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Cartilage defects can be difficult to treat; therefore, tissue engineering of cartilage is emerging as a promising potential therapy. One interesting area of research explores the delivery of cells to the cartilage defect via scaffold-based cell delivery vehicles and microsurgery. This study explores the use of novel poly(glycerol sebacate) methacrylate (PGSm)-polymerised high internal phase emulsion (polyHIPE) microspheres as scaffolds with embedded cells for cartilage tissue engineering. Porous microsphere scaffolds (100 µm–1 mm diameter) were produced from emulsions consisting of water and a methacrylate-based photocurable resin of poly(glycerol sebacate). These resins were used in conjunction with a T-junction fluidic device and an ultraviolet (UV) curing lamp to produce porous microspheres with a tuneable size. This technique produced biodegradable PGSm microspheres with similar mechanical properties to cartilage. We further explore these microspheres as scaffolds for three-dimensional culture of chondrocytes. The microspheres proved to be very efficient scaffolds for primary chondrocyte culture and were covered by a dense extracellular matrix (ECM) network during the culture period, creating a tissue disk. The presence of glycosaminoglycans (GAGs) and collagen-II was confirmed, highlighting the utility of the PGSm microspheres as a delivery vehicle for chondrocytes. A number of imaging techniques were utilised to analyse the tissue disk and develop methodologies to characterise the resultant tissue. This study highlights the utility of porous PGSm microspheres for cartilage tissue engineering.

Advances in nanotechnology have been exploited to develop new biomaterials including nanocrystalline hydroxyapatite (nHA) with physical properties close to those of natural bone mineral. While clinical data are encouraging, relatively little is understood regarding bone cells’ interactions with synthetic graft substitutes based on this technology. The aim of this research was therefore to investigate the in vitro response of both osteoblast cell lines and primary osteoblasts to an nHA paste. Cellular metabolic activity was assessed using the cell viability reagent PrestoBlue and quantitative, real-time PCR was used to determine gene expression related to osteogenic differentiation. A potential role of calcium-sensing receptor (CaSR) in the response of osteoblastic cells to nHA was also investigated. Indirect contact of the nHA paste with human osteoblastic cells (Saos-2, MG63, primary osteoblasts) and human bone marrow-derived mesenchymal stem cells enhanced the cell metabolic activity. The nHA paste also stimulated gene expression of runt-related transcription factor 2, collagen 1, alkaline phosphatase, and osteocalcin, thereby indicating an osteogenic response. CaSR was not involved in nHA paste-induced increases in cellular metabolic activity. This investigation demonstrated that the nHA paste has osteogenic properties that contribute to clinical efficacy when employed as an injectable bone graft substitute.

The regeneration of large bone defects remains clinically challenging. The aim of our study was to use a rat model to use nasal chondrocytes to engineer a hypertrophic cartilage tissue which could be remodelled into bone in vivo by endochondral ossification.\ud \ud Primary adult rat nasal chondrocytes were isolated from the nasal septum, the cell numbers expanded in monolayer culture and the cells cultured in vitro on polyglycolic acid scaffolds in chondrogenic medium for culture periods of 5–10 weeks. Hypertrophic differentiation was assessed by determining the temporal expression of key marker genes and proteins involved in hypertrophic cartilage formation. The temporal changes in the genes measured reflected the temporal changes observed in the growth plate. Collagen II gene expression increased 6 fold by day 7 and was then significantly downregulated from day 14 onwards. Conversely, collagen X gene expression was detectable by day 14 and increased 100-fold by day 35. The temporal increase in collagen X expression was mirrored by increases in alkaline phosphatase gene expression which also was detectable by day 14 with a 30-fold increase in gene expression by day 35. Histological and immunohistochemical analysis of the engineered constructs showed increased chondrocyte cell volume (31–45 μm), deposition of collagen X in the extracellular matrix and expression of alkaline phosphatase activity. However, no cartilage mineralisation was observed in in vitro culture of up to 10 weeks. On subcutaneous implantation of the hypertrophic engineered constructs, the grafts became vascularised, cartilage mineralisation occurred and loss of the proteoglycan in the matrix was observed. Implantation of the hypertrophic engineered constructs into a rat cranial defect resulted in angiogenesis, mineralisation and remodelling of the cartilage tissue into bone. Micro-CT analysis indicated that defects which received the engineered hypertrophic constructs showed 38.48% in bone volume compared to 7.01% in the control defects.\ud \ud Development of tissue engineered hypertrophic cartilage to use as a bone graft substitute is an exciting development in regenerative medicine. This is a proof of principal study demonstrating the potential of nasal chondrocytes to engineer hypertrophic cartilage which will remodel into bone on in vivo transplantation. This approach to making engineered hypertrophic cartilage grafts could form the basis of a new potential future clinical treatment for maxillofacial reconstruction.

