Katja F. Duesterdieck-Zellmer

Gene and protein expression of cartilage canal and osteochondral junction chondrocytes and full-thickness cartilage in early equine osteochondrosis ☆

The objective of this study was to investigate the expression of several regulatory factors associated with cartilage maturation in horses with early osteochondrosis (OC) compared to normal controls. The hypothesis was that expression levels of Indian hedgehog (Ihh), parathyroid hormone-related peptide (PTH-rP), vascular endothelial growth factor (VEGF), platelet-derived growth factor-A (PDGF-A), and matrix metalloproteinase-13 and -3 (MMP-13, -3) would be increased in OC. Articular cartilage and osteochondral samples were collected from the femoropatellar joints from seven OC and eight normal young (1-6 months) horses after euthanasia and snap frozen or suspended in 4% paraformaldehyde. Laser capture microdissection was used to capture cells surrounding cartilage canals and the osteochondral junction. Total RNA was isolated from whole cartilage and laser-captured cells. Equine-specific Ihh, PTH-rP, VEGF, PDGF-A, MMP-13, and MMP-3 mRNA expression levels were evaluated by real-time (RT)-PCR. Spatial tissue protein expression was determined by immunohistochemistry. In laser-captured samples, there was significantly increased MMP-13 and PDGF-A gene expression in chondrocytes adjacent to cartilage canals and increased PDGF-A gene expression in osteochondral junction chondrocytes of OC-affected foals. In full-thickness cartilage samples, there was significantly increased Ihh, MMP-3, and MMP-13 gene expression in OC samples, while PTH-rP protein expression was significantly higher along the osteochondral junction. The results suggest that pathways involving cartilage maturation and ossification are altered in early OC and may be associated with disease pathogenesis.

Use of Plain Radiography in the Diagnosis, Surgical Management, and Postoperative Treatment of Obstructive Urolithiasis in 25 Goats and 2 Sheep

Objective To describe use of plain radiography for diagnosis, surgical management, and postoperative treatment of obstructive urolithiasis in small ruminants. Study Design Retrospective case series. Animals Small ruminants (n = 27; 25 goats, 2 sheep). Methods Medical records (January 2002–November 2011) and radiographs for all small ruminants diagnosed with obstructive urolithiasis and having plain abdominal radiographs were reviewed. Signalment, surgical procedures, radiographic findings, ultrasonographic findings, position of calculi, and how plain radiography influenced surgical management and postoperative treatment were recorded. Results Radiopaque urinary calculi were detected in 23 (85%) plain radiographic studies. Location of uroliths determined by plain radiography included: cystic only (n = 5), distal to the sigmoid flexure and cystic (5), subischial (5), distal to the sigmoid flexure only (3), sigmoid flexure (3), and subischial and cystic (2). In 8 of these animals, postoperative radiographs revealed residual calculi in the urethra and were essential for their targeted removal by urethrotomy in 7 animals. Conclusions In regions where radiopaque calculi (calcium carbonate, calcium oxalate, silica) are commonly encountered in small ruminants, plain radiographs are recommended to determine the appropriate surgical approach(es) and to confirm resolution of the obstruction.OBJECTIVE To describe use of plain radiography for diagnosis, surgical management, and postoperative treatment of obstructive urolithiasis in small ruminants. STUDY DESIGN Retrospective case series. ANIMALS Small ruminants (n = 27; 25 goats, 2 sheep). METHODS Medical records (January 2002-November 2011) and radiographs for all small ruminants diagnosed with obstructive urolithiasis and having plain abdominal radiographs were reviewed. Signalment, surgical procedures, radiographic findings, ultrasonographic findings, position of calculi, and how plain radiography influenced surgical management and postoperative treatment were recorded. RESULTS Radiopaque urinary calculi were detected in 23 (85%) plain radiographic studies. Location of uroliths determined by plain radiography included: cystic only (n = 5), distal to the sigmoid flexure and cystic (5), subischial (5), distal to the sigmoid flexure only (3), sigmoid flexure (3), and subischial and cystic (2). In 8 of these animals, postoperative radiographs revealed residual calculi in the urethra and were essential for their targeted removal by urethrotomy in 7 animals. CONCLUSIONS In regions where radiopaque calculi (calcium carbonate, calcium oxalate, silica) are commonly encountered in small ruminants, plain radiographs are recommended to determine the appropriate surgical approach(es) and to confirm resolution of the obstruction.

