptures in canines

Analysis of Motion Patterns for Pain Estimation of Horses

This paper focuses on the automated analysis of motion patterns for relating an individual's behaviour with its pain experience. Reliable, automated behaviour analysis can improve video observation considerably, i.e. by lessening the work load of human operators, decreasing human error and by increasing anonymity and privacy. Possible applications are observation of traffic, public places and public transport. A new potential application is the early detection of pathologically relevant events, e.g. animal diseases (e.g. colics in horses), the automated post-surgical pain assessment of animals and similar applications. The challenge in the horses scenario is that they are flight animals that can not afford much of visible pain behaviour. Our approach is built on top of state of the art methods for object detection and tracking. From the object motion we derive motion patterns and respective features which we analyse by machine-learning methods. In the this paper we will present atypical behaviour detection (i.e. pain estimation) in animal videos, for which we have acquired a large video database. It could be shown that the condition of the horse can be analysed and classified by means of local histograms

Hypoxia and mesenchymal stromal cells as key drivers of initial fracture healing in an equine in vitro fracture hematoma model.

Fractures in horses-whether simple fractures with just one clean break, or incomplete greenstick with stress fractures, or complications such as shattered bones can all be either minimal or even catastrophic. Thus, improvement in fracture healing is a hallmark in equine orthopedics. The fracture healing process implements a complex sequence of events including the initial inflammatory phase removing damaged tissue, re-establishment of vessels and mesenchymal stromal cells, a soft and hard callus phase closing the fracture gap as well as the remodeling phase shaping the bone to a scar-free tissue. Detailed knowledge on processes in equine fracture healing in general and on the initial phase in particular is apparently very limited. Therefore, we generated equine in vitro fracture hematoma models (FH models) to study time-dependent changes in cell composition and RNA-expression for the most prominent cells in the FH model (immune cells, mesenchymal stromal cells) under conditions most closely adapted to the in vivo situation (hypoxia) by using flow cytometry and qPCR. In order to analyze the impact of mesenchymal stromal cells in greater detail, we also incubated blood clots without the addition of mesenchymal stromal cells under the same conditions as a control. We observed a superior survival capacity of mesenchymal stromal cells over immune cells within our FH model maintained under hypoxia. Furthermore, we demonstrate an upregulation of relevant angiogenic, osteogenic and hypoxia-induced markers within 48 h, a time well-known to be crucial for proper fracture healing

A large animal model for standardized testing of bone regeneration strategies

Abstract Background The need for bone graft substitutes including those being developed to be applied together with new strategies of bone regeneration such as tissue engineering and cell-based approaches is growing. No large animal model of bone regeneration has been accepted as a standard testing model. Standardization may be the key to moving systematically towards better bone regeneration. This study aimed to establish a model of bone regeneration in the sheep that lends itself to strict standardization and in which a number of substances can be tested within the same animal. To this end the caudal border of the ovine scapula was used as a consistent bed of mineralized tissue that provided sufficient room for a serial alignment of multiple experimental drill holes. Results The findings show that for the sake of standardization, surgery should be restricted to the middle part of the caudal margin, an area at least 80 mm proximal from the Glenoid cavity, but not more than 140 mm away from it, in the adult female Land Merino sheep. A distance of 5 mm from the caudal margin should also be observed. Conclusions This standardized model with defined uniform defects and defect sites results in predictable and reproducible bone regeneration processes. Defects are placed unilaterally in only one limb of the animal, avoiding morbidity in multiple limbs. The fact that five defects per animal can be evaluated is conducive to intra-animal comparisons and reduces the number of animals that have to be subject to experimentation