Ligamentum teres reconstruction using autogenous semitendinosus tendon with toggle technique in rabbits.

Background: Ligamentum teres (LT) has traditionally been considered a vestigial or redundant structure in humans; however, based on new studies and the evolution of hip arthroscopy, the LT injury has been viewed as a source of hip pain. Therefore, LT reconstruction can be beneficial in some cases. Rabbits have been frequently used as a model for cranial cruciate ligament reconstruction but few studies are available for ligamentum teres reconstruction. Objective: To evaluate the semitendinosus tendon to replace ligamentum teres with the toggle technique, using rabbits as an experimental model. Methods: Twenty-six female Norfolk rabbits with approximately 3 months of age were divided into two equal groups after excision of ligamentum teres (LT) from the right hip joint: G1-no reconstruction of LT and capsulorrhaphy; G2-double-bundle reconstruction of the LT using semitendinosus tendon autograft. In both groups, the LT was removed from the right hip joint. In G2 the autograft was harvested from the left hind limb of the same rabbit. The rabbits were evaluated clinically at different time intervals; before surgery (M1), 48 h (M2), 15 days (M3), 30 days (M4) and 90 days (M5) after surgery. Results: The rabbits supported their limbs on the ground in both the groups. As complications of the procedure, four hip joints showed subluxations in the radiographic evaluation of G1; three at M4 and one at M5. In G2; two luxations of hip joints at M3 and one subluxation at M4 were seen. On ultrasound, irregular articular surface was seen in 30.8% of the rabbits that had subluxation of hip joints. Gross evaluation identified tendon graft integrity in 76.92% of the rabbits. Histological analysis revealed graft adhesion to the bone in the early phase comprised of sharpey-like collagen fibers. Conclusion: The double-bundle reconstruction of the LT using autologous semitendinosus tendon associated with the toggle rod shows an early phase of tendon graft ligamentization at 90 days post-operatively in young rabbits, but biomechanical bias suffered by the tendon during gait must be considered.

A three-dimensional (3D), serum-free, Collagen Type I system for chondrogenesis of canine bone marrow-derived multipotent stromal cells (cMSCs).

The dog is an underrepresented large animal translational model for orthopedic cell-based tissue engineering. While chondrogenic differentiation of canine multipotent stromal cells (cMSCs) has been reported using the classic micromass technique, cMSCs respond inconsistently to this method. The objectives of this study were to develop a three-dimensional (3D), serum-free, Collagen Type I system to facilitate cMSC chondrogenesis and, once established, to determine the effect of chondrogenic growth factors on cMSC chondrogenesis. Canine MSCs were polymerized in 100 µL Collagen Type I gels (5 mg/mL) at 1 x 106 cells/construct. Constructs were assessed using morphometry, live/dead staining, and histology in 10 various chondrogenic media. Four media were selected for additional in-depth analyses via lactate dehydrogenase release, total glycosaminoglycan content, qPCR (COL1A1, COL2A, SOX9, ACAN, BGLAP and SP7), immunofluorescence, and TUNEL staining. In the presence of dexamethasone and transforming growth factor-ß3 (TGF-ß3), both bone morphogenic protein-2 (BMP-2) and basic fibroblast growth factor (bFGF) generated larger chondrogenic constructs, although BMP-2 was required to achieve histologic characteristics of chondrocytes. Chondrogenic medium containing dexamethasone, TGF-ß3, BMP-2 and bFGF led to a significant decrease in lactate dehydrogenase release at day 3 and glycosaminoglycan content was significantly increased in these constructs at day 3, 10, and 21. Both osteogenic and chondrogenic transcripts were induced in response to dexamethasone, TGF-ß3, BMP-2 and bFGF. Collagen Type II and X were detected in all groups via immunofluorescence. Finally, TUNEL staining was positive in constructs lacking BMP-2 or bFGF. In conclusion, the 3D, serum-free, Collagen Type-I assay described herein proved useful in assessing cMSC differentiation and will serve as a productive system to characterize cMSCs or to fabricate tissue engineering constructs for clinical use.

