Jennifer J. Warnock

Cell-based Meniscal Tissue Engineering: A Case for Synoviocytes

BackgroundAvascular 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/purposesThus, 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.MethodsAn 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.ResultsBased 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.ConclusionsThe 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-β1, 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.

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

BackgroundThe 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.ResultsHematocrit, 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.ConclusionsAlterations 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.

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.

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.

Use of computed tomography to compare two femoral head and neck excision ostectomy techniques as performed by two novice veterinarians.

OBJECTIVES To compare the results of femoral head and neck excision (FHNE) ostectomy performed by two novice veterinarians using an osteotome and mallet or microsagittal saw. METHODS In this ex vivo cadaveric study, hindlimbs of eight canine cadavers were randomized to FHNE with osteotome or micro sagittal saw as performed by two recently graduated veterinarians. The hindimbs were imaged by computed tomography (CT) before and after the osteotomy. Post FHNE CT images were evaluated by a board certified radiologist blinded to the ostectomy technique for assessment of the number of bone fragments, fissures, smoothness of osteotomy margination, and volume of residual femoral neck. RESULTS Femoral head and neck excision performed with the osteotome produced more peri-ostectomy bone fragments, cortical fissures, irregular margins, and residual femoral neck volume, compared with osteotomy using a saw. CLINICAL RELEVANCE Compared to FHNE performed with a sagittal saw, osteotome FHNE resulted in a greater bone trauma and residual neck bone volume, which would require post-ostectomy modification in a clinical setting.

Augmentation of diaphyseal fractures of the radius and ulna in toy breed dogs using a free autogenous omental graft and bone plating

OBJECTIVES Evaluation of the short-term outcome, duration of bone healing, and complications following bone plate fixation in dogs weighing ≤6 kg, with and without the use of a free autogenous greater omental graft (OG). MATERIALS AND METHODS A retrospective clinical study reviewed the medical records of 25 dogs of body weight <6 kg with mid to distal diaphyseal fractures of the radius and ulna (29 fractures) treated with open reduction bone plate fixation. Thirteen out of 29 fractures were implanted with an additional 2-3 cm³ OG lateral, cranial, and medial to the fracture site, adjacent to the bone plate. RESULTS Median time to radiographic healing in OG fractures (n = 11) was 70 days (range 28-98) compared to 106 days (range: 56-144) in non-OG grafted fractures (n = 14). The OG dogs had no major complications; minor complications included oedema, erythema, and mild osteopenia. Six of the eight non-OG dogs for which follow-up could be obtained developed osteopenia necessitating implant removal, four of which re-fractured the radius one to five months after implant removal, with one dog re-fracturing the limb a second time and resulting in amputation. Telephone follow-up of owners of OG dogs (n = 11) three to 15 months (median 10) post-surgery did not identify any signs of lameness or other complications. Owners of the non-OG dogs (n = 8) reported that there were not any signs of lameness six to 48 months (median 36) post-surgery. CLINICAL RELEVANCE Free autogenous omental grafting of diaphyseal fractures of the radius and ulna was associated with radial and ulnar healing with minimal complications in dogs weighing less than 6 kg.

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

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

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

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

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

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