Canine Chondrocytes Research Articles

The Effectiveness of Specific Collagen Peptides on Osteoarthritis in Dogs-Impact on Metabolic Processes in Canine Chondrocytes

In clinical trials over the past decade, the beneficial effect of orally administered collagen peptides in osteoarthritic dogs has been clearly demonstrated [1] [2] [3]. Although a statistically significant improvement in the lameness and vitality of dogs in general has been documented, the mode of action of the collagen peptide treatment is still under discussion. A previous study [3] indicated that the reduction in lameness and increased mobility in dogs after collagen peptide treatment were associated with a statistically significantly lowered plasma content of MMP-3, which is involved in collagen degradation. In addition, the content of the MMP-antagonist TIMP-1 increased slightly after collagen peptide supplementation, suggesting a direct impact on the cartilage metabolism, particularly on the decrease of extracellular matrix degradation. Based on these findings, the impact of specific collagen peptides (PETA-GILE?) on cartilage metabolism was tested in canine chondrocytes in the current investigation. In addition to the biosynthesis of various matrix molecules (type II collagen, aggrecan and elastin), the RNA profile of inflammatory cytokines and degenerative matrix molecules was investigated. The results showed clearly that the supplementation of specific collagen peptides reduced catabolic processes, as indicated by a statistically significant decrease in inflammatory cytokines and proteases in canine chondrocytes compared with untreated control experiments. In addition, a statistically significantly enhanced biosynthesis of type II collagen, elastin, and aggrecan was observed. Hence, the current data supports the suggested anti-inflammatory effect of specific collagen peptides, but also clearly demonstrates a pronounced stimulatory impact on matrix molecule synthesis. A combination of both observed effects might help to explain the previously reported clinical improvements after collagen peptide supplementation. Furthermore, the beneficial effect of the specific collagen peptides was also confirmed in case reports on osteoarthritic dogs that demonstrated decreased lameness and increased vitality in the affected animals after PETAGILE treatment.

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties

Animal cells have served as highly controllable model systems for furthering cartilage tissue engineering practices in pursuit of treating osteoarthritis. Although successful strategies for animal cells must ultimately be adapted to human cells to be clinically relevant, human chondrocytes are rarely employed in such studies. In this study, we evaluated the applicability of culture techniques established for juvenile bovine and adult canine chondrocytes to human chondrocytes obtained from fresh or expired osteochondral allografts. Human chondrocytes were expanded and encapsulated in 2% agarose scaffolds measuring ∅3–4mm×2.3mm, with cell seeding densities ranging from 15 to 90×106cells/mL. Subsets of constructs were subjected to transient or sustained TGF-β treatment, or provided channels to enhance nutrient transport. Human cartilaginous constructs physically resembled native human cartilage, and reached compressive Young’s moduli of up to ~250kPa (corresponding to the low end of ranges reported for native knee cartilage), dynamic moduli of ~950kPa (0.01Hz), and contained 5.7% wet weight (%/ww) of glycosaminoglycans (≥ native levels) and 1.5%/ww collagen. We found that the initial seeding density had pronounced effects on tissue outcomes, with high cell seeding densities significantly increasing nearly all measured properties. Transient TGF-β treatment was ineffective for adult human cells, and tissue construct properties plateaued or declined beyond 28 days of culture. Finally, nutrient channels improved construct mechanical properties, presumably due to enhanced rates of mass transport. These results demonstrate that our previously established culture system can be successfully translated to human chondrocytes.

In vitro effects of triamcinolone acetonide and in combination with hyaluronan on canine normal and spontaneous osteoarthritis articular cartilage.

The purposes of this study were to examine the cartilage degradation effects of triamcinolone acetonide (TA) on normal and osteoarthritic (OA) primary canine chondrocytes and cartilage explants and to examine the cartilage degradation effects of TA in combination with low-molecular-weight hyaluronan (LMWHA). To assess the effects of these drugs on cell culture, 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and real-time PCR were used to measure chondrotoxicity and determine gene expression, respectively. Uronic acid and hydroxyproline remaining in cartilage and histopathology were used to estimate the effects of these drugs on cartilage explants. In chondrocyte cultures, TA reduced chondrocyte viability in a concentration-dependent manner. LMWHA 2.5mg/ml combined with TA at IC20 (0.09mg/ml) could increase the viability of normal chondrocytes when compared with TA-treated alone. TA at IC20 induced down-regulation of ACAN and induced up-regulation of ADAMTS5 in canine normal chondrocytes. TA at IC20 (0.11mg/ml) up-regulated ADAMTS5, MMP2, MMP3, MMP13, and ACAN expression in canine OA chondrocytes. In explant culture, TA at 1.25, 2.5, and 5mg/ml increased the severity of structural damage, chondrocyte loss and cluster formation, and proteoglycan loss in OA cartilage. LMWHA could decrease the chondrotoxicity of TA at IC20 only in normal chondrocytes, as observed by chondrocyte viability. The combination of LMWHA and TA did not show clearly beneficial effects in all other normal and OA samples. Consequently, using TA alone or in combination with LMWHA in OA cartilage should be of concern because it may lead to cartilage destruction.

