Uta Delling

Stem cell-based tissue engineering in veterinary orthopaedics

Regenerative medicine is one of the most intensively researched medical branches, with enormous progress every year. When it comes to translating research from bench to bedside, many of the pioneering innovations are achieved by cooperating teams of human and veterinary medical scientists. The veterinary profession has an important role to play in this new and evolving technology, holding a great scientific potential, because animals serve widely as models for human medicine and results obtained from animals may serve as preclinical results for human medicine. Regenerative veterinary medicine utilizing mesenchymal stromal cells (MSC) for the treatment of acute injuries as well as chronic disorders is gradually turning into clinical routine. As orthopaedic disorders represent a major part of all cases in veterinary clinical practice, it is not surprising that they are currently taking a leading role in MSC therapies. Therefore, the purpose of this paper is to give an overview on past and current achievements as well as future perspectives in stem cell-based tissue engineering in veterinary orthopaedics.

Longitudinal evaluation of effects of intra-articular mesenchymal stromal cell administration for the treatment of osteoarthritis in an ovine model.

In this study, the therapeutic effect of intra-articularly injected autologous mesenchymal stromal cells (MSCs) was evaluated in an ovine osteoarthritis (OA) model using consecutive magnetic resonance imaging (MRI), radiography, and macroscopic and histologic postmortem examination. In 12 sheep, OA was induced by bilateral, lateral meniscectomy. After 6 weeks, 20 × 106 bone marrow-derived MSCs (50% MSCs were superparamagnetic iron oxide particle labeled) were injected intra-articularly into one knee joint. The contralateral knee served as negative control. MR images were acquired before OA induction, immediately before and after MSC injection, and 1, 4, 8, and 12 weeks thereafter using a 0.5T unit and a T2* gradient echo sequence. Radiographs were obtained before OA induction, at MSC injection, and 12 weeks thereafter. The MRI scoring system included articular cartilage, bone, joint capsule, and synovial fluid evaluation. The radiographic scoring system included the joint space and bone. Postmortem evaluation entailed macroscopic and histologic assessment. Longitudinal MRI revealed a significant deceleration of OA progression in MSC-treated joints. However, at the conclusion of the study, there was no significant difference in the degree of OA detected by MRI, radiography, and postmortem evaluation between the treatment and control group. The degree of OA on MRI varied among the 12 animals at the time of injection, but there was no difference between the left and right limb. In conclusion, intra-articular MSCs decreased OA progression. However, no significant treatment effects were seen at the conclusion of the study at 12 weeks. This somewhat contradicts previously published results. Nevertheless, the choice of OA model, outcome measures, or lack of additional medication might explain the differences. Our results indicate that OA might benefit from intra-articular MSC injection, but further studies are needed to refine patient selection and injection parameters for a more substantially improved outcome.

Comparative Labeling of Equine and Ovine Multipotent Stromal Cells With Superparamagnetic Iron Oxide Particles for Magnetic Resonance Imaging In Vitro.

The purpose of this study was to evaluate the use of three different superparamagnetic iron oxide (SPIO) particles for labeling of ovine and equine bone marrow (BM)-derived multipotent stromal cells (MSCs) in vitro. MSCs were obtained from five adult sheep and horses, respectively. After three passages (p3), cells were labeled with either 1) Molday ION Rhodamine B, 2) Endorem, 3) Resovist, or 4) remained unlabeled as control. Labeling efficiency, marker retention, and long-term detectability in MRI until p7 were evaluated. Further, proliferation capacity and trilineage differentiation as indicators for potential impact on stromal cell characteristics were assessed. MSCs of both species were successfully labeled with all three SPIO products. A high, exclusively intracellular, iron uptake was achieved by Molday ION Rhodamine B only. Labeling with Resovist led to prominent extracellular iron presence; labeling with Endorem was less efficient. During MRI, all labeled cells showed strong hypointense signals, contrary to unlabeled controls. Resovist induced the largest areas of hypointense signals, followed by Molday ION Rhodamine B and Endorem. MRI signal detectability decreased from p4 to p7. Proliferation, adipogenic, and osteogenic differentiation potential were not reduced by cell labeling compared to unlabeled cells. Chondrogenic differentiation capacity decreased with increasing amount of iron associated with the cells. Among the three products, Resovist and Molday were identified as promising labeling agents. While Resovist achieved superior results in most of the assessed parameters, Molday ION Rhodamine B ensured intracellular iron uptake without extracellular SPIO complexes and consistent hypointense signals on MRI.

In Vivo Tracking and Fate of Intra-Articularly Injected Superparamagnetic Iron Oxide Particle-Labeled Multipotent Stromal Cells in an Ovine Model of Osteoarthritis:

In this study, superparamagnetic iron oxide (SPIO) particle-labeled mesenchymal stromal cells (MSCs) were injected intra-articularly into osteoarthritic knee joints. Their fate and distribution were evaluated using magnetic resonance imaging (MRI) and macroscopic and histologic postmortem examination. Osteoarthritis was induced in 12 sheep by bilateral meniscectomy. After 6 weeks, one knee joint received 10 × 106 SPIO-labeled MSCs (Molday Ion Rhodamine B). Contralateral knees received a control injection of a) PBS, b) SPIO in PBS, c) 10 × 106 nonvital SPIO-labeled MSCs in PBS, or d) no injection. MR images were acquired immediately after injection and 1, 4, 8, and 12 weeks thereafter using a 0.5-T unit and a T2* sequence. Signal intensity of synovial fluid and synovial lining was assessed semiquantitatively using a scoring system. Viable SPIO-labeled MSCs produced a strong hypointense signal in the synovial fluid immediately after injection, but normal signal intensity of the synovial fluid was observed 1 week later. Synovial lining maintained its hypointensity throughout the study period. Nonvital SPIO-labeled MSCs induced hypointense signals of the synovial fluid; synovial lining appeared weak and inconsistently hypointense in the following weeks. Pure SPIO produced a strong hyperintense signal in the synovial fluid at the time of injection only. Histologically, in all knee joints receiving viable SPIO-labeled MSCs, SPIO particles were detected (Prussian blue) within the synovial lining, dorsal fat pad, and neomeniscus tissue, but not in osteochondral samples. Few SPIO particles were detected in joints injected with nonvital SPIO-labeled MSCs. Immunohistologically, no increased cell death (TUNEL) was observed in the area of detected SPIO particles, but we did observe potential chondrogenic cell differentiation (Safranin O or S100β). We conclude that viable SPIO-labeled MSCs remain detectable within the joint for 12 weeks and attach themselves to some but not all diseased joint structures.