Injuries to articular cartilage are frequently difficult to repair, in part because of the poor regenerative capacity of this tissue. To date, no successful system for complete regeneration of the most challenging cartilage defects has been demonstrated. The aim of this work was to develop functionalised hydrogels at the nanoscale by Layer-by-Layer (LbL) assembly to promote cartilage healing. Hydrogels, based on sodium alginate (NaAlg) and gelatin (G), were prepared by an external gelation method consisting of CaCl2 diffusion and genipin addition for G crosslinking. Successively, hydrogels were coated with G to obtain a positive charge on the surface, then functionalised by LbL assembly to create 16 nanolayers, based on poly(styrene sulfonate)/poly(allyl amine) (PSS/PAH), including a specific peptide sequence (CTATVHL) and transforming growth factors β1 (TGF-β1). Physico-chemical properties were evaluated by XPS, ATR-FTIR and rheological analyses while in vitro cytocompatibility was studied using bovine articular chondrocytes (BAC). XPS spectra showed N1s and S2p peaks, indicating that PAH and PSS have been introduced with success. ATR-FTIR indicated the specific PAH and PSS absorption peaks. Finally, the biomolecule incorporation influenced positively the processes of BAC adhesion and proliferation, and glycosamynoglycan secretion. The functionalised alginate-based hydrogels described here are ideally suited to chondral regeneration in terms of their integrity, stability, and cytocompatibility.

Fibroblasts are the major cellular component of connective tissue and experience mechanical perturbations due to matrix remodelling and interstitial fluid movement. Transforming growth factor β1 (TGF-β1) can promote differentiation of fibroblasts in vitro to a contractile myofibroblastic phenotype characterised by the presence of α-smooth muscle actin (α-SMA) rich stress fibres. To study the role of mechanical stimulation in this process, we examined the response of primary human fibroblasts to physiological levels of fluid movement and its influence on fibroblast differentiation and responses to TGF-β1. We reported that in both oral and dermal fibroblasts, physiological levels of fluid flow induced widespread changes in gene expression compared to static cultures, including up-regulation of genes associated with TGFβ signalling and endocytosis. TGF-β1, activin A and markers of myofibroblast differentiation including α-SMA and collagen IA1 were also increased by flow but surprisingly the combination of flow and exogenous TGF-β1 resulted in reduced differentiation. Our findings suggest this may result from enhanced internalisation of caveolin and TGF-β receptor II. These findings suggest that a) low levels of fluid flow induce myofibroblast differentiation and b) fluid flow antagonises the fibroblast response to pro-differentiation signals such as TGF-β1. We propose that this may be a novel mechanism by which mechanical forces buffer responses to chemical signals in vivo, maintaining a context-specific fibroblast phenotype. J. Cell. Biochem. 118: 878-890, 2017. © 2016 Wiley Periodicals, Inc.

Introduction: Cannabinoids have shown to reduce joint damage in animal models of arthritis and reduce matrix\ud metalloproteinase expression in primary human steoarthritic (OA) chondrocytes. The actions of cannabinoids are mediated by a number of receptors, including cannabinoid receptors 1 and 2 (CB1 and CB2), Gprotein-coupled receptors 55 and 18 (GPR55 and GPR18), transient receptor potential vanilloid-1 (TRPV1), and peroxisome proliferator-activated receptors alpha and gamma (PPARa and PPARc). However, to date very few studies have investigated the expression and localization of these receptors in human chondrocytes, and expression during degeneration, and thus their potential in clinical applications is unknown.\ud Methods: Human articular cartilage from patients with symptomatic OA was graded histologically and the expression\ud and localization of cannabinoid receptors within OA cartilage and underlying bone were determined\ud immunohistochemically. Expression levels across regions of cartilage and changes with degeneration were investigated.\ud Results: Expression of all the cannabinoid receptors investigated was observed with no change with grade of\ud degeneration seen in the expression of CB1, CB2, GPR55, PPARa, and PPARc. Conversely, the number of chondrocytes\ud within the deep zone of cartilage displaying immunopositivity for GPR18 and TRPV1 was significantly\ud decreased in degenerate cartilage. Receptor expression was higher in chondrocytes than in osteocytes in the\ud underlying bone.\ud Conclusions: Chondrocytes from OA joints were shown to express a wide range of cannabinoid receptors even\ud in degenerate tissues, demonstrating that these cells could respond to cannabinoids. Cannabinoids designed to\ud bind to receptors inhibiting the catabolic and pain pathways within the arthritic joint, while avoiding psychoactive effects, could provide potential arthritis therapies.\ud Key words: articular cartilage; cannabinoid receptors; cannabinoids; osteoarthritis