Two fluoroquinolones and their combinations with hyaluronan: comparison of effects on canine chondrocyte culture

Fluoroquinolones (FQs) are frequently used for septic arthritis. Increased antibacterial activity has been associated with mammalian cell cytotoxicity that may increase the risk of developing osteoarthritis. This study compared the direct effects of two different FQs, enrofloxacin (Enro) and marbofloxacin (Mar), on normal primary canine chondrocytes and inflammatory-stimulated chondrocytes, in addition to their administration in combination with hyaluronan (HA). Cell viability, cell apoptosis, s-GAG production, and expression patterns of inflammatory, extracellular matrix (ECM) component and protease genes were measured. Enro co-culturing with HA could modify s-GAG synthesis compared with the negative control group. Co-treatment with both FQs and HA significantly decreased cell viability and induced more total apoptotic cell death compared with the negative control and pre-IL-1β-stimulated group. Enro regulated IL-1β-stimulated cells to overexpress IL-1β, TNF, and MMP3, whereas Mar induced upregulation of PTGS2 and NFKB1 and enhanced the expression of ECM component genes HAS1, COL2A1, and ACAN as well as TIMP1 and MMP9. Simultaneous use of HA with Enro can effectively reduce the expression of IL-1β, TNF, and MMP3 in pre-IL-1β-stimulated chondrocytes. These results suggest the beneficial effects of HA in reducing the adverse effects of Enro treatment at the transcriptional level.

Carbon nanotubes: a promising tissue engineering approach for in vitro cultivation & differentiation of primary canine articular chondrocytes

Development of biocompatible materials has great potential in biomedical engineering both for in vitro studies as well as for in vivo applications. Two- and three-dimensional carbon nanotube (CNT) substrates imitating and providing an extracellular matrix-like structure are promising constructs as cell-supporting scaffolds. Lately, they have received considerable interest in tissue engineering; however, cellular responses to nanoscale stimuli need to be better understood. Here, we present the preliminary results on the effect of CNT-based scaffolds on the proliferation and arrangement of primary canine chondrocytes (PCCs). We aim to develop scaffolding materials for the in vitro cultivation of normal and neoplastic cells with the ultimate objective of using them for applications such as tissue implants in cartilage repair and tissue regeneration after surgical intervention. In the proposed studies we aim to use an aerogel network of CNTs that has been drawn from a vertically aligned array as a synthetic substrate for the growth and alignment of primary canine chondrocytes. This aerogel consists of CNTs that are aligned parallel to the major axis of the CNTs; they have exceptionally low densities, are electrically and thermally conductive whilst maintaining very high tensile strength and elasticity. We are studying the cell growth, adhesion, morphology, viability and metabolism of cells seeded onto CNT substrates. Preliminary results to date have revealed that PCCs are capable of proliferating on CNT-based scaffolds, although the cell viability seems to be slightly decreased in comparison to the conventional 2D cell culture. Moreover, our nanosubstrates are able to induce directional cell growth of PCCs via aligning cells along CNTs. The latter is essential for in vivo application of nanosubstrates in tissue regeneration.

Long-term storage and preservation of tissue engineered articular cartilage.

With limited availability of osteochondral allografts, tissue engineered cartilage grafts may provide an alternative treatment for large cartilage defects. An effective storage protocol will be critical for translating this technology to clinical use. The purpose of this study was to evaluate the efficacy of the Missouri Osteochondral Allograft Preservation System (MOPS) for room temperature storage of mature tissue engineered grafts, focusing on tissue property maintenance during the current allograft storage window (28 days). Additional research compares MOPS to continued culture, investigates temperature influence, and examines longer-term storage. Articular cartilage constructs were cultured to maturity using adult canine chondrocytes, then preserved with MOPS at room temperature, in refrigeration, or kept in culture for an additional 56 days. MOPS storage maintained desired chondrocyte viability for 28 days of room temperature storage, retaining 75% of the maturity point Young's modulus without significant decline in biochemical content. Properties dropped past this time point. Refrigeration maintained properties similar to room temperature at 28 days, but proved better at 56 days. For engineered grafts, MOPS maintained the majority of tissue properties for the 28-day window without clearly extending that period as it had for native grafts. These results are the first evaluating engineered cartilage storage.

Suppression of dedifferentiation and hypertrophy in canine chondrocytes through lentiviral vector expression of Sox9 and induced pluripotency stem cell factors.

Prolonged in vitro culture of primary articular chondrocytes results in dedifferentiation to a fibroblast-like cell with reduced expression of the Sox9 transcription factor and the extracellular matrix protein collagen II. The ability to genetically-modify chondrocytes to allow both proliferation and maintenance of an articular phenotype may provide increased numbers of appropriate cells for regeneration of large cartilage defects. Canine chondrocytes were expanded in monolayer culture and transduced with a lentiviral vector expressing Sox9 or in combination with a multicistronic lentiviral vector expressing the four induced pluripotency stem (iPS) cell factors, Oct4, Klf4, Sox2 and c-Myc (OSKM). 3D pellet cultures of transduced cells in the presence of TGFβ-3 revealed increased pellet size and higher levels of total glycosaminoglycan in both Sox9 and Sox9+ OSKM co-transduced chondrocytes compared to untransduced and green fluorescent protein expressing controls. Immunohistochemical detection of Sox9 and collagen II was evident in transduced cells (Sox9, OSKM, or Sox9+ OSKM) with very low levels in untransduced chondrocytes, demonstrating a dedifferentiated state (P < 0.01). The marker for chondrocyte hypertrophy, collagen X was highly expressed in Sox9 transduced chondrocytes but lower in OSKM or Sox9+ OSKM cells (P < 0.05). A combination of Sox9 and OSKM gene delivery to canine chondrocytes allows continuous proliferation in monolayer culture with a higher expression of col2a1 without an increase in the hypertrophy marker collagen X in 3D pellet cultures.

Evaluation of nanofiber-based polyglycolic acid scaffolds for improved chondrocyte retention and in vivo bioengineered cartilage regeneration.

In conventional studies on the regeneration of auricle-shaped cartilage in autogenous models using large animals, there were problems with the cartilage regeneration induction capacity and long-term retention of geometric shape. In this study, the authors sought to improve on outcome in these regards: a nonwoven fabric of polyglycolic acid was developed through nanotechnology, and the effect of nanofiber diameter on in vitro cell-seeding efficiency and the in vivo response after implantation in an autogenous large-animal model were evaluated. Canine chondrocytes were isolated and seeded onto polyglycolic acid fabric ranging from 0.5 to 20 μm in average diameter. Cell seeding efficiency was highest for mid-range polyglycolic acid fibers (average diameter, 0.8, 3.0, and 7.0 μm). Flat and auricle-shaped scaffolds were constructed using polypropylene structural support, sandwiching a nonwoven polyglycolic acid fabric that contained autogenous chondrocytes together with basic fibroblast growth factor-laden particles and an exterior fibrin sealant. Scaffolds were then implanted autogenously and evaluated at 5 and 20 weeks. Biomechanical strength was optimal for polyglycolic acid fiber diameters of 0.8 to 3.0 μm. Optimal cell maintenance and neocartilage response were seen with polyglycolic acid fiber diameters in the same mid-range for nanofiber constructs. These findings demonstrate the potential for nanoscale modulation of auricle-shaped cartilage regeneration in a large-animal model.

Ion channel modulation of chondrocyte cell volume regulation (1182.1)

Healthy chondrocytes exist with depolarised resting membrane potentials (Vm); critical for volume regulation. Chondrocytes express several cell volume regulation channels, including Transient Receptor Potential (TRP) V4, V5 and V6 and epithelial sodium channels. We investigated mechanisms of cell volume regulation in healthy chondrocytes. Patch clamp electrophysiology was used to measure canine chondrocyte Vm (Vm=‐12±3mV; n=21). Hypotonic challenge caused a significant Vm hyperpolarisation of 11±1mV (n=9; p&lt;0.05). We hypothesised this was due to Ca2+ influx activating a Ca2+‐activated potassium channel (KCa). Studies propose TRPV4 as a possible component of osmotically‐activated Ca2+ influx so we investigated this channel. However, the TRPV4 inhibitor, RN1734, failed to prevent hypotonicity‐induced hyperpolarisation (ΔVm=‐19±4mV, n=4; p&lt;0.05). Application of a TRPV4 agonist, 4αPDD, caused no significant change in Vm (n=12), whole cell current (n=7) or cell volume (n=8). Furthermore, application of iberiotoxin or 2‐APB completely abolished the hypotonicity‐induced hyperpolarisation. This leads us to conclude that Ca2+ increase in response to hypotonicity is likely to result from an, as yet, unidentified range of ion channels and/or Ca2+ stores and the KCa channel responsible for the hyperpolarisation is a large‐KCa channel.Grant Funding Source: Supported by European Union Seventh Framework Programme(FP7/2007‐2013) under grant agreement 305815

TNF/TNFR1 pathway and endoplasmic reticulum stress are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes

Background and purposeQuinolones cause obvious cartilaginous lesions in juvenile animals by chondrocyte apoptosis, which results in the restriction of their use in pediatric and adolescent patients. Studies showed that chondrocytes can be induced to produce TNFα, and the cisternae of the endoplasmic reticulum in quinolone-treated chondrocytes become dilated. We investigated whether TNF/TNFR1 pathway and endoplasmic reticulum stress (ERs) are involved in ofloxacin (a typical quinolone)-induced apoptosis of juvenile canine chondrocytes. Experimental approachCanine juvenile chondrocytes were treated with ofloxacin. Cell survival and apoptosis rates were determined with MTT method and flow cytometry, respectively. The gene expression levels of the related signaling molecules (TNFα, TNFR1, TRADD, FADD and caspase-8) in death receptor pathways and main apoptosis-related molecules (calpain, caspase-12, GADD153 and GRP78) in ERs were measured by qRT-PCR. The gene expression of TNFR1 was suppressed with its siRNA. The protein levels of TNFα, TNFR1 and caspase-12 were assayed using Western blotting. Key resultsThe survival rates decreased while apoptosis rates increased after the chondrocytes were treated with ofloxacin. The mRNA levels of the measured apoptosis-related molecules in death receptor pathways and ERs, and the protein levels of TNFα, TNFR1 and caspase-12 increased after the chondrocytes were exposed to ofloxacin. The downregulated mRNA expressions of TNFR1, Caspase-8 and TRADD, and the decreased apoptosis rates of the ofloxacin-treated chondrocytes occurred after TNFR1–siRNA interference. Conclusions and implicationsOfloxacin-induced chondrocyte apoptosis in a time- and concentration-dependent fashion. TNF/TNFR1 pathway and ERs are involved in ofloxacin-induced apoptosis of juvenile canine chondrocytes in the early stage.

Growth Factor Priming Differentially Modulates Components of the Extracellular Matrix Proteome in Chondrocytes and Synovium-Derived Stem Cells

To make progress in cartilage repair it is essential to optimize protocols for two-dimensional cell expansion. Chondrocytes and SDSCs are promising cell sources for cartilage repair. We previously observed that priming with a specific growth factor cocktail (1 ng/mL transforming growth factor-β1, 5 ng/mL basic fibroblast growth factor, and 10 ng/mL platelet-derived growth factor-BB) in two-dimensional culture, led to significant improvement in mechanical and biochemical properties of synovium-derived stem cell (SDSC)-seeded constructs. The current study assessed the effect of growth factor priming on the proteome of canine chondrocytes and SDSCs. In particular, growth factor priming modulated the proteins associated with the extracellular matrix in two-dimensional cultures of chondrocytes and SDSCs, inducing a partial dedifferentiation of chondrocytes (most proteins associated with cartilage were down-regulated in primed chondrocytes) and a partial differentiation of SDSCs (some collagen-related proteins were up-regulated in primed SDSCs). However, when chondrocytes and SDSCs were grown in pellet culture, growth factor-primed cells maintained their chondrogenic potential with respect to glycosaminoglycan and collagen production. In conclusion, the strength of the label-free proteomics technique is that it allows for the determination of changes in components of the extracellular matrix proteome in chondrocytes and SDSCs in response to growth factor priming, which could help in future tissue engineering strategies.

Modulation of inflammation and oxidative stress in canine chondrocytes

To determine whether oxidative stress could be induced in canine chondrocytes in vitro. Chondrocytes obtained from healthy adult mixed-breed dogs. Harvested chondrocytes were maintained at 37°C with 5% CO2 for 24 hours. To assess induction of oxidative stress, 2 stimuli were used: hydrogen peroxide and a combination of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). To determine the effect of hydrogen peroxide, a set of chondrocyte-seeded plates was incubated with control medium alone or hydrogen peroxide (100, 200, or 300μM) for 24 hours. For inhibition of oxidative stress, cells were incubated for 24 hours with N-acetylcysteine (NAC; 10mM) before exposure to hydrogen peroxide. Another set of chondrocyte-seeded plates was incubated with control medium alone or with IL-1β (10 ng/mL) and TNF-α (1 ng/mL) for 24 hours. Supernatants were obtained for measurement of prostaglandin E2 production, and cell lysates were used for measurement of superoxide dismutase (SOD) activity and reduced-glutathione (GSH) concentration. Chondrocytes responded to the oxidative stressor hydrogen peroxide with a decrease in SOD activity and GSH concentration. Exposure to the antioxidant NAC caused an increase in SOD activity in hydrogen peroxide-stressed chondrocytes to a degree comparable with that in chondrocytes not exposed to hydrogen peroxide. Similarly, NAC exposure induced significant increases in GSH concentration. Activation with IL-1β and TNF-α also led to a decrease in SOD activity and increase in prostaglandin E2 production. Canine chondrocytes responded to the oxidative stress caused by exposure to hydrogen peroxide and cytokines. Exposure to oxidative stress inducers could result in perturbation of chondrocyte and cartilage homeostasis and could contribute to the pathophysiology of osteoarthritis. Use of antioxidants, on the other hand, may be helpful in the treatment of arthritic dogs.

Chondrocyte Behavior on Micropatterns Fabricated Using Layer-by-Layer Lift-Off: Morphological Analysis.

Cell patterning has emerged as an elegant tool in developing cellular arrays, bioreactors, biosensors, and lab-on-chip devices and for use in engineering neotissue for repair or regeneration. In this study, micropatterned surfaces were created using the layer-by-layer lift-off (LbL-LO) method for analyzing canine chondrocytes response to patterned substrates. Five materials were chosen based on our previous studies. These included: poly(dimethyldiallylammonium chloride) (PDDA), poly(ethyleneimine) (PEI), poly(styrene sulfonate) (PSS), collagen, and chondroitin sulfate (CS). The substrates were patterned with these five different materials, in five and ten bilayers, resulting in the following multilayer nanofilm architectures: (PSS/PDDA)5, (PSS/PDDA)10; (CS/PEI)4/CS, (CS/PEI)9/CS; (PSS/PEI)5, (PSS/PEI)10; (PSS/Collagen)5, (PSS/Collagen)10; (PSS/PEI)4/PSS, (PSS/PEI)9/PSS. Cell characterization studies were used to assess the viability, longevity, and cellular response to the configured patterned multilayer architectures. The cumulative cell characterization data suggests that cell viability, longevity, and functionality were enhanced on micropatterned PEI, PSS, collagen, and CS multilayer nanofilms suggesting their possible use in biomedical applications.

Benzamil sensitive ion channels contribute to volume regulation in canine chondrocytes.

Background and PurposeChondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na+ transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be.Experimental ApproachCanine chondrocytes were harvested from dogs killed for unassociated welfare reasons. We used immunohistochemistry and patch-clamp electrophysiology to investigate ENaC expression and video microscopy to analyse the effects of pharmacological inhibition of ENaC/Deg on cell volume regulation.Key ResultsImmunofluorescence showed that canine chondrocytes expressed ENaC protein. Single-channel recordings demonstrated expression of a benzamil-sensitive Na+ conductance (9 pS), and whole-cell experiments show this to be approximately 1.5 nS per cell with high selectivity for Na+. Benzamil hyperpolarized chondrocytes by approximately 8 mV with a pD2 8.4. Chondrocyte regulatory volume decrease (RVI) was inhibited by benzamil (pD2 7.5) but persisted when extracellular Na+ ions were replaced by Li+.Conclusion and ImplicationsOur data suggest that benzamil inhibits RVI by reducing the influx of Na+ ions through ENaC/Deg-like ion channels and present ENaC/Deg as a possible target for pharmacological modulation of chondrocyte volume.

