Florien Jenner

Differential gene expression of the intermediate and outer interzone layers of developing articular cartilage in murine embryos.

Nascent embryonic joints, interzones, contain a distinct cohort of progenitor cells responsible for the formation of the majority of articular tissues. However, to date the interzone has largely been studied using in situ analysis for candidate genes in the context of the embryo rather than using an unbiased genome-wide expression analysis on isolated interzone cells, leaving significant controversy regarding the exact role of the intermediate and outer interzone layers in joint formation. Therefore, in this study, using laser capture microdissection (three biological replicates), we selectively harvested the intermediate and outer interzones of mouse embryos at gestational age 15.5 days, just prior to cavitation, when the differences between the layers should be most profound. Microarray analysis (Agilent Whole Mouse Genome Oligo Microarrays) was performed and the differential gene expression between the intermediate interzone cells and outer interzone cells was examined by performing a two-sided paired Students t-test and pathway analysis. One hundred ninety-seven genes were differentially expressed (≥ 2-fold) between the intermediate interzone and the outer interzone with a P-value ≤ 0.01. Of these, 91 genes showed higher expression levels in the intermediate interzone and 106 were expressed higher in the outer interzone. Pathway analysis of differentially expressed genes suggests an important role for inflammatory processes in the interzone layers, especially in the intermediate interzone, and hence in joint and articular cartilage development. The high representation of genes relevant to chondrocyte hypertrophy and endochondral ossification in the outer interzone suggests that it undergoes endochondral ossification.

Core decompression of the equine navicular bone: an in vivo study in healthy horses.

OBJECTIVE To determine the physiologic response of the equine navicular bone to core decompression surgery in healthy horses. STUDY DESIGN Experimental in vivo study. MATERIALS Healthy adult horses (n=6). METHODS Core decompression was completed by creating three 2.5-mm-diameter drill channels into the navicular bone under arthroscopic control. The venous (P(V)), arterial (P(A)), articular (P(DIPJ)), and intraosseous pressures (IOP) were recorded before and after decompression drilling. Each IOP measurement consisted of a baseline (IOP(B)) and a stress test (intramedullary injection of saline solution, IOP(S)) recording. Lameness was assessed subjectively and using force plate gait analysis. Fluorochrome bone labeling was performed. Horses were euthanatized at 12 weeks. Navicular bone mineral density (BMD) was measured, and bone histology evaluated. RESULTS Peak IOP (IOP(max)) after stress testing was significantly (P<.05) reduced immediately after core decompression; however, the magnitude of these effects was decreased at 3 and 6 weeks after decompression. A significant (P<.05) correlation existed between IOP(max) and BMD. No lameness was observed beyond the first week after surgery. Substantial remodeling and neovascularization was evident adjacent the surgery sites. CONCLUSION Navicular bone core decompression surgery reduced IOP(max), and, with the exception of a mild short-lived lameness, caused no other adverse effects in healthy horses during the 12-week study period.Objective: To determine the physiologic response of the equine navicular bone to core decompression surgery in healthy horses. Study Design: Experimental in vivo study. Materials: Healthy adult horses (n=6). Methods: Core decompression was completed by creating three 2.5-mm-diameter drill channels into the navicular bone under arthroscopic control. The venous (PV), arterial (PA), articular (PDIPJ), and intraosseous pressures (IOP) were recorded before and after decompression drilling. Each IOP measurement consisted of a baseline (IOPB) and a stress test (intramedullary injection of saline solution, IOPS) recording. Lameness was assessed subjectively and using force plate gait analysis. Fluorochrome bone labeling was performed. Horses were euthanatized at 12 weeks. Navicular bone mineral density (BMD) was measured, and bone histology evaluated. Results: Peak IOP (IOPmax) after stress testing was significantly (P<.05) reduced immediately after core decompression; however, the magnitude of these effects was decreased at 3 and 6 weeks after decompression. A significant (P<.05) correlation existed between IOPmax and BMD. No lameness was observed beyond the first week after surgery. Substantial remodeling and neovascularization was evident adjacent the surgery sites. Conclusion: Navicular bone core decompression surgery reduced IOPmax, and, with the exception of a mild short-lived lameness, caused no other adverse effects in healthy horses during the 12-week study period.