Bioactive glasses are known to stimulate bone healing, and the incorporation of strontium has the potential to increase their potency. In this study, calcium oxide in the 45S5 bioactive glass composition was partially (50%, Sr50) or fully (100%, Sr100) substituted with strontium oxide on a molar basis. The effects of the substitution on bioactive glass properties were studied, including density, solubility, and in vitro cytotoxicity. Stimulation of osteogenic differentiation was investigated using mesenchymal stromal cells obtained from rat bone marrow. Strontium substitution resulted in altered physical properties including increased solubility. Statistically significant reductions in cell viability were observed with the addition of bioactive glass powders to culture medium. Specifically, addition of ≥ 13.3 mg/ml of 45S5 bioactive glass or Sr50, or ≥ 6.7 mg/ml of Sr100, resulted in significant inhibition. Real-time PCR analyses detected the upregulation of genes associated with osteoblastic differentiation in the presence of all bioactive glass compositions. Some genes, including Alpl and Bglap, were further stimulated in the presence of Sr50 and Sr100. It was concluded that strontium-substituted bioactive glasses promoted osteogenesis in a differentiating bone cell culture model and, therefore, have considerable potential for use as improved bioactive glasses for bone tissue regeneration. © 2015 The Authors. Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.

Infection is the major reason of GTR/GBR membrane failure in clinical application. In this work, we developed GTR/GBR nanofiber membranes with localized drug delivery function to prevent infection. Metronidazole (MNA), an antibiotic, was successfully incorporated into electrospun polycaprolactone (PCL) nanofibers at different concentrations (0, 1, 5, 10, 20, 30, and 40 wt% polymer). To obtain the optimum anti-infection membrane, we systematically investigated the physical-chemical and mechanical properties of the nanofiber membranes with different drug contents. The interaction between PCL and MNA was identified by molecular dynamics simulation. MNA released in a controlled, sustained manner over 2 weeks and the antibacterial activity of the released MNA remained. The incorporation of MNA improved the hydrophilicity and in vitro biodegradation rate of PCL nanofibers. The nanofiber membranes allowed cells to adhere to and proliferate on them and showed excellent barrier function. The membrane loaded with 30% MNA had the best comprehensive properties. Analysis of subcutaneous implants demonstrated that MNA-loaded nanofibers evoked a less severe inflammatory response than pure PCL nanofibers. These results demonstrate the potential of MNA-loaded nanofiber membranes as GTR/GBR membrane with antibacterial and anti-inflammatory function for extensive biomedical applications.

Autologous bone and rib cartilage are considered the gold standard for bone and cartilage reconstruction of craniofacial defects. However, there may be insufficient donor tissue for effective reconstruction of large and/or complex shaped defects. Tissue engineering technologies offer real potential to provide engineered autologous cartilage and bone tissues for transplantation. Tissue engineering tissue grafts requires: (1) an appropriate source of autologous cells (such as primary cells or mesenchymal cells); (2) a biomimetic scaffold to provide a three-dimensional shaped support for the cells and “biological instructions” to promote appropriate cell differentiation and good tissue formation; (3) an effective bioreactor culture system to provide the appropriate nutrient and gaseoues exchange and physical stimulation of the developing tissue. This review will consider the current position of tissue engineering to generate bone and cartilage grafts and the current hurdles and problems which need to be overcome.

Biomaterials composed of hydroxyapatite (HA) are currently used for the treatment of bone defects resulting from trauma or surgery. However, hydroxyapatite supplied in the form of a paste is considered a very convenient medical device compared to the materials where HA powder and liquid need to be mixed immediately prior to the bone treatment during surgery. In this study we have tested a series of hydroxyapatite (HA) pastes with varying microstructure and different rheological behaviour to evaluate their injectability and biocompatibility. The particle morphology and chemical composition were evaluated using HRTEM, XRD and FTIR. Two paste-types were compared, with the HA particles of both types being rod shaped with a range of sizes between 20 and 80 nm while differing in the particle aspect ratio and the degree of roundness or sharpness. The pastes were composed of pure HA phase with low crystallinity. The rheological properties were evaluated and it was determined that the pastes behaved as shear-thinning, non-Newtonian liquids. The difference in viscosity and yield stress between the two pastes was investigated. Surprisingly, mixing of these pastes at different ratios did not alter viscosity in a linear manner, providing an opportunity to produce a specific viscosity by mixing the two materials with different characteristics. Biocompatibility studies suggested that there was no difference in vitro cell response to either paste for primary osteoblasts, bone marrow mesenchymal stromal cells, osteoblast-like cells, and fibroblast-like cells. This class of nanostructured biomaterial has significant potential for use as an injectable bone graft substitute where the properties may be tailored for different clinical indications.

Poly[2-ethyl(2-pyrrolidone) methacrylate] and hyaluronic acid hydrogels were synthesized via free-radical polymerization of 2-ethyl(2-pyrrolidone) methacrylate, hyaluronic acid and different crosslinkers. The ability of these hydrogels to induce apatite formation by incubating in simulated body fