Differential roles of putative arginine fingers of AAA + ATPases Rvb1 and Rvb2.

The evolutionarily conserved AAA + ATPases Rvb1 and Rvb2 proteins form a heteromeric complex (Rvb1/2) required for assembly or remodeling of macromolecular complexes in essential cellular processes ranging from chromatin remodeling to ribosome biogenesis. Rvb1 and Rvb2 have a high degree of sequence and structural similarity, and both contain the classical features of ATPases of their clade, including an N-terminal AAA + subdomain with the Walker A motif, an insertion domain that typically interacts with various binding partners, and a C-terminal AAA + subdomain containing a Walker B motif, the Sensor I and II motifs, and an arginine finger. In this study, we find that despite the high degree of structural similarity, Rvb1 and Rvb2 have distinct active sites that impact their activities and regulation within the Rvb1/2 complex. Using a combination of biochemical and genetic approaches, we show that replacing the homologous arginine fingers of Rvb1 and Rvb2 with different amino acids not only has distinct effects on the catalytic activity of the complex, but also impacts cell growth, and the Rvb1/2 interactions with binding partners. Using molecular dynamics simulations, we find that changes near the active site of Rvb1 and Rvb2 cause long-range effects on the protein dynamics in the insertion domain, suggesting a molecular basis for how enzymatic activity within the catalytic site of ATP hydrolysis can be relayed to other domains of the Rvb1/2 complex to modulate its function. Further, we show the impact that the arginine finger variants have on snoRNP biogenesis and validate the findings from molecular dynamics simulations using a targeted genetic screen. Together, our results reveal new aspects of the regulation of the Rvb1/2 complex by identifying a relay of long-range molecular communication from the ATPase active site of the complex to the binding site of cofactors. Most importantly, our findings suggest that despite high similarity and cooperation within the same protein complex, the two proteins have evolved with unique properties critical for the regulation and function of the Rvb1/2 complex. Significance: AAA ATPases constitute a large family of proteins involved in various essential cellular functions in living organisms in all kingdoms of life. Members of this family typically form homo or hetero multimers that convert the energy from ATP hydrolysis to mechanical work. How the conserved features of AAA ATPases relay the energy from ATP hydrolysis to other functional domains of the complex remains largely unknown. Here, using arginine finger variants of Rvb1 and Rvb2, two evolutionarily conserved closely related AAA + ATPases that form a heterohexameric complex, we reveal how individual protomers in a heteromeric complex can uniquely contribute to the overall function of the complex and how changes in the ATP binding site can be relayed to distal functional domains.

Assembly chaperone Nas6 selectively destabilizes 26S proteasomes with defective regulatory particle-core particle interfaces.

The 26S proteasome is a 66-subunit-chambered protease present in all eukaryotes that maintains organismal health by degrading unneeded or defective proteins. Defects in proteasome function or assembly are known to contribute to the development of various cancers, neurodegeneration, and diabetes. During proteasome biogenesis, a family of evolutionarily conserved chaperones assembles a hexameric ring of AAA+ family ATPase subunits contained within the proteasomal regulatory particle (RP) and guide their docking onto the surface of the proteolytic core particle (CP). This RP-CP interaction couples the substrate capture and unfolding process to proteolysis. We previously reported a mutation in the proteasome that promoted dissociation of the RP and CP by one of these chaperones, Nas6. However, the nature of the signal for Nas6-dependent proteasome disassembly and the generality of this postassembly proteasome quality control function for Nas6 remain unknown. Here, we use structure-guided mutagenesis and in vitro proteasome disassembly assays to demonstrate that Nas6 more broadly destabilizes 26S proteasomes with a defective RP-CP interface. We show that Nas6 can promote dissociation of mature proteasomes into RP and CP in cells harboring defects on either side of the RP-CP interface. This function is unique to Nas6 and independent from other known RP assembly chaperones. Further biochemical experiments suggest that Nas6 may exploit a weakened RP-CP interface to dissociate the RP from the CP. We propose that this postassembly role of Nas6 may fulfill a quality control function in cells by promoting the recycling of functional subcomplexes contained within defective proteasomes.