Greater trochanter osteotomy as a component of cementless total hip replacement: Five cases in four dogs.

OBJECTIVE: To describe the surgical technique and report the long-term outcome of greater trochanteric osteotomy (GTO) as a component of cementless total hip replacement (THR) in dogs with severe medialization of the greater trochanter or chronic craniodorsal hip luxation. STUDY DESIGN: Short case series. ANIMALS: Four dogs treated with five THRs. METHODS: Data collected from medical records included signalment, indication for THR, duration of clinical signs, body weight, pre- and post-operative radiographic assessment, surgical templating, osteotomy technique, THR implant selection, surgical time, complications, and long-term clinical and radiographic follow-up. Clinical outcomes were determined based on in-hospital history, orthopedic examination, and radiographic evaluation. RESULTS: All five surgical procedures resulted in satisfactory long-term clinical results at follow-up a median of 48.2 months (range, 34-56 months) after THR. There were no minor complications and one major complication. One dog experienced post-operative luxation unrelated to the GTO and was successfully treated with a cup revision. CONCLUSION AND CLINICAL RELEVANCE: GTO was effective in facilitating cementless THR in dogs with either severe medialization of the greater trochanter or chronic craniodorsal luxation.

Total hip replacement in dogs with contralateral pelvic limb amputation: A retrospective evaluation of 13 cases.

OBJECTIVE: To report the clinical characteristics, surgical management, and medium-term outcomes of total hip replacement (THR) performed in dogs with previous contralateral pelvic limb amputation. ANIMALS: Thirteen client-owned dogs. STUDY DESIGN: Multi-institutional retrospective clinical study. METHODS: Data recorded from medical records included signalment, indication for amputation and THR, and surgical complications. Implant positioning and complications were assessed on radiographs. Clinical outcomes were evaluated during follow-up examinations by one of the authors and through a mobility- and lifestyle-based questionnaire completed by owners. RESULTS: All 13 dogs had satisfactory clinical results at follow-up a median of 3 months (range, 2-36) after THR. No postoperative luxation was recorded. Four dogs had minor complications that did not require additional treatment. The only major complication was one failure of osseointegration of a cementless acetabular cup, and it was successfully revised. CONCLUSION: Total hip replacement resulted in satisfactory clinical results and acceptable morbidity in this population. CLINICAL SIGNIFICANCE: Total hip replacement should be considered in dogs with severe coxofemoral joint disease and contralateral pelvic limb amputation.

Characterization of a pluripotent stem cell-derived matrix with powerful osteoregenerative capabilities.

Approximately 10% of fractures will not heal without intervention. Current treatments can be marginally effective, costly, and some have adverse effects. A safe and manufacturable mimic of anabolic bone is the primary goal of bone engineering, but achieving this is challenging. Mesenchymal stem cells (MSCs), are excellent candidates for engineering bone, but lack reproducibility due to donor source and culture methodology. The need for a bioactive attachment substrate also hinders progress. Herein, we describe a highly osteogenic MSC line generated from induced pluripotent stem cells that generates high yields of an osteogenic cell-matrix (ihOCM) in vitro. In mice, the intrinsic osteogenic activity of ihOCM surpasses bone morphogenic protein 2 (BMP2) driving healing of calvarial defects in 4 weeks by a mechanism mediated in part by collagen VI and XII. We propose that ihOCM may represent an effective replacement for autograft and BMP products used commonly in bone tissue engineering.

Effect of proximal abducting ulnar osteotomy (PAUL) on frontal plane thoracic limb alignment: An ex vivo canine study.