Implante de condrócitos homólogos em defeitos osteocondrais de cães: padronização da técnica e avaliação histopatológica

Padronizou-se a metodologia para cultura de condrócitos em cães e avaliou-se seu implante em lesões osteocondrais, utilizando-se a membrana biossintética de celulose (MBC) como revestimento. Dez cães, adultos e clinicamente sadios, foram submetidos à artrotomia das articulações fêmoro-tíbio-patelares. Defeitos de 4mm de diâmetro e profundidade foram induzidos no sulco troclear de ambos os membros. MBC foi aplicada na base e na superfície das lesões. Os defeitos do membro direito foram preenchidos com condrócitos homólogos cultivados formando o grupo-tratado (GT); os do membro esquerdo, sem implante celular, foram designados grupo-controle (GC). A evolução pós-operatória foi analisada com especial interesse nos processos de reparação da lesão, por meio de histomorfometria e imuno-histoquímica para colágeno tipo II e sulfato de condroitina. A cultura de condrócitos homólogos apresentou alta densidade e taxa de viabilidade. Observou-se integridade do tecido neoformado com a cartilagem adjacente na avaliação histológica, em ambos os grupos. Na imuno-histoquímica, verificou-se predomínio de colágeno tipo II no GT. Morfometricamente, não houve diferença significativa entre o tecido fibroso e o fibrocartilaginoso entre os grupos. A cultura de condrócitos homólogos de cães foi exequível. O tecido neoformado apresentou qualidade discretamente superior associado ao implante homólogo de condrócitos, contudo não promoveu reparação por cartilagem hialina.

In vitro evaluation of chondrosarcoma cells and canine chondrocytes on layer-by-layer (LbL) self-assembled multilayer nanofilms

Short-term cell–substrate interactions of two secondary chondrocyte cell lines (human chondrosarcoma cells, canine chondrocytes) with layer-by-layer self-assembled multilayer nanofilms were investigated for a better understanding of cellular-behaviour dependence on a number of nanofilm layers. Cell–substrate interactions were studied on polyelectrolyte multilayer nanofilms (PMNs) of eleven different biomaterials. Surface characterization of PMNs performed using AFM showed increasing surface roughness with increasing number of layers for most of the biomaterials. LDH-L and MTT assays were performed on chondrosarcoma cells and canine chondrocytes, respectively. A major observation was that 10-bilayer nanofilms exhibited lesser cytotoxicity towards human chondrosarcoma cells than their 5-bilayer counterparts. In the case of canine chondrocytes, BSA enhanced cell metabolic activity with increasing number of layers, underscoring the importance of the multilayer nanofilm architecture on cellular behaviour.

PDLA/PLLA and PDLA/PCL nanofibers with a chitosan-based hydrogel in composite scaffolds for tissue engineered cartilage

Osteoarthritis (OA) is the most prevalent musculoskeletal disease in humans, causing pain, loss of joint motility and function, and severely reducing the standard of living of patients. Cartilage tissue engineering attempts to repair the damaged tissue of individuals suffering from OA by providing mechanical support to the joint as new tissue regenerates. The aim of this study was to create composite three dimensional scaffolds comprised of electrospun poly(D,L-lactide)/poly(L-lactide) (PDLA/PLLA) or poly(D,L-lactide)/polycaprolactone (PDLA/PCL) with salt leached pores and an embedded chitosan hydrogel to determine the potential of these scaffolds for cartilage tissue engineering. PDLA/PLLA-hydrogel scaffolds displayed the largest compressive moduli followed by PDLA/PCL-hydrogel scaffolds. Dynamic mechanical tests showed that the PDLA/PLLA scaffolds had no appreciable recovery while PDLA/PCL scaffolds did exhibit some recovery. Primary canine chondrocytes produced both collagen type II and proteoglycans (primary components of extracellular matrix in cartilage) while being cultured on scaffolds composed of electrospun PDLA/PCL. As a result, a composite electrospun embedded hydrogel scaffold shows promise for treating individuals suffering from OA.

