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.

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.

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.

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.

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.

Cell-based meniscal tissue engineering: a case for synoviocytes.

BACKGROUND: Avascular meniscal injuries are largely incapable of healing; the most common treatment remains partial meniscectomy despite the risk of subsequent osteoarthritis. Meniscal responses to injury are partially mediated through synovial activity and strategies have been investigated to encourage healing through stimulating or transplanting adjacent synovial lining. However, with their potential for chondrogenesis, synovial fibroblast-like stem cells hold promise for meniscal cartilage tissue engineering. QUESTIONS/PURPOSES: Thus, specific purposes of this review were to (1) examine how the synovial intima and synoviomeniscal junction affect current meniscal treatment modalities; and (2) examine the components of tissue engineering (cells, scaffolds, bioactive agents, and bioreactors) in the specific context of how cells of synovial origin may be used for meniscal healing or regeneration. METHODS: An online bibliographic search through PubMed was performed in March 2010. Studies were subjectively evaluated and reviewed if they addressed the question posed. Fifty-four resources were initially retrieved, which offered information on the chondrogenic potential of synovial-based cells that could prove valuable for meniscal fibrocartilage engineering. RESULTS: Based on the positive effects of adjoining synovium on meniscal healing as used in some current treatment modalities, the chondrogenic potential of fibroblast-like stem cells of synovial origin make this cell source a promising candidate for cell-based tissue engineering strategies. CONCLUSIONS: The abundance of autologous synovial lining, its ability to regenerate, and the potential of synovial-derived stem cells to produce a wide spectrum of chondral matrix components make it an ideal candidate for future meniscal engineering investigations.

Evaluation of in vitro growth factor treatments on fibrochondrogenesis by synovial membrane cells from osteoarthritic and nonosteoarthritic joints of dogs.

OBJECTIVE: To determine the in vitro effects of selected growth factors on fibrochondrogenesis by synovial membrane cells from nonosteoarthritic (normal) and osteoarthritic joints of dogs. ANIMALS: 5 dogs with secondary osteoarthritis of shoulder or stifle joints and 6 dogs with normal joints. PROCEDURES: Synovial membrane cells were harvested from normal and osteoarthritic joints and cultured in monolayer with or without (control) basic fibroblast growth factor, transforming growth factor-ß1, and insulin-like growth factor-1. In the cultured cells, fibrochondrogenesis was measured by use of a real-time reverse transcriptase PCR assay to determine relative expressions of collagen I, collagen II, and aggrecan genes and of 3 genes involved in embryonic chondrogenesis: Sry-type homeobox protein-9 (SOX-9), frizzled-motif associated with bone development (Frzb), and regulator of G-protein signaling-10 (RGS-10). Tissue collagen content was measured via a hydroxyproline assay, and sulfated glycosaminoglycan content was measured via a 1,9-dimethylmethylene blue assay. Cellularity was determined via a double-stranded DNA assay. Immunohistochemical analysis for collagens I and II was also performed. RESULTS: In vitro collagen synthesis was enhanced by growth factor stimulation. Although osteoarthritic-joint synoviocytes could undergo a fibrocartilage-like phenotypic shift, their production of collagenous extracellular matrix was less than that of normal-joint synoviocytes. Gene expressions of SOX-9 and RGS-10 were highest in the osteoarthritic-joint cells; Frzb expression was highest in growth factor treated cells. CONCLUSIONS AND CLINICAL RELEVANCE: Autogenous synovium may be a viable cell source for meniscal tissue engineering. Gene expressions of SOX-9 and RGS-10 may be potential future targets for in vitro enhancement of chondrogenesis.

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.

Arthroscopy of the antebrachiocarpal joint in dogs.

A technique for arthroscopy of the antebrachiocarpal joint in dogs is described. Both antebrachiocarpal joints in 9 dog cadavers were examined arthroscopically and grossly to refine the technique and determine structures that could be seen. Two arthroscope portals were evaluated in each joint. The antebrachiocarpal synovium, ulnar carpal bone, distal portion of the ulna, medial and lateral collateral ligaments, accessory carpal bone, intercarpal ligament of the radial and ulnar carpal bone, distal portion of the radius, radial carpal bone, palmar process of the radial carpal bone, ligaments of the accessory carpal bone, palmar radiocarpal ligament, and palmar ulnocarpal ligament were visible and accessible to instruments. Arthroscopy was also performed in 5 client-owned dogs, allowing diagnosis of hyperextension injuries (n = 2), septic arthritis (2), and immune-mediated arthropathy (1). Arthroscopy of the antebrachiocarpal joint was found to be a useful adjunct to standard diagnostic modalities.

Surgical management of cricopharyngeal dysphagia in dogs: 14 cases (1989-2001).

OBJECTIVE: To determine outcome of and complications associated with cricopharyngeal myotomy or myectomy for treatment of cricopharyngeal dysphagia (CPD) in dogs. DESIGN: Retrospective study. ANIMALS: 14 dogs. PROCEDURE: Medical records of dogs with CPD that underwent cricopharyngeal myotomy or myectomy were examined. Follow-up information was obtained through telephone interviews with owners and referring veterinarians and clinical examinations when feasible. RESULTS: 16 surgical procedures were performed on the 14 dogs. Dysphagia was completely resolved immediately after surgery in 1 dog, and clinical signs did not recur (follow-up time of 8 years); a second dog also had immediate complete resolution of dysphagia, but follow-up time was only 10 days. Three dogs had transient complete resolution with a mean time to recurrence of dysphagia of 12.3 weeks (range, 2 to 36 weeks). Three dogs had permanent partial resolution. Six dogs had no improvement after surgery. Eight of the 14 dogs were euthanatized because of problems related to CPD, including persistent dysphagia (n = 8) and aspiration pneumonia (5). CONCLUSIONS AND CLINICAL RELEVANCE: The failure rate for dogs undergoing surgical treatment of CPD may be high, particularly if concurrent aspiration pneumonia or malnutrition is not addressed prior to surgery. For those dogs with concurrent diseases, more aggressive medical management, such as enteral tube feeding, may be warranted rather than surgery. In dogs with CPD complicated by other anatomic or functional conditions, such as myasthenia gravis, laryngeal paralysis, and esophageal stricture, surgery may also not be indicated.

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.

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.

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.

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.

Effects of growth factors on equine synovial fibroblasts seeded on synthetic scaffolds for avascular meniscal tissue engineering.

Across species, the avascular portion of the knee meniscus cannot heal spontaneously if severely injured. The most common treatment is meniscectomy which results in osteoarthritis. The objective of this study was to assess the fibrochondrogenic potential of equine fibroblast-like synoviocytes (FLS) seeded on scaffolds under the influence of growth factors in vitro to determine the potential of developing a novel cell-based repair strategy. Cultured FLS were seeded onto synthetic scaffolds in a rotating bioreactor under the influence of three growth factor regimens: none, basic fibroblast growth factor (bFGF) alone, and bFGF plus transforming growth factor (TGF-beta(1)) and insulin-like growth factor (IGF-1). Constructs were analyzed for mRNA expression and production of fibrochondroid extracellular matrix constituents. Type II collagen and aggrecan mRNA were significantly higher in growth factor-treated groups (p<0.05). Despite sub-optimal extracellular matrix production, FLS can exhibit fibrochondral characteristics and may have potential for cell-based tissue engineering for avascular meniscal regeneration.