OBJECTIVE: To determine the effect of proximal abducting ulnar osteotomy (PAUL) on frontal plane thoracic limb alignment in standing and recumbent positions. STUDY DESIGN: Ex vivo cadaveric study. SAMPLE POPULATION: Canine thoracic limbs (n = 15 limb pairs). METHODS: Limbs were acquired from healthy Labrador retrievers that had been euthanized for reasons unrelated to this study. A limb press was used to obtain standing and recumbent caudocranial radiographs before and after PAUL. Foot lateralization and rotation were directly measured in standing position. Mechanical joint angles were determined using full limb radiographic montages and the center of rotation of angulation (CORA) method for pre-PAUL (Pre), 2-mm PAUL (PAUL2), and 3-mm PAUL (PAUL3). Data are reported as mean ± SD and 95% CI. Mixed linear modeling was used to identify differences in limb alignment values and foot position, with significance established at P ≤ .004. RESULTS: There were differences in five of 12 limb alignment values pre-PAUL and post-PAUL in standing and recumbent positions. In the standing position, there was an increase in mechanical medial proximal radioulnar angle (Pre, 80.6° ± 2.5°; PAUL2, 82.6° ± 2.4°; PAUL3, 84° ± 2.4°) and a decrease in elbow compression angle (Pre, 1.4° ± 1.3°; PAUL2, 1° ± 0.9°; PAUL3, 0.8° ± 1°). There was a movement of mechanical humeral radioulnar angle (Pre, -8.9° ± 2.8°; PAUL2, -6.1° ± 2.7°; PAUL3, -5.2 ± 2.7°), mechanical thoracic humeral angle (Pre, 3.9° ± 1.7°; PAUL2, 2.4° ± 1.4°; PAUL3, 2.6° ± 1.5°), and elbow mechanical axis deviation (Pre, 1.9% ± 1.1%; PAUL2, 0.9% ± 1.1%; PAUL3, 0.4% ± 1.4%) toward a value of "0" representing coaxial alignment of the limb. The foot underwent lateralization (Pre, 1.4 ± 0.6 cm; PAUL2, 1.8 ± 0.7 cm; PAUL3, 2.3 ± 0.7 cm) and external rotation (Pre, 10.5° ± 4.7°; PAUL2, 13.7° ± 5.1°; PAUL3, 16° ± 6.6°). CONCLUSION: In the ex vivo setting, PAUL resulted in translation of the mechanical axis of the thoracic limb from a medial to lateral direction through alterations in limb alignment values associated with the elbow, humerus, and proximal radius/ulna. CLINICAL SIGNIFICANCE: Additional studies are required to determine whether PAUL alters thoracic limb alignment in client-owned dogs.

Toward zonally tailored scaffolds for osteochondral differentiation of synovial mesenchymal stem cells.

Synovium-derived mesenchymal stem cells (SMSCs) are an emerging cell source for regenerative medicine applications, including osteochondral defect (OCD) repair. However, in contrast to bone marrow MSCs, scaffold compositions which promote SMSC chondrogenesis/osteogenesis are still being identified. In the present manuscript, we examine poly(ethylene) glycol (PEG)-based scaffolds containing zonally-specific biochemical cues to guide SMSC osteochondral differentiation. Specifically, SMSCs were encapsulated in PEG-based scaffolds incorporating glycosaminoglycans (hyaluronan or chondroitin-6-sulfate [CSC]), low-dose of chondrogenic and osteogenic growth factors (TGFß1 and BMP2, respectively), or osteoinductive poly(dimethylsiloxane) (PDMS). Initial studies suggested that PEG-CSC-TGFß1 scaffolds promoted enhanced SMSC chondrogenic differentiation, as assessed by significant increases in Sox9 and aggrecan. Conversely, PEG-PDMS-BMP2 scaffolds stimulated increased levels of osteoblastic markers with significant mineral deposition. A "Transition" zone formulation was then developed containing a graded mixture of the chondrogenic and osteogenic signals present in the PEG-CSC-TGFß1 and PEG-PDMS-BMP2 constructs. SMSCs within the "Transition" formulation displayed a phenotypic profile similar to hypertrophic chondrocytes, with the highest expression of collagen X, intermediate levels of osteopontin, and mineralization levels equivalent to "bone" formulations. Overall, these results suggest that a graded transition from PEG-CSC-TGFß1 to PEG-PDMS-BMP2 scaffolds elicits a gradual SMSC phenotypic shift from chondrocyte to hypertrophic chondrocyte to osteoblast-like. As such, further development of these scaffold formulations for use in SMSC-based OCD repair is warranted. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2019-2029, 2019.