Evaluation of diffusion of triamcinolone acetonide from the distal interphalangeal joint into the navicular bursa in horses

OBJECTIVE To determine whether triamcinolone acetonide diffuses from the distal interphalangeal joint (DIPJ) to the navicular bursa, diffusion is direct or systemic, and addition of sodium hyaluronan has an effect on diffusion in horses. ANIMALS 11 adult horses without forelimb lameness. PROCEDURES 1 randomly chosen forelimb DIPJ of each horse received an injection of 10 mg of triamcinolone acetonide plus 20 mg of sodium hyaluronan (group 1), and the contralateral forelimb DIPJ received an injection of 10 mg of triamcinolone acetonide plus 2 mL of lactated Ringers solution (group 2). Synovial fluid samples were taken from both forelimb navicular bursae and 1 hind limb navicular bursa (systemic control group) at 6 hours. Triamcinolone acetonide concentrations in synovial fluid were quantified by use of high-performance liquid chromatography plus tandem mass spectrometry. Data were logarithmically transformed, and contrast analysis was performed on the 3 groups. RESULTS Triamcinolone acetonide was detected in navicular bursal samples in all groups. Groups 1 and 2 had significantly greater concentrations of triamcinolone acetonide than the systemic control group. There was no significant difference between groups 1 and 2. CONCLUSIONS AND CLINICAL RELEVANCE Triamcinolone acetonide diffused directly from the DIPJ into the navicular bursa in clinically normal horses, and diffusion was not affected by addition of hyaluronan. Injection into the DIPJ with triamcinolone acetonide or a triamcinolone acetonide-hyaluronan combination can potentially be used for treatment of navicular syndrome, but further studies are needed to determine whether triamcinolone acetonide diffuses similarly in horses with navicular syndrome.

Elasticity and breaking strength of synthetic suture materials incubated in various equine physiological and pathological solutions

REASONS FOR PERFORMING STUDY Selection of suture material in equine surgery is often based on costs or subjective factors, such as the surgeons personal experience, rather than objective facts. The amount of objective data available on durability of suture materials with regard to specific equine physiological conditions is limited. OBJECTIVES To evaluate the effect of various equine physiological and pathological fluids on the rate of degradation of a number of commonly used suture materials. STUDY DESIGN In vitro material testing. METHODS Suture materials were exposed in vitro to physiological fluid, followed by biomechanical analysis. Three absorbable suture materials, glycolide/lactide copolymer, polyglactin 910 and polydioxanone were incubated at 37°C for 7, 14 or 28 days in phosphate-buffered saline, equine serum, equine urine and equine peritoneal fluid from an animal with peritonitis. Five strands of each suture material type were tested to failure in a materials testing machine for each time point and each incubation medium. Yield strength, strain and Youngs modulus were calculated, analysed and reported. RESULTS For all suture types, the incubation time had a significant effect on yield strength, percentage elongation and Youngs modulus in all culture media (P<0.0001). Suture type was also shown significantly to influence changes in each of yield strength, percentage elongation and Youngs modulus in all culture media (P<0.0001). While the glycolide/lactide copolymer demonstrated the highest Day 0 yield strength, it showed the most rapid degradation in all culture media. For each of the 3 material characteristics tested, polydioxanone showed the least variation across the incubation period in each culture medium. CONCLUSIONS The duration of incubation and the type of fluid have significant effects on the biomechanical properties of various suture materials. These findings are important for evidence-based selection of suture material in clinical cases.

3D co-culture of meniscal cells and mesenchymal stem cells in collagen type I hydrogel on a small intestinal matrix - a pilot study towards equine meniscus tissue engineering.

Meniscal injuries are the most frequently encountered soft tissue injuries in the equine stifle joint. Due to the inherent limited repair potential of meniscal tissue, meniscal injuries do not only affect the meniscus itself but also lead to impaired joint homeostasis and secondary osteoarthritis. The presented study compares 3D coculture constructs of primary equine mesenchymal stem cells (MSC) and meniscus cells (MC) seeded on three different scaffolds-a cell-laden collagen type I hydrogel (Col I gel), a tissue-derived small intestinal matrix scaffold (SIS-muc) and a combination thereof-for their qualification to be applied for meniscus tissue engineering. To investigate cell attachment of primary MC and MSC on SIS-muc matrix SEM pictures were performed. For molecular analysis, lyophilized samples of coculture constructs with different cell ratios (100% MC, 100% MSC, and 50% MC and 50% MSC, 20% MC, and 80% MSC) were digested and analyzed for DNA and GAG content. Active matrix remodeling of 3D coculture models was indicated by matrix metalloproteinases detection. For comparison of tissue-engineered constructs with the histologic architecture of natural equine menisci, paired lateral and medial menisci of 15 horses representing different age groups were examined. A meniscus phenotype with promising similarity to native meniscus tissue in its GAG/DNA expression in addition to Col I, Col II, and Aggrecan production was achieved using a scaffold composed of Col I gel on SIS-muc combined with a coculture of MC and MSC. The results encourage further development of this scaffold-cell combination for meniscus tissue engineering.