Iatrogenic cartilage injury associated with the use of stainless-steel cannulas and silicone-guarded cannulas for canine stifle arthroscopy.

OBJECTIVE: To determine the ability to reduce iatrogenic cartilage injury (IACI) during canine stifle arthroscopy by using a silicone arthroscope cannula guard. STUDY DESIGN: Ex vivo canine cadaver experimental study. ANIMALS: Paired canine stifles from 14 cadavers (≥20 kg). METHODS: Stifles (N = 28) were assigned to unguarded traditional or silicone-guarded arthroscopy. Stifle arthroscopy and full joint exploration with meniscal probing was performed by a second-year surgery resident (I.C.) in fourteen canine cadavers, alternating between left and right stifles for guarded vs unguarded arthroscopy. After arthroscopy, stifles were disarticulated, and india ink assay was performed to identify IACI. Total IACI number, lesion length and area, duration of procedure, and procedure difficulty score were recorded for each stifle. RESULTS: Unguarded arthroscopy resulted in more total IACI per joint (unguarded 5.2 ± 3.0, guarded 2.4 ± 1.4; P = .02), larger IACI area (unguarded 5.2 ± 4.2 mm2 , guarded 2.3 ± 1.5 mm2 ; P = .02), and IACI length (unguarded 13.6 ± 6.9 mm, guarded 8.6 ± 5.9 mm; P = .03). No difference was identified in duration of procedure (unguarded 11.8 ± 5.2 minutes, guarded 13.8 ± 4.3 minutes; P = .79) or procedure difficulty score (unguarded 1.7 ± 0.6, guarded 1.6 ± 0.6 P = .73). CONCLUSION: Silicone-guarded arthroscope cannulas decreased IACI number and size during canine cadaveric stifle arthroscopy without increasing duration of procedure or surgical difficulty. CLINICAL SIGNIFICANCE: Silicone-guarded arthroscope cannulas may be safer than traditional cannulas for novice veterinary surgeons performing stifle arthroscopy.

An Allosteric Interaction Network Promotes Conformation State-Dependent Eviction of the Nas6 Assembly Chaperone from Nascent 26S Proteasomes.

The 26S proteasome is the central ATP-dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation-specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP binding by the ATPase subunit Rpt3 promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly-coupled allosteric signals promote chaperone eviction and provides a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.

An Investigation of a Novel Tendon Transfer Surgery for High Median-Ulnar Nerve Palsy in a Chicken Model.

PURPOSE: The state-of-the-art tendon transfer surgery for high median-ulnar nerve palsy involves directly suturing four finger flexor tendons to one wrist extensor muscle. This couples finger flexion limiting the patient's ability to grasp objects. Therefore, we propose a new approach to attach a novel passive implant to the extensor digitorum longus tendon in order to create a differential mechanism in situ. The implant is expected to enable the fingers to adapt to an object's shape during grasping. Chickens have been used as a model in tendon research, but studies have primarily focused on the digital flexor tendon mechanism. Thus, the aim of this study was to explore the feasibility of the chicken model for extensor tendon research and to validate the surgical technique for a new approach to tendon transfer surgery. MATERIALS AND METHODS: Twenty-nine chickens were randomly divided into three groups: implant (n = 12), sham (n = 10), and control (n = 7). Postoperative healing and complications were documented. RESULTS: Surgery was successful in all chickens. All animals healed appropriately by Day 16 postoperatively. Chickens in the implant group experienced significantly more intermittent toe-knuckling gait than the sham group (p = 0.001). CONCLUSIONS: The described surgical technique allowed for successful application of a novel implantable passive mechanism in a live chicken model. In combination with previous work, findings from the present study further validated a novel tendon-transfer surgery for high median-ulnar nerve palsy. Based on the degree of intermittent abnormal gait experienced by the implant group, refinement to the implant design is warranted in future studies.