Minimally invasive synovium harvest for potential use in meniscal tissue engineering

Tissue engineering is being investigated as a means for treating avascular meniscal injury or total meniscal loss in human and veterinary patients. The purpose of this study was to determine if an arthroscopic tissue shaver can be used to collect viable synoviocytes for in vitro culture during therapeutic stifle arthroscopy, with the long term goal of producing autologous meniscal fibrocartilage for meniscal tissue engineering. Synovium was harvested arthroscopically from 13 dogs with naturally occurring cranial cruciate ligament deficiency and obtained from 5 dogs with patellar luxation via arthrotomy. Cells harvested via arthroscopy and arthrotomy were treated with a chondrogenic growth factor protocol and analyzed for meniscal-like matrix constituents including collagens type I, II, and glycosaminoglycans. Arthrotomy and Arthroscopic origin cells formed contracted tissues containing collagen I, II and small amounts of GAG. These surgical methods provide clinically relevant access to synoviocytes for potential use in meniscal tissue engineering.

Wnt/β‐catenin signaling of cartilage canal and osteochondral junction chondrocytes and full thickness cartilage in early equine osteochondrosis

The objective of this study was to elucidate gene and protein expression of Wnt signaling molecules in chondrocytes of foals having early osteochondrosis (OC) versus normal controls. The hypothesis was that increased expression of components of Wnt signaling pathway in osteochondral junction (OCJ) and cartilage canal (CC) chondrocytes would be found in early OC when compared to controls. Paraffin‐embedded osteochondral samples (7 OC, 8 normal) and cDNA from whole cartilage (7 OC, 10 normal) and chondrocytes surrounding cartilage canals and osteochondral junctions captured with laser capture microdissection (4 OC, 6 normal) were obtained from femoropatellar joints of 17 immature horses. Equine‐specific Wnt signaling molecule mRNA expression levels were evaluated by two‐step real‐time qPCR. Spatial tissue protein expression of β‐catenin, Wnt‐11, Wnt‐4, and Dkk‐1 was determined by immunohistochemistry. There was significantly decreased Wnt‐11 and increased β‐catenin, Wnt‐5b, Dkk‐1, Lrp6, Wif‐1, Axin1, and SC‐PEP gene expression in early OC cartilage canal chondrocytes compared to controls. There was also significantly increased β‐catenin gene expression in early OC osteochondral junction chondrocytes compared to controls. Based on this study, abundant gene expression differences in OC chondrocytes surrounding cartilage canals suggest pathways associated with catabolism and inhibition of chondrocyte maturation are targeted in early OC pathogenesis.

Synoviocyte neotissues towards in vitro meniscal tissue engineering

Meniscal injuries are a common cause of pain and osteoarthritis in dogs. We describe here the production of synoviocyte-derived autologous neotissues for potential application in meniscal tissue engineering, via two different culture techniques: contracted or tensioned synthesis of synoviocyte neotissues. Synoviocytes were obtained during routine stifle arthroscopy and cultured from 14 dogs with naturally occurring osteoarthritis of the stifle. Neotissues were analyzed for meniscal-like matrix components and their gene expression, inflammatory gene expression, and cell viability. Tension improved cell viability, and, independent of cell viability, fibrochondrogenic activity by promoting expression of collagen type 1 and aggrecan genes and attenuating gene expression of IL-6. Through this mechanism tension increased collagen protein content and chondrogenic index of neotissues. Alpha smooth muscle actin was present in all neotissues and was responsible for grossly visible contractile behavior. Application of tension to synoviocytes may be a viable culture method towards in vitro meniscal tissue engineering.