Knot Security of 5 Metric (USP 2) Sutures: Influence of Knotting Technique, Suture Material, and Incubation Time for 14 and 28 Days in Phosphate Buffered Saline and Inflamed Equine Peritoneal Fluid

Objectives To evaluate knot security for 3 knot types created in 3 commonly used 5 metric suture materials incubated in physiological and pathological fluids. Study Design In vitro mechanical study. Sample Population Knotted suture loops (n = 5/group). Methods Loops of 3 different suture materials (glycolide/lactide copolymer; polyglactin 910; polydioxanone) were created around a 20 mm rod using 3 knot types (square [SQ], surgeons [SK], and triple knot [TK]) and were tested to failure in distraction (6 mm/min) after tying (day 0) and after being incubated for 14 and 28 days in phosphate buffered saline (PBS) or inflamed peritoneal fluid. Failure load (N) and mode were recorded and compared. Results For polydioxanone, significant differences in force to knot failure were found between SQ and SK/TK but not between SK and TK. The force required to break all constructs increased after incubation in phosphate buffered saline (PBS). With glycolide/lactide copolymer no differences in force to knot failure were observed. With polyglactin 910, a significant difference between SQ and TK was observed, which was not seen between the other knot types. Incubation in inflamed peritoneal fluid caused a larger and more rapid decrease in force required to cause knot failure than incubation in PBS. Conclusions Mechanical properties of suture materials have significant effects on knot security. For polydioxanone, SQ is insufficient to create a secure knot. Additional wraps above a SK confer extra stability in some materials, but this increase may not be clinically relevant or justifiable. Glycolide/lactide copolymer had excellent knot security.OBJECTIVES To evaluate knot security for 3 knot types created in 3 commonly used 5 metric suture materials incubated in physiological and pathological fluids. STUDY DESIGN In vitro mechanical study. SAMPLE POPULATION Knotted suture loops (n = 5/group). METHODS Loops of 3 different suture materials (glycolide/lactide copolymer; polyglactin 910; polydioxanone) were created around a 20 mm rod using 3 knot types (square [SQ], surgeons [SK], and triple knot [TK]) and were tested to failure in distraction (6 mm/min) after tying (day 0) and after being incubated for 14 and 28 days in phosphate buffered saline (PBS) or inflamed peritoneal fluid. Failure load (N) and mode were recorded and compared. RESULTS For polydioxanone, significant differences in force to knot failure were found between SQ and SK/TK but not between SK and TK. The force required to break all constructs increased after incubation in phosphate buffered saline (PBS). With glycolide/lactide copolymer no differences in force to knot failure were observed. With polyglactin 910, a significant difference between SQ and TK was observed, which was not seen between the other knot types. Incubation in inflamed peritoneal fluid caused a larger and more rapid decrease in force required to cause knot failure than incubation in PBS. CONCLUSIONS Mechanical properties of suture materials have significant effects on knot security. For polydioxanone, SQ is insufficient to create a secure knot. Additional wraps above a SK confer extra stability in some materials, but this increase may not be clinically relevant or justifiable. Glycolide/lactide copolymer had excellent knot security.

Laser Capture Microdissection of Murine Interzone Cells: Layer Selection and Prediction of RNA Yield