A canine in vitro model for evaluation of marrow-derived mesenchymal stromal cell-based bone scaffolds.

Tissue engineered bone grafts based on bone marrow mesenchymal stromal cells (MSCs) are being actively developed for craniomaxillofacial (CMF) applications. As for all tissue engineered implants, the bone-regenerating capacity of these MSC-based grafts must first be evaluated in animal models prior to human trials. Canine models have traditionally resulted in improved clinical translation of CMF grafts relative to other animal models. However, the utility of canine CMF models for evaluating MSC-based bone grafts rests on canine MSCs (cMSCs) responding in a similar manner to scaffold-based stimuli as human MSCs (hMSCs). Herein, cMSC and hMSC responses to polyethylene glycol (PEG)-based scaffolds were therefore compared in the presence or absence of osteoinductive polydimethylsiloxane (PDMS). Notably, the conjugation of PDMS to PEG-based constructs resulted in increases in both cMSC and hMSC osteopontin and calcium deposition. Based on these results, cMSCs were further used to assess the efficacy of tethered bone morphogenic protein 2 (BMP2) in enhancing PEG-PDMS scaffold osteoinductivity. Addition of low doses of tethered BMP2 (100 ng/mL) to PEG-PDMS systems increased cMSC expression of osterix and osteopontin compared to both PEG-PDMS and PEG-BMP2 controls. Furthermore, these increases were comparable to effects seen with up to five-times higher BMP2 doses noted in literature. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2382-2393, 2018.

Articular cartilage scores in cranial cruciate ligament-deficient dogs with or without bucket handle tears of the medial meniscus.

OBJECTIVE: To compare articular cartilage scores in cranial cruciate ligament (CCL)-deficient dogs with or without concurrent bucket handle tears (BHT) of the medial meniscus. STUDY DESIGN: Retrospective case series. ANIMALS: Client-owned dogs treated with arthroscopy and tibial plateau leveling osteotomy or extracapsular repair for complete CCL rupture (290 stifles from 264 dogs). METHODS: Medical records and arthroscopic images were reviewed. Medial femoral condyle (MFC) and medial tibial plateau (MTP) cartilage was scored using the modified Outerbridge scale. Periarticular osteophytosis (PAO) and injury to the medial meniscus were recorded. Data were analyzed using Student's t-tests, Wilcoxon rank-sum test, and Fisher's exact test for changes in the stifle based on meniscal condition, body weight, and duration of lameness. RESULTS: PAO, MFC, and MTP articular cartilage scores were not significantly different in dogs with or without BHT. There were no significant differences in MFC or MTP scores when dogs were evaluated based on bodyweight and the presence or absence of a BHT. However, PAO formation was significantly increased in dogs weighing >13.6 kg and concurrent meniscal injury vs. dogs weighing <13.6 kg and concurrent meniscal injury (P < .001). Significantly more stifles with chronic lameness (40 of 89; 44.9%) had the highest PAO score of 2 reported compared to only 42 of 182 stifles (23.1%) with acute lameness (P < .001). CONCLUSION: The presence of a BHT of the medial meniscus was not associated with more severe arthroscopic articular cartilage lesions in the medial joint compartment at the time of surgery.

The Effect of Sliding Humeral Osteotomy (SHO) on Frontal Plane Thoracic Limb Alignment: An Ex Vivo Canine Cadaveric Study.