Tiludronate concentrations and cytologic findings in synovial fluid after intravenous regional limb perfusion with tiludronate in horses

Anecdotal accounts of tiludronate administration via intravenous regional limb perfusion (IVRLP) exist despite a lack of information regarding safety for synovial structures in the perfused area. The objective of this study was to determine whether tiludronate concentrations in synovial structures after IVRLP with low dose (0.5 mg, LDT) or high dose (50 mg, HDT) tiludronate remain below a value demonstrated in vitro to be safe for articular cartilage (<19,000 ng/ml), and to determine effects of tiludronate on synovial fluid cytology variables compared to saline perfused control limbs. Using a randomized controlled experimental study design, horses received IVRLP with LDT (n = 6) or HDT (n = 6) in one forelimb and IVRLP with saline in the contralateral limb. Synovial fluid cytology variables and tiludronate concentrations were evaluated in navicular bursae (NB), and distal interphalangeal (DIP) and metacarpophalangeal (MCP) joints one week before and 30–45 min after IVRLP, and in DIP and MCP joints 24 h after IVRLP. Data were analyzed with 2-way rmANOVA (p < 0.05). Highest measured synovial fluid tiludronate concentrations occurred 30–45 min post-perfusion. Mean tiludronate concentrations were lower in LDT limbs (MCP = 39.6 ± 14.3 ng/ml, DIP = 118.1 ± 66.6 ng/ml, NB = 82.1 ± 30.2 ng/ml) than in HDT limbs (MCP = 3,745.1 ± 1,536.6 ng/ml, DIP = 16,274.0 ± 5,460.2 ng/ml, NB = 6,049.3 ± 1,931.7 ng/ml). Tiludronate concentration was >19,000 ng/ml in DIP joints of two HDT limbs. Tiludronate was measurable only in synovial fluid from HDT limbs 24 h post-perfusion. There were no differences in synovial fluid cytology variables between control and treated limbs. Conclusions. In some horses, IVRLP with HDT may result in synovial fluid concentrations of tiludronate that may have adverse effects on articular cartilage, based on in vitro data. IVRLP with LDT is unlikely to promote articular cartilage degradation. Further studies to determine a safe and effective dose for IVRLP with tiludronate are needed.

Growth factor treated tensioned synoviocyte neotissues: towards meniscal bioscaffold tissue engineering.

Meniscal injury is a common cause of osteoarthritis, pain, and disability in dogs and humans, but tissue-engineered bioscaffolds could be a treatment option for meniscal deficiency. The objective of this study was to compare meniscus-like matrix histology, composition, and biomechanical properties of autologous tensioned synoviocyte neotissues (TSN) treated with fetal bovine serum (TSNfbs) or three chondrogenic growth factors (TSNgf). Fourth passage canine synoviocytes from 10 dogs were grown in hyperconfluent monolayer culture, formed into TSN, and then cultured for 3 weeks with 17.7% FBS or three human recombinant TSNgf (bFGF, TGF-β1, and IGF-1). Cell viability was determined with laser microscopy. Histological architecture and the composition of fibrocartilage matrix were evaluated in TSN by staining tissues for glycosaminoglycan (GAG), α-smooth muscle actin, and collagen 1 and 2; quantifying the content of GAG, DNA, and hydroxyproline; and measuring the gene expression of collagens type 1α and 2α, the GAG aggrecan, and transcription factor Sry-type Homeobox Protein-9 (SOX9). Biomechanical properties were determined by materials testing force-deformation curves. The TSN contained components and histological features of mensical fibrocartilage extracellular matrix. Growth factor-treated TSN had higher DNA content but lower cell viability than TSNfbs. TSNgf had greater fibrocartilage-like matrix content (collagen 2 and GAG content with increased collagen 2α and SOX9 gene expression). Additionally, TSNgf collagen was more organized histologically and so had greater tensile biomechanical properties. The results indicate the potential of TSN when cultured with growth factors as implantable bioscaffolds for the treatment of canine meniscal deficiency.

Culture of canine synoviocytes on porcine intestinal submucosa scaffolds as a strategy for meniscal tissue engineering for treatment of meniscal injury in dogs.