Effects of Low-Temperature Hydrogen Peroxide Gas Plasma Sterilization on In Vitro Cytotoxicity of Poly(ϵ-Caprolactone) (PCL)

Our objective was to determine whether low-temperature hydrogen peroxide (H2O2) gas plasma sterilization of porous three-dimensional poly(ϵ-caprolactone) (PCL) constructs significantly inhibits cellular metabolism of canine chondrocytes. Porous cylindrical constructs were fabricated using fused deposition modeling and divided into four sterilization groups. Two groups were sterilized with low-temperature H2O2 gas plasma (LTGP) and constructs from one of those groups were subsequently rinsed with Dulbecco’s Modified Essential Media (LTGPDM). Constructs in the other two groups were disinfected with either 70% isopropyl alcohol or exposure to UV light. Canine chondrocytes were seeded in 6-well tissue-culture plates and allowed to adhere prior to addition of PCL. Cellular metabolism was assessed by adding resazurin to the tissue-culture wells and assessing conversion of this substrate by viable cells to the fluorescent die resorufin. This process was performed at three times prior to addition of PCL and at four times after addition of PCL to the tissue-culture wells. Metabolism was not significantly different among the different tissue-culture wells at any of the 3 times prior to addition of PCL. Metabolism was significantly different among the treatment groups at 3 of 4 times after addition of PCL to the tissue culture wells. Metabolism was significantly lower with constructs sterilized by LTGP than all other treatment groups at all 3 of these times. We conclude that LTGP sterilization of PCL constructs resulted in significant cytotoxicity to canine chondrocytes when compared to PCL constructs disinfected with either UV light exposure or 70% isopropyl alcohol.

In vitro Effects of Polysaccharide Gel Extracted from Durian Rinds (Durio zibethinus L.) on the Enzymatic Activities of MMP-2, MMP-3 and MMP-9 in Canine Chondrocyte Culture

Osteoarthritis (OA) is a major joint disease found in animals, especially dogs, with 20% rate of incidence. The alternative therapy using medicinal plants has been of interest for a long time. Polysaccharide gel (PG) extracted from durian rinds (Durio zibethinus L.) was first isolated in 1998 for use in pharmaceuticals with its efficiency in open wound healing and antimicrobial activity. In this study, different concentrations of PG (1-100 g/ml) were used to evaluate the effect on canine chondrocytes through enzymatic activities of matrix metalloproteinase 2 (MMP-2), MMP-3 and MMP-9 concerning OA. The results showed that both proteolytic activities of MMP-2 and MMP-9 were inhibited significantly using the extract starting at a concentration of 50 g/ml. For MMP-3, enzymatic activity could not be inhibited by the PG extract in canine chondrocytes at any concentration level (1-100 g/ml). This could suggest that PG extract has a strong potential to inhibit MMP-2 and MMP-9 activities.

Contribution of the Epithelial Sodium Channel to Chondrocyte Regulatory Volume Increase

Chondrocytes are the cells of articular cartilage, responsible for the production and maintenance of the extracellular milieu. They exist in a constantly changing osmotic environment and to survive such osmotic changes they must be able to quickly and effectively regulate their volume. Cells use regulatory volume increase (RVI) to oppose osmotic shrinkage. It has previously been shown that the epithelial sodium channel (ENaC) is important to this process in rat hepatocytes1. Here we investigate the possible contribution of ENaC to RVI mechanisms in canine chondrocytes.Chondrocytes were isolated from cartilage according to standard methods2. Amiloride sensitive single-channel activity reversed at a membrane potential of −1±5mV (n = 5), mean conductance was 9±0.4pS (n = 5) and kinetics were slow. The calculated ENa under these conditions was −6mV, which coupled to the very small conductance would be consistent with this channel being an ENaC. Channel open probability in control conditions was 0.3±0.06 and decreased by 97±2% after application of the ENaC inhibitor, amiloride (10μM; n = 3).Upon exposure to hypertonic solution, cell volume decreased significantly by 35±3% (n = 5; p<0.001). Within 20 minutes of reaching their smallest size, cells under control conditions had returned to 92±4% of their original volume, not significantly different to starting volume (p=0.07). When 100nM benzamil, a specific ENaC inhibitor, was added to the hypertonic solution, cells shrank by 41±3% (n = 5) and were unable to return to their original volume.These data suggest that ENaC contributes to RVI in canine chondrocytes.1. F. Wehner, C. Bohmer, H. Heinzinger et al., Cellular Physiology and Biochemistry 10 (5-6), 335 (2000).2. R. Lewis, K. Asplin, G. Bruce et al., J Cell Physiol 226 (8) (2011).