OBJECTIVE: To determine the effect of sliding humeral osteotomy (SHO) on frontal plane thoracic limb alignment in standing and recumbent limb positions. STUDY DESIGN: Canine cadaveric study. SAMPLE POPULATION: Canine thoracic limbs (n=15 limb pairs). METHODS: Limbs acquired from healthy Labrador Retrievers euthanatized for reasons unrelated to this study were mounted in a limb press and aligned in a standing position followed by axial loading at 30% body weight. Frontal plane radiography was performed in standing and recumbent positions pre- and post-SHO. In the standing position, lateralization of the foot was measured pre- and post-SHO using a textured grid secured to the limb press base plate. Twelve thoracic limb alignment values (mean ± SD and 95% CI) were determined using the center of rotation of angulation (CORA) method were compared using linear mixed models to determine if significant differences existed between limb alignment values pre- or post-SHO, controlling for dog, limb, and limb position. RESULTS: Six of 12 standing or recumbent alignment values were significantly different pre- and post-SHO. SHO resulted in decreased mechanical lateral distal humeral angle and movement of the mechanical humeral radio-ulnar angle, radio-ulnar metacarpal angle, thoracic humeral angle, and elbow mechanical axis deviation toward coaxial limb alignment. In the standing position, the foot underwent significant lateralization post-SHO. CONCLUSION: SHO resulted in significant alteration in frontal plane thoracic limb alignment. Additional studies are necessary to determine if the changes reported using our ex vivo model occur following SHO in vivo.

Fragmentation of the Medial Coronoid Process in Toy and Small Breed Dogs: 13 Elbows (2000-2012).

Fragmentation of the medial coronoid process (FCP) is an uncommon cause of thoracic limb lameness in toy and small breed dogs. Arthroscopic findings and treatment remains poorly described. The objective of this study was to describe the arthroscopic findings and short-term outcome following arthroscopic treatment in toy and small breed dogs with FCP. Medical records were retrospectively reviewed. Arthroscopic findings were available from 13 elbows (12 dogs). Outcome data ≥4 wk postoperatively were available for nine elbows. Owner satisfaction scores were available for 10 elbows. Common preoperative findings included lameness, elbow pain, and imaging abnormalities consistent with FCP. Displaced FCP was the most common FCP lesion identified. Cartilage lesions at the medial coronoid process were identified in 92.3% of elbows (n = 12), with a median Outerbridge score of 4 (range 1-5). Concurrent cartilage lesions of the medial humeral condyle were identified in 76.9% of elbows (n = 10). Seven of nine elbows had full or acceptable function postoperatively. Median owner outcome satisfaction was 91% (range 10-100). FCP should be considered a cause of thoracic limb lameness in toy and small breed dogs. Arthroscopy can be safely and effectively used to diagnose and treat FCP in these breeds.

Outcome Following Surgical Correction of Grade 4 Medial Patellar Luxation in Dogs: 47 Stifles (2001-2012).

Grade 4/4 medial patellar luxation (MPL) is a complex disease of the canine stifle that often requires surgical realignment of the patella to resolve clinical lameness. Outcome following surgery remains poorly described. Medical records were retrospectively reviewed for surgical correction of grade 4 MPL. Signalment and exam findings, surgical procedures performed, complications, and clinical outcome were reported. Data was statistically analyzed for association with major complication occurrence and unacceptable function following surgery. Forty-seven stifles from 41 dogs were included. The surgical procedures most frequently utilized for patellar realignment were the combination of femoral trochleoplasty, tibial tuberosity transposition, and joint capsule modification. Median in-hospital veterinary examination was performed at 69 days (range 30-179 days) following surgery. Full function was reported for 42.6% of cases (n=20). Acceptable function was reported for 40.4% of cases (n=19). Unacceptable function was reported for 17% of cases (n=8). The overall complication rate was 25.5% (n=12), with revision surgery for major complications required in 12.8% of cases (n=6). Corrective osteotomies were associated with major complications (P < 0.001). In general, pelvic limb function improves following surgical correction of grade 4 MPL; however, a return to full function should be considered guarded.

In vitro biomechanical comparison of 3.5 mm LC-DCP/intramedullary rod and 5 mm clamp-rod internal fixator (CRIF)/intramedullary rod fixation in a canine femoral gap model.