Application of Stem Cells for the Treatment of Joint Disease in Horses

Stem cells in the form of mesenchymal stromal cells derived from various sources have been identified to have the potential of supporting the therapy of joint disease in the horse, and preliminary data has been published about the clinical application of stem cells in horses suffering from clinical joint disease. Furthermore, the horse is recognized to be the ideal large animal model for the preclinical study of cell therapy in joints. The advantage of this species in this respect is the size of the joints, which makes surgical applications practically feasible in analogy to human surgery. Additionally, the horse is the only model species with a cartilage thickness in the knee joint comparable to that of humans. Especially the fact that horses develop clinical joint disease discerns this species from other large animal models like small ruminants. The therapy of clinical disease in model animal species represents the ideal situation for preclinical studies of novel therapeutic strategies. Here, we describe the experimental and clinical approaches to joint disease in the horse.

Magnetic resonance imaging of pulp in normal and diseased equine cheek teeth.

A retrospective analysis of 619 upper and lower cheek teeth from 62 horses was performed. Based on clinical findings, as well as radiographic and magnetic resonance (MR) imaging findings, the teeth were classified into five groups. There were 20 teeth with abnormal MR imaging signals as well as clinical alterations and 599 healthy teeth. Using MR imaging, the appearance of pulp in diseased and disease-free teeth was compared, and the appearance of abnormal pulp was studied. Subsequently, the ability of MR imaging to diagnose pulpitis and pulp necrosis in teeth with normal external appearance was investigated. In horses with clinically verified dental disease, abnormal MR imaging findings were confirmed in the pulp of all affected teeth. An enlarged blurred pulp image with a lower signal intensity was observed only in clinically diseased teeth and was a reliable criterion for diagnosing dental disease on MR imaging. On the other hand, partial or complete absence of pulp in all MR imaging sequences was observed in both diseased and nondiseased teeth. These data demonstrate that pulp changes in equine cheek teeth can be evaluated using MR imaging.

Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses

Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 °C vs. room temperature; −20 °C vs. −80 °C), four time frames (24 h vs. 48 h; 48 h vs. 72 h), and (III) three MSC concentrations (5 × 106, 10 × 106, 20 × 106 MSC/ml). Cell viability (Trypan Blue exclusion; percent and total number viable cell), proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6) and differentiation protocols were performed in duplicates. Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability). In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and “transport” at 4 °C for 24 h (70.6% vs. control group 75.3%); this was not significant. Contrary, viability was unacceptably low (<40%) for all freezing protocols at −20 °C or −80 °C, particularly with bone marrow supernatant or plasma and DMSO. In part III, various cell concentrations also had no significant influence on any of the evaluated parameters. Chondrogenic differentiation showed a trend towards being decreased for all transport conditions, compared to control cells. Discussion. In this study, transport conditions were not found to impact viability, proliferation or ability for trilineage differentiation of MSCs, most likely due to the small sample size and large number of comparisons. The unusual low viability after all freezing protocols is in contrast to previous equine studies. Potential causes are differences in the freezing, but also in thawing method. Also, the selected container (glass syringe) may have impacted viability. Future research may be warranted into the possibly negative effect of transport on chondrogenic differentiation.

Evaluation of a Training Model to Teach Veterinary Students a Technique for Injecting the Jugular Vein in Horses

In this study, a newly-developed model for training veterinary students to inject the jugular vein in horses was evaluated as an additional tool to supplement the current method of teaching. The model was first validated by 19 experienced equine veterinarians, who judged the model to be a realistic and valuable tool for learning the technique. Subsequently, it was assessed using 24 students who were divided randomly into two groups. The injection technique was taught conventionally in a classroom lecture and a live demonstration to both groups, but only group 1 received additional training on the new model. All participants filled out self-assessment questionnaires before and after group 1 received training on the model. Finally, the proficiency of both groups was assessed using an objective structured clinical evaluation (OSCE) on live horses. Students from group 1 showed significantly improved confidence after their additional training on the model and also showed greater confidence when compared to group 2 students. In the OSCE, group 1 had a significantly better score compared to group 2: the median (with inter-quartile range) was 15 (0.7) vs. 11.5 (2.8) points out of 15, respectively. The training model proved to be a useful tool to teach veterinary students how to perform jugular vein injections in horses in a controlled environment, without time limitations or animal welfare concerns. The newly developed training model offers an inexpensive, efficient, animal-sparing way to teach this clinical skill to veterinary students.

Surgical removal of a dermoid cyst from the bony part of the nasolacrimal duct in a Scottish highland cattle heifer

The ophthalmologic, radiologic and surgical findings of a 2-year-old Scottish Highland Cattle heifer with a dermoid cyst within the bony part of the nasolacrimal duct and the successful treatment are presented.