Objective: Articular chondrocytes originate from a distinct cohort of progenitor cells located in the so-called interzone in embryonic developing joints. We conducted this study 1) to determine if it is possible to identify the intermediate and outer interzone layers histologically (using cresyl violet) without adjunct in situ hybridization or immunohistochemistry; 2) to establish whether sufficient amounts of RNA can be harvested from each interzone layer of individual embryos to allow gene expression analysis; 3) to develop measurements that can provide an estimation of the RNA yield prior to costly amplification steps. Methods: Cells from the outer (OI) and intermediate (II) interzone of the nascent femorotibial joint and the epiphyseal cartilage (EC) of femur and tibia of murine embryos of 13.5 and 15.5 days gestation were harvested using laser capture microdissection (LCM). Subsequently, microarray analysis was performed to confirm appropriate layer selection. The surface area harvested and the grey value (gv) of photomicrographs taken during LCM was measured and the corresponding relative optical density (ROD) was calculated and degree and significance of the correlation with RNA yield were determined. Results: Cells from the OI, II and EC could be identified histologically using cresyl violet staining and were successfully harvested with LCM yielding sufficient amounts of RNA for linear amplification and microarray analysis. The RNA yield correlated significantly with the tissue surface area harvested, the mean gv and the corresponding ROD. Conclusions: This study provides a technique for selective laser capture microdissection and subsequent microarray analysis of murine interzone cells of the intermediate and outer layer and presents a method to estimate the RNA yield by measuring the tissue area harvested and calculating the ROD. We recommend to harvest a minimum of 1×106 μm2 of 13.5 days murine embryos and 3×106 μm2 of 15.5 days murine embryos to obtain approximately 10 ng total RNA that can be used for linear T7-based amplification and subsequent microarray analysis.

Magnetic resonance microscopy atlas of equine embryonic development.

REASONS FOR PERFORMING STUDY Equine embryogenesis post implantation is not well studied, and only two-dimensional illustrations are available. A thorough appreciation of the complex three-dimensional relationship between tissues and organs and their development is, however, crucial for understanding physiological and pathological mechanisms. OBJECTIVES The goals were 2-fold: 1) to establish a freely accessible online atlas as a reference tool for the scientific and pedagogic communities; and 2) to create a framework for integration of data with known spatiotemporal distribution, such as gene expression or cell lineage. STUDY DESIGN Descriptive anatomical study. METHODS Magnetic resonance microscopy was performed on embryos of 28, 32, 35, 37, 39, 40, 42, 45, 50 and 65 days gestation using a 9.4 T magnet. Equine embryos were staged according to the Carnegie system. Acquired images were optimised using histogram optimisation and processed for easy online access. RESULTS Magnetic resonance microscopy protocols for imaging of equine embryos and fetuses were developed. The wider spread of signal intensity values achieved by histogram equalisation increased visual contrast considerably. Despite their longer gestation, equine conceptuses appeared to reach the various Carnegie staging benchmarks earlier than human embryos. CONCLUSIONS The equine atlas is designed to serve as an online reference tool for research and teaching. POTENTIAL RELEVANCE The equine atlas may serve as a foundation and scaffold for improved anatomical labelling, spatial and temporal data integration and further understanding of physiological and pathophysiological processes involved in development and disease.

Core Decompression of the Equine Navicular Bone: An In Vitro Biomechanical Study

OBJECTIVE To determine the effect of core decompression surgery and bone mineral density (BMD) on the mechanical properties of equine navicular bones. STUDY DESIGN Experimental, in vitro study. SAMPLE POPULATION Fore limb navicular bones (n=36 pairs) from sound 2-5-year-old horses with no radiographic abnormalities of the distal aspect of the forelimbs. METHODS Navicular BMD was measured using dual energy X-ray absorptiometry. One randomly assigned navicular bone from each pair served as control. The contralateral test specimen was allocated to 1 of 6 treatment groups defined by drill bit size (3.2 versus 2.5 mm diameter) and by the number of drill channels (1, 2, or 3) created in the proximal border of the bone. Bones were then tested until failure in 3-point bending. Data were statistically analyzed using ANOVA and regression analysis. RESULTS There were significant (P<.001) positive correlations between BMD and biomechanical data. A significant (P<.001) reduction in breaking strength was noted between intact and drilled bone pairs; however, the diameter and number of decompression channels did not significantly (P>.05) influence the extent of the reduction in mechanical strength. CONCLUSION In vitro core decompression significantly decreases the breaking strength of the equine navicular bone.

Inflammation-induced transgene expression in genetically engineered equine mesenchymal stem cells.

Osteoarthritis, a chronic and progressive degenerative joint disorder, ranks amongst the top five causes of disability. Given the high incidence, associated socioeconomic costs and the absence of effective disease‐modifying therapies of osteoarthritis, cell‐based treatments offer a promising new approach. Owing to their paracrine, differentiation and self‐renewal abilities, mesenchymal stem cells (MSCs) have great potential for regenerative medicine, which might be further enhanced by targeted gene therapy. Hence, the development of systems allowing transgene expression, particularly when regulated by natural disease‐dependent occuring substances, is of high interest.