OBJECTIVE: To compare the biomechanical properties of clamp rod internal fixation (CRIF)/rod and LC-DCP/rod constructs in a canine femoral gap model. STUDY DESIGN: Cadaveric biomechanical study. SAMPLE POPULATION: Canine femora (n = 10 pair). METHODS: Femora with 40 mm ostectomies were assigned to LC-DCP/rod or CRIF/rod treatment groups. Five construct pairs had 4-point bending and 5 pairs had torsional loading. Construct stiffness, strength, and bending angle at failure or permanent angular deformation (torsional loading) were determined. Statistical comparisons were performed using Student t tests; significance was set at P ≤ .05. RESULTS: There was significantly greater permanent angular deformation, or residual twist, in the CRIF/rod constructs (23.1 ± 0.89°) compared with LC-DCP/rod constructs (7.47 ± 2.08°). Whereas there was no significant difference in torsional stiffness of these constructs at torsional loads <4.92 N m (P = .819), LC-DCP/rod constructs had significantly greater torsional stiffness (0.303 ± 0.079 N m/°) and strength (11.546 ± 2.79 N m) than CRIF/rod construct stiffness (0.06 ± 0.013 N m/°) and strength (6.078 ± 0.527 N m) at torsional loads >4.92 N m. Differences in stiffness and strength in 4-point bending were not statistically significant. CONCLUSIONS: LC-DCP/rod constructs had significantly less permanent angular deformation than CRIF/rod constructs. CRIF/rod constructs became less stiff as torsional load was increased, thus the LC-DCP/rod constructs had significantly greater torsional stiffness and strength under high torsional loads. LC-DCP/rod and CRIF/rod constructs performed similarly under 4-point bend loading conditions.

Thoracic limb alignment in healthy labrador retrievers: evaluation of standing versus recumbent frontal plane radiography.

OBJECTIVE: To report thoracic limb alignment values in healthy dogs; to determine if limb alignment values are significantly different when obtained from standing versus recumbent radiographic projections. STUDY DESIGN: Prospective cross-sectional study. ANIMALS: Labrador Retrievers (n = 45) >15 months of age. METHODS: Standing and recumbent radiographs were obtained and limb montages were randomized before analysis by a single investigator blinded to dog, limb, and limb position. Twelve limb alignment values were determined using the CORA methodology. Measurements were performed in triplicate and intra-observer variability was evaluated by intra-class correlation coefficient (ICC). Limb alignment values were reported as mean ± SD and 95% confidence intervals. Linear mixed models were used to determine if significant associations existed between limb alignment values and limb, limb position, gender, age, weight, and body condition score. RESULTS: There were significant differences in standing and recumbent limb alignment values for all values except elbow mechanical axis deviation (eMAD). Limb, gender, age, body weight, and body condition score had no effect. ICC values ranged from 0.522 to 0.758, indicating moderate to substantial agreement for repeated measurements by a single investigator. CONCLUSIONS: Limb alignment values are significantly different when determined from standing versus recumbent radiographs in healthy Labrador Retrievers.

Stabilization of the CORA based leveling osteotomy for treatment of cranial cruciate ligament injury using a bone plate augmented with a headless compression screw.

OBJECTIVE: To report the effectiveness of a bone plate/headless compression screw (HCS) construct in preventing tibial plateau angle (TPA) shift postoperatively and to describe radiographic healing of the osteotomy. STUDY DESIGN: Case series. ANIMALS: Dogs (n = 31). METHODS: Records of dogs diagnosed with cranial cruciate ligament (CCL) injury treated with the center of rotation of angulation (CORA) based leveling osteotomy (CBLO) stabilized with a bone plate augmented with a HCS were reviewed. Breed, age, weight, and gender were recorded. Radiographs were reviewed for determination of preoperative tibial plateau angle (PreTPA), postoperative TPA (PostTPA), patellar tendon angle (PTA) postoperatively, and TPA at final evaluation (FinalTPA). Difference between PostTPA and FinalTPA was used to define any TPA shift. Radiographic healing at final evaluation was graded based on a 5-point scale. RESULTS: Mean time to final recheck was 88 days (range 49-237 days) with mean ± SD PreTPA = 28.6 ± 4.8°; PostTPA = 9.2 ± 2.2°; FinalTPA = 9.7 ± 2.6°; and TPA Shift = 0.52 ± 1.61°. There was no significant difference between PostTPA and FinalTPA (P = .084, power > 0.80). Mean postoperative PTA was 89.9 ± 1.7°. There were 2 implant related complications; 1 HCS migration and 1 HCS failure. CONCLUSION: Stabilization of the CBLO using a bone plate augmented with a HCS was effective in maintaining PostTPA and achieving satisfactory radiographic healing.