Evaluation of the clinical effects of diet and physical rehabilitation in dogs following tibial plateau leveling osteotomy.

OBJECTIVE To assess clinical effects of an omega-3 fatty acid and protein-enriched diet, physical rehabilitation, or both in dogs following tibial plateau leveling osteotomy (TPLO) and arthroscopic surgery for cranial cruciate ligament (CCL) disease. DESIGN Randomized, prospective clinical trial. ANIMALS 48 dogs with unilateral CCL disease. PROCEDURES Dogs were randomly assigned to receive a dry omega-3 fatty acid and protein-enriched dog food formulated to support joint health (test food [TF]), a dry food formulated for maintenance of adult dogs (control food [CF]), TF plus rehabilitation (TF-R), or CF plus rehabilitation (CF-R). Data collected over 6 months included body weight, body condition score, ground reaction force data, tibial plateau angle, limb circumference measurements, subjective pain and lameness scores assigned by surgeons and dog owners, and daily activity measured by accelerometry. RESULTS Peak vertical force and vertical impulse were greater after surgery for dogs in the TF groups than in the CF groups; peak vertical force was greater after surgery in dogs that underwent rehabilitation than in those that did not. Owner scores indicated lower frequencies of lameness and signs of pain during some activities for the TF group, compared with other groups, and for the TF-R and CF-R groups, compared with the CF group. Sedentary time decreased and time spent in light-to-moderate or vigorous activity increased in all groups over time. Rehabilitation was significantly associated with greater time spent in light-to-moderate activity, regardless of diet. CONCLUSIONS AND CLINICAL RELEVANCE Feeding the TF and providing physical rehabilitation during the first 6 months after TPLO were associated with improvements in some indices of clinical outcome and function in dogs. Significant interactions between time and some outcome variables were observed, indicating further research is warranted.

Assessment of the effects of diet and physical rehabilitation on radiographic findings and markers of synovial inflammation in dogs following tibial plateau leveling osteotomy.

OBJECTIVE To determine the effects of an omega-3 fatty acid and protein-enriched diet, physical rehabilitation, or both on radiographic findings and markers of synovial inflammation in dogs following tibial plateau leveling osteotomy and arthroscopic surgery for treatment of cranial cruciate ligament disease. DESIGN Randomized, prospective clinical trial. ANIMALS 48 dogs with unilateral cranial cruciate ligament disease. PROCEDURES Dogs were randomly assigned to receive a dry omega-3 fatty acid and protein-enriched dog food formulated to support joint health (test food [TF]), a dry food formulated for adult canine maintenance (control food [CF]), TF plus rehabilitation, or CF plus rehabilitation after surgery. Synovial fluid prostaglandin (PG) E2 and interleukin-1ß concentrations, radiographic osteoarthritis scores, osteotomy site healing, and patellar ligament thickness were assessed at predetermined time points up to 6 months after surgery. RESULTS Dogs that received CF had significantly higher PGE2 concentrations over time following surgery than did dogs that received TF, regardless of rehabilitation status. Synovial fluid interleukin-1ß concentrations did not change over time in any groups. Diet and rehabilitation were both associated with osteoarthritis scores, with significantly lower scores over time for dogs that received TF versus CF and for dogs that underwent rehabilitation versus those that did not. Proportions of dogs with complete osteotomy healing 8 and 24 weeks after surgery were significantly lower for dogs that received TF than for dogs that received CF, regardless of rehabilitation status. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested that feeding the TF can result in lower synovial fluid PGE2 concentrations and that both the TF and rehabilitation can reduce progression of osteoarthritis in the 6 months following tibial plateau leveling osteotomy; clinical relevance of slower osteotomy healing in dogs fed the TF was unclear.

Comparison of iatrogenic articular cartilage injury in canine stifle arthroscopy versus medial parapatellar mini-arthrotomy in a cadaveric model.