Meniscal injury is a common cause of canine lameness. Tissue engineered bioscaffolds may be a treatment option for dogs suffering from meniscal damage. The aim of this study was to compare in vitro meniscal-like matrix formation and biomechanical properties of porcine intestinal submucosa sheets (SIS), used in canine meniscal regenerative medicine, to synoviocyte-seeded SIS bioscaffold (SSB), cultured with fetal bovine serum (SSBfbs) or chondrogenic growth factors (SSBgf). Synoviocytes from nine dogs were seeded on SIS and cultured for 30days with 17.7% fetal bovine serum or recombinant chondrogenic growth factors (IGF-1, TGFβ1 and bFGF). The effect on fibrochondrogenesis was determined by comparing mRNA expression of collagen types Iα and IIα, aggrecan, and Sry-type homeobox protein-9 (SOX9) as well as protein expression of collagens I and II, glycosaminoglycan (GAG), and hydroxyproline. The effect of synoviocyte seeding and culture conditions on biochemical properties was determined by measuring peak load, tensile stiffness, resilience, and toughness of bioscaffolds. Pre-culture SIS contained 13.6% collagen and 2.9% double-stranded DNA. Chondrogenic growth factor treatment significantly increased SOX9, collagens I and IIα, aggrecan gene expression (P<0.05), and histological deposition of fibrocartilage extracellular matrix (GAG and collagen II). Culture with synoviocytes increased SIS tensile peak load at failure, resilience, and toughness of bioscaffolds (P<0.05). In conclusion, culturing SIS with synoviocytes prior to implantation might provide biomechanical benefits, and chondrogenic growth factor treatment of cultured synoviocytes improves in vitro axial meniscal matrix formation.

Age-related differential gene and protein expression in postnatal cartilage canal and osteochondral junction chondrocytes

Wnt/β-catenin, Indian hedgehog (Ihh)/Parathyroid-related peptide (PTHrP) and retinoid signaling pathways regulate cartilage differentiation, growth, and function during development and play a key role in endochondral ossification. The objective of this study was to elucidate the gene and protein expression of signaling molecules of these regulatory pathways in chondrocytes surrounding cartilage canals and the osteochondral junction during neonatal and pre-adolescent development. This study revealed cell-specific and age-related differences in gene and protein expression of signaling molecules of these regulatory pathways. A trend for higher gene expression of PTHrP along the cartilage canals and Ihh along the osteochondral junction suggests the presence of paracrine feedback in articular-epiphyseal cartilage. Differential expression of canonical (β-catenin, Wnt-4, Lrp4, Lrp6) and noncanonical Wnt signaling (Wnt-5b, Wnt-11) and their inhibitors (Dkk1, Axin1, sFRP3, sFRP5, Wif-1) surrounding the cartilage canals and osteochondral junction provides evidence of the complex interactions occurring during endochondral ossification.

Comparison of Growth Factor Treatments on the Fibrochondrogenic Potential of Canine Fibroblast-Like Synoviocytes for Meniscal Tissue Engineering

OBJECTIVE To determine the in vitro effects of differing growth factor treatments on the fibrochondrogenic potential of fibroblast-like synoviocytes from cruciate ligament deficient femorotibial joints of dogs. STUDY DESIGN In vitro study. SAMPLE POPULATION Synoviocytes from dogs (n = 8) with naturally occurring cruciate ligament insufficiency. METHODS Synoviocytes were cultured in monolayer and synthesized into tensioned synoviocyte bioscaffolds (TSB) suspended in media containing TGF-β3, or FGF-2, TGF-β1, and IGF-I. The 1,9-dimethylmethylene blue (DMMB) assay and toluidine blue stain assessed glycosaminoglycan content; hydroxyproline assay, and collagen I and II immunohistochemistry assessed collagen content. Biomechanical properties were determined by materials testing/force-deformation curves. RESULTS All tissue cultures formed tensioned fibrous tissue-like constructs. Mean tissue cellularity and cellular viability was significantly greater in the triple growth factor-treated TSB by 0.09% and 44%, respectively. Percentage collagen content, and relative gene expression for collagen I, II, and aggrecan was not significantly different between groups. Median percentage of GAG content was significantly greater in triple growth factor-treated TSB by 1.6%. Biomechanical properties were not different in compression. Triple growth factor-treated TSB were significantly stronger in toughness, peak load to failure, and stiffness in tension. CONCLUSIONS TGF-β3 cultured bioscaffolds failed to outperform triple growth factor-treated TSB. Architectural extracellular matrix (ECM) organization and cellularity likely explained the differences between groups. TGF-β3 alone cannot be recommended at this time for in vitro formation of autologous fibrocartilage bioscaffolds for meniscal deficiency.