An intrapericardial technique for PDA ligation: surgical description and clinical outcome in 35 dogs.

A number of surgical techniques have been reported for dissection and ligation of patent ductus arteriosi (PDAs) in dogs. The objectives of this study were to provide a detailed description of an intrapericardial technique for PDA dissection and ligation and to report the clinical outcome of that technique in dogs. Medical records of 35 dogs were retrospectively reviewed for signalment, clinical signs, echocardiographic findings, surgical time, intra- and postoperative complications, and completeness of ductal closure. Median surgery time was 60 min (range, 35-125 min). Neither intraoperative nor postoperative complications occurred. Within 48 hr of surgery, the continuous left basilar heart murmur was absent in all dogs, and complete echocardiographic closure was confirmed in 29 of 32 dogs. Residual flow was identified echocardiographically in three dogs within 48 hr of surgery. Residual flow was decreased in one dog at 1 mo, which resolved within 33 mo. One dog had mild residual flow postoperatively but did not return for follow-up. The intrapericardial technique was successful for PDA dissection and ligation and had a lower rate (6%) of echocardiographic residual flow compared with previously reported techniques.

Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling.

BACKGROUND: Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D) and invasion of three-dimensional (3D) collagen matrices and, in particular, evaluated the role of MT1-MMP in this process. RESULTS: LPA stimulated while S1P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (approximatly 700 microm over 48 hours) in response to either lipid. siRNA targeting of LPA1 and Rac1, or S1P1, Rac1, and Cdc42 specifically inhibited LPA- or S1P-induced HT1080 invasion, respectively. Analysis of LPA-induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (approximately 125 microm vs. approximately 45 microm) and velocity of invasion (approximately 0.09 microm/min vs. approximately 0.03 microm/min) only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs)-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT)-MMPs. Furthermore, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process. CONCLUSION: LPA is a fundamental regulator of MT1-MMP-dependent tumor cell invasion of 3D collagen matrices. In contrast, S1P appears to act as an inhibitory stimulus in most cases, while stimulating only select tumor lines. MT1-MMP is required only when tumor cells navigate 3D barriers and not when cells migrate on 2D substrata. We demonstrate that tumor cells require coordinate regulation of LPA/S1P receptors and Rho GTPases to migrate, and additionally, require MT1-MMP in order to invade collagen matrices during neoplastic progression.

Coregulation of vascular tube stabilization by endothelial cell TIMP-2 and pericyte TIMP-3.

The endothelial cell (EC)-derived tissue inhibitor of metalloproteinase-2 (TIMP-2) and pericyte-derived TIMP-3 are shown to coregulate human capillary tube stabilization following EC-pericyte interactions through a combined ability to block EC tube morphogenesis and regression in three-dimensional collagen matrices. EC-pericyte interactions strongly induce TIMP-3 expression by pericytes, whereas ECs produce TIMP-2 in EC-pericyte cocultures. Using small interfering RNA technology, the suppression of EC TIMP-2 and pericyte TIMP-3 expression leads to capillary tube regression in these cocultures in a matrix metalloproteinase-1 (MMP-1)-, MMP-10-, and ADAM-15 (a disintegrin and metalloproteinase-15)-dependent manner. Furthermore, we show that EC tube morphogenesis (lumen formation and invasion) is primarily controlled by the TIMP-2 and -3 target membrane type (MT) 1 MMP. Additional targets of these inhibitors include MT2-MMP and ADAM-15, which also regulate EC invasion. Mutagenesis experiments reveal that TIMP-3 requires its proteinase inhibitory function to induce tube stabilization. Overall, these data reveal a novel role for both TIMP-2 and -3 in the pericyte-induced stabilization of newly formed vascular networks that are predisposed to undergo regression and reveal specific molecular targets of the inhibitors regulating these events.