OBJECTIVE: To assess iatrogenic articular cartilage injury (IACI) resulting from arthroscopy versus medial parapatellar mini-arthrotomy of the stifle. STUDY DESIGN: Paired comparison of canine cadaver stifles treated with arthroscopy or mini-arthrotomy ANIMALS: Paired canine stifles from 14 cadavers (≥20 kg). METHODS: Stifles (N = 28) were assigned to arthroscopy or arthrotomy. Full stifle joint exploration and meniscal probing were performed. Joints were disarticulated and India ink assay performed. IACI was defined as sharply delineated lesions with India ink uptake. Incidence, number, and lesion area in defects articular cartilage, incision length, surgery duration, and joint structures visualized were recorded. RESULTS: Arthroscopy resulted in greater IACI than mini-arthrotomy, including incidence of IACI (arthroscopy: 13 stifles, mini-arthrotomy: 4 stifles; P = .009), number of IACI per stifle (arthroscopy: 3.4 ± 2.90, mini-arthrotomy: 0.9 ± 1.96; P = .04), and IACI area (arthroscopy: 5.9 ± 7.58 mm2 , mini-arthrotomy: 1.7 ± 4.50 mm2 ; P = .003). Incision length was shorter with arthroscopy (1.0 ± 0.38 cm) versus mini-arthrotomy (5.3 ± 0.61 cm; P < .0001). Surgical duration was not significantly different between groups (arthroscopy: 12.5 ± 3.49 minutes, mini-arthrotomy: 11.05 ± 1.60 minutes; P = .21). Visualization of articular structures was incomplete in 14/14 mini-arthrotomy stifles and 1/14 arthroscopy stifles (P < .001). CONCLUSION: Incidence, number, and area of IACI were greater in the arthroscopy group versus the mini-arthrotomy group. Mini-arthrotomy resulted in a longer incision and incomplete joint visualization. Methods of preventing IACI and clinical significance of IACI warrant further investigation.

Comparison of radiography and computed tomography to evaluate fractures of the canine tarsus.

Detection and accurate classification of traumatic tarsal fractures are important for identifying cases requiring surgical intervention. The aim of this prospective, experimental, methods comparison study was to directly compare the accuracy, sensitivity, and specificity of tarsal computed tomography (CT), ten-view and two-view digital radiographs for detecting traumatic fractures of the canine tarsus. The working hypothesis was that tarsal fractures would be detected with higher accuracy, sensitivity, and specificity using CT imaging compared to radiography, and a ten-view would be superior to a two-view radiographic study. Ten cadaver hind limbs of medium to large dogs received a CT scan and ten-view radiographic study before and after induction of fractures with a hydraulic press. All bones included in the radiographic images were assessed for fractures by two observers and gross dissection was used as the gold standard. The two-view radiographic study (dorsoplantar, lateromedial) was created from the ten-view study and reviewed 2 years later. All limbs sustained fractures, the most common locations were the talus and calcaneus (n = 7). The sensitivity of CT was greater than ten-view radiographic study (77% vs. 57%), while the specificity was similar (97% vs. 98%). The sensitivity and specificity of the ten-view and two-view radiograph studies were similar (57% vs. 55%; both 98%). Computed tomography images were reassessed postdissection to determine if failure to identify fractures resulted from observer error. Overall, CT was better than radiography for detecting fractures of the canine tarsus, however there was little improvement with ten-view compared to two-view radiographic studies.

Evaluation of the Diagnostic Accuracy of Conventional 2-Dimensional and 3-Dimensional Computed Tomography for Assessing Canine Sacral and Pelvic Fractures by Radiologists, Orthopedic Surgeons, and Veterinary Medical Students.