MMP-1 activation by serine proteases and MMP-10 induces human capillary tubular network collapse and regression in 3D collagen matrices.

Previous work has shown that endothelial cell (EC)-derived matrix metalloproteinases (MMPs) regulate regression of capillary tubes in vitro in a plasmin- and MMP-1 dependent manner. Here we report that a number of serine proteases can activate MMP-1 and cause capillary tube regression; namely plasma kallikrein, trypsin, neutrophil elastase, cathepsin G, tryptase and chymase. Plasma prekallikrein failed to induce regression without coactivators such as high molecular weight kininogen (HMWK) or coagulation Factor XII. The addition of trypsin, the neutrophil serine proteases (neutrophil elastase and cathepsin G) and the mast cell serine proteases (tryptase and chymase) each caused MMP-1 activation and collagen type I proteolysis, capillary tubular network collapse, regression and EC apoptosis. Capillary tube collapse is accompanied by collagen gel contraction, which is strongly related to the wound contraction that occurs during regression of granulation tissue in vivo. We also report that proMMP-10 protein expression is markedly induced in ECs undergoing capillary tube morphogenesis. Addition of each of the serine proteases described above led to activation of proMMP-10, which also correlated with MMP-1 activation and capillary tube regression. Treatment of ECs with MMP-1 or MMP-10 siRNA markedly delayed capillary tube regression, whereas gelatinase A (MMP-2), gelatinase B (MMP-9) and stromelysin-1 (MMP-3) siRNA-treated cells behaved in a similar manner to controls and regressed normally. Increased expression of MMP-1 or MMP-10 in ECs using recombinant adenoviral delivery markedly accelerated serine protease-induced capillary tube regression. ECs expressing increased levels of MMP-10 activated MMP-1 to a greater degree than control ECs. Thus, MMP-10-induced activation of MMP-1 correlated with tube regression and gel contraction. In summary, our work demonstrates that MMP-1 zymogen activation is mediated by multiple serine proteases and MMP-10, and that these events are central to EC-mediated collagen degradation and capillary tube regression in 3D collagen matrices.

Pneumoperitoneum in dogs and cats: 39 cases (1983-2002).

OBJECTIVE: To identify the most common causes of pneumoperitoneum in dogs and cats and determine history, clinical features, and outcome of affected animals. DESIGN: Retrospective study. ANIMALS: 31 dogs and 8 cats. PROCEDURES: Medical records were reviewed for signalment; history; abnormal physical, clinicopathologic, and radiographic findings; results of cytologic analysis and bacterial culture of abdominal fluid; gross and histologic findings at surgery or necropsy; and outcome. RESULTS: Pneumoperitoneum was classified as spontaneous in 25 animals and traumatic in 14. Causes of traumatic pneumoperitoneum included vehicular impact, gunshot wounds, abdominal dog bite wounds, and iatrogenic pneumothorax. Spontaneous pneumoperitoneum was caused by gastrointestinal tract perforation in 23 animals; underlying causes included neoplasia, nonsteroidal anti-inflammatory drug administration, and corticosteroid administration. Two animals developed spontaneous pneumoperitoneum after bladder rupture. Animals with spontaneous pneumoperitoneum were significantly older and had clinical signs of longer duration than those with traumatic pneumoperitoneum. Sixteen animals survived, including 15 of 23 animals that underwent surgery. Animals that survived had significantly higher serum albumin concentrations than did animals that died or were euthanatized. CONCLUSIONS AND CLINICAL RELEVANCE: Although pneumoperitoneum is most often attributable to perforation of a hollow viscus, other causes do exist. Early exploration is recommended for diagnosis and treatment of the underlying condition.