OBJECTIVE: To determine, using 3 groups of evaluators of varying experience reading orthopedic CT studies, if 3-dimensional computed tomography (3D-CT) provides a more accurate and time efficient method for diagnosis of canine sacral and pelvic fractures, and displacements of the sacroiliac and coxofemoral joints compared with 2-dimensional computed tomography (2D-CT). STUDY DESIGN: Retrospective clinical and prospective study. ANIMALS: Dogs (n = 23): 12 dogs with traumatic pelvic fractures, 11 canine cadavers with pelvic trauma induced by a lateral impactor. METHODS: All dogs had a 2D-CT exam of the pelvis and subsequent 3D-CT reconstructions from the 2D-CT images. Both 2D-CT and 3D-CT studies were anonymized and randomly presented to 2 veterinary radiologists, 2 veterinary orthopedic surgeons, and 2 veterinary medical students. Evaluators classified fractures using a confidence scale and recorded the duration of evaluation for each modality and case. RESULTS: 3D-CT was a more time-efficient technique for evaluation of traumatic sacral and pelvic injuries compared with 2D-CT in all evaluator groups irrespective of experience level reading orthopedic CT studies. However, for radiologists and surgeons, 2D-CT was the more accurate technique for evaluating sacral and pelvic fractures. CONCLUSION: 3D-CT improves sacral and pelvic fracture diagnosis when added to 2D-CT; however, 3D-CT has a reduced accuracy for evaluation of sacral and pelvic fractures if used without concurrent evaluation of 2D-CT images.

Fibrochondrogenic potential of synoviocytes from osteoarthritic and normal joints cultured as tensioned bioscaffolds for meniscal tissue engineering in dogs.

Meniscal tears are a common cause of stifle lameness in dogs. Use of autologous synoviocytes from the affected stifle is an attractive cell source for tissue engineering replacement fibrocartilage. However, the diseased state of these cells may impede in vitro fibrocartilage formation. Synoviocytes from 12 osteoarthritic ("oaTSB") and 6 normal joints ("nTSB") were cultured as tensioned bioscaffolds and compared for their ability to synthesize fibrocartilage sheets. Gene expression of collagens type I and II were higher and expression of interleukin-6 was lower in oaTSB versus nTSB. Compared with nTSB, oaTSB had more glycosaminoglycan and alpha smooth muscle staining and less collagen I and II staining on histologic analysis, whereas collagen and glycosaminoglycan quantities were similar. In conclusion, osteoarthritic joint-origin synoviocytes can produce extracellular matrix components of meniscal fibrocartilage at similar levels to normal joint-origin synoviocytes, which makes them a potential cell source for canine meniscal tissue engineering.

Comparison of meniscal fibrochondrocyte and synoviocyte bioscaffolds toward meniscal tissue engineering in the dog.

Tissue engineering is a promising field of study toward curing the meniscal deficient stifle; however the ideal cell type for this task is not known. We describe here the extraction of synoviocytes and meniscal fibrochondrocytes from arthroscopic debris from six dogs, which were cultured as tensioned bioscaffolds to synthesize meniscal-like fibrocartilage sheets. Despite the diseased status of the original tissues, synoviocytes and meniscal fibrochondrocytes had high viability at the time of removal from the joint. Glycosaminoglycan and collagen content of bioscaffolds did not differ. Meniscal fibrochondrocyte bioscaffolds contained more type II collagen, but collagen deposition was disorganized, with only 30-40% of cells viable. The collagen of synoviocyte bioscaffolds was organized into sheets and bands and 80-90% of cells were viable. Autologous, diseased meniscal fibrochondrocytes and synoviocytes are plausible cell sources for future meniscal tissue engineering research, however cell viability of meniscal fibrochondrocytes in the tensioned bioscaffolds was low.

Comparison of 2 fluid ingress/egress systems for canine stifle arthroscopy using computed tomography.

OBJECTIVE: To evaluate volume of extra-articular fluid egress and complications associated with 2 fluid ingress/egress techniques for stifle arthroscopy. STUDY DESIGN: Ex vivo study. STUDY SUBJECTS: Canine cadavers (n = 14). METHODS: Four cadavers (8 stifles) were used to validate 3D computed tomographic (CT) methods to quantify stifle joint intra- and extra-articular volumes of iodinated contrast medium. Ten canine cadavers (20 stifles) had preoperative CT, followed by stifle arthroscopy using a 10% solution of iodinated contrast enhanced ingress fluid delivered by pressure bag (2PB) or by arthroscopic peristaltic pump (3FP). All 3FP limbs had an additional fluid egress portal placed by cannula and obturator. Arthroscopy was limited to 20 minutes/joint. The volume of the contrast medium egress into the soft tissues was measured on postoperative 3D CT reconstructed images. RESULTS: Mean percentage of total ingress fluids administered that remained in the joint and extra-articular tissues postoperatively was 8.8 ± 1.2% in 3FP and 33.2 ± 8% in 2PB (P = .014). Two 3FP joints had 4-5 mm egress obturator tracks on the proximal medial trochlear ridge. Two 2PB joints had severe joint collapse from extracapsular fluid precluding further examination. Intermittent visual blurring by joint fluid mixing or fat pad fragmentation/dissolution was noted in 2PB joints. CONCLUSIONS: A superior technique was not identified: 2PB had greater egress fluid tissue accumulation, whereas 3FP had better viewing of intra-articular structures with less tissue egress fluid accumulation; however, cartilage damage was induced with the egress obturator.

Culture of equine fibroblast-like synoviocytes on synthetic tissue scaffolds towards meniscal tissue engineering: a preliminary cell-seeding study.

Introduction. Tissue engineering is a new methodology for addressing meniscal injury or loss. Synovium may be an ideal source of cells for in vitro meniscal fibrocartilage formation, however, favorable in vitro culture conditions for synovium must be established in order to achieve this goal. The objective of this study was to determine cellularity, cell distribution, and extracellular matrix (ECM) formation of equine fibroblast-like synoviocytes (FLS) cultured on synthetic scaffolds, for potential application in synovium-based meniscal tissue engineering. Scaffolds included open-cell poly-L-lactic acid (OPLA) sponges and polyglycolic acid (PGA) scaffolds cultured in static and dynamic culture conditions, and PGA scaffolds coated in poly-L-lactic (PLLA) in dynamic culture conditions. Materials and Methods. Equine FLS were seeded on OPLA and PGA scaffolds, and cultured in a static environment or in a rotating bioreactor for 12 days. Equine FLS were also seeded on PGA scaffolds coated in 2% or 4% PLLA and cultured in a rotating bioreactor for 14 and 21 days. Three scaffolds from each group were fixed, sectioned and stained with Masson's Trichrome, Safranin-O, and Hematoxylin and Eosin, and cell numbers and distribution were analyzed using computer image analysis. Three PGA and OPLA scaffolds from each culture condition were also analyzed for extracellular matrix (ECM) production via dimethylmethylene blue (sulfated glycosaminoglycan) assay and hydroxyproline (collagen) assay. PLLA coated PGA scaffolds were analyzed using double stranded DNA quantification as areflection of cellularity and confocal laser microscopy in a fluorescent cell viability assay. Results. The highest cellularity occurred in PGA constructs cultured in a rotating bioreactor, which also had a mean sulfated glycosaminoglycan content of 22.3 µg per scaffold. PGA constructs cultured in static conditions had the lowest cellularity. Cells had difficulty adhering to OPLA and the PLLA coating of PGA scaffolds; cellularity was inversely proportional to the concentration of PLLA used. PLLA coating did not prevent dissolution of the PGA scaffolds. All cell scaffold types and culture conditions produced non-uniform cellular distribution. Discussion/Conclusion. FLS-seeding of PGA scaffolds cultured in a rotating bioreactor resulted in the most optimal cell and matrix characteristics seen in this study. Cells grew only in the pores of the OPLA sponge, and could not adhere to the PLLA coating of PGA scaffold, due to the hydrophobic property of PLA. While PGA culture in a bioreactor produced measureable GAG, no culture technique produced visible collagen. For this reason, and due to the dissolution of PGA scaffolds, the culture conditions and scaffolds described here are not recommended for inducing fibrochondrogenesis in equine FLS for meniscal tissue engineering.

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-ß3, or FGF-2, TGF-ß1, 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-ß3 cultured bioscaffolds failed to outperform triple growth factor-treated TSB. Architectural extracellular matrix (ECM) organization and cellularity likely explained the differences between groups. TGF-ß3 alone cannot be recommended at this time for in vitro formation of autologous fibrocartilage bioscaffolds for 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, TGFß1 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.

In vitro synthesis of tensioned synoviocyte bioscaffolds for meniscal fibrocartilage tissue engineering.

BACKGROUND: Meniscal injury is a common cause of lameness in the dog. Tissue engineered bioscaffolds may be a treatment option for meniscal incompetency, and ideally would possess meniscus- like extracellular matrix (ECM) and withstand meniscal tensile hoop strains. Synovium may be a useful cell source for meniscal tissue engineering because of its natural role in meniscal deficiency and its in vitro chondrogenic potential. The objective of this study is to compare meniscal -like extracellular matrix content of hyperconfluent synoviocyte cell sheets ("HCS") and hyperconfluent synoviocyte sheets which have been tensioned over wire hoops (tensioned synoviocyte bioscaffolds, "TSB") and cultured for 1 month. RESULTS: Long term culture with tension resulted in higher GAG concentration, higher chondrogenic index, higher collagen concentration, and type II collagen immunoreactivity in TSB versus HCS. Both HCS and TSB were immunoreactive for type I collagen, however, HCS had mild, patchy intracellular immunoreactivity while TSB had diffuse moderate immunoreactivity over the entire bisocaffold. The tissue architecture was markedly different between TSB and HCS, with TSB containing collagen organized in bands and sheets. Both HCS and TSB expressed alpha smooth muscle actin and displayed active contractile behavior. Double stranded DNA content was not different between TSB and HCS, while cell viability decreased in TSB. CONCLUSIONS: Long term culture of synoviocytes with tension improved meniscal- like extra cellular matrix components, specifically, the total collagen content, including type I and II collagen, and increased GAG content relative to HCS. Future research is warranted to investigate the potential of TSB for meniscal tissue engineering.

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.

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.

The effect of agility exercise on eicosanoid excretion, oxidant status, and plasma lactate in dogs.

BACKGROUND: The objective was to determine the effects of agility exercise on dogs of different skill levels with respect to urinary eicosanoids, urinary 15F2t-isoprostane (lipid peroxidation marker) and hematological/biochemical changes in plasma. Fifteen adult dogs had blood and urine samples obtained prior to, immediately and 4-hours following an agility exercise. RESULTS: Hematocrit, red blood cells (RBC), albumin, and hemoglobin increased following exercise, with greatest increases correlating to increased skill group (novice, intermediate, masters); at 4-hours post-exercise, hematocrit, RBC, and hemoglobin were decreased. Phosphorus increased following exercise with the greatest increase in novice and intermediates. Plasma lactate increased 3.6-fold in masters, 3.2-fold in intermediates, and 1.2-fold in novice dogs. Urine thromboxane B2 (TXB2) more than tripled 4-hours post-exercise while 6-keto prostaglandin F1α (PGF1α, prostacyclin metabolite), prostaglandin E2 metabolites (13,14-dihydro-15-keto-prostaglandin A2 and 13,14-dihydro-15-keto-prostaglandin E2), and 13,14-dihydro-15-keto prostaglandin F2α were unaffected as determined by a competitive enzyme immunoassay and standardized by division with urine creatinine. Urine 15F2t-isoprostane increased insignificantly. CONCLUSIONS: Alterations in the plasma post-exercise were likely due to hemoconcentration from insensible water loss, splenic contraction and sympathetic stimulation while 4-hours later autohemodilution reduced RBC parameters. Elevations in plasma lactate and urinary TXB2 correlated with advanced skill level/speed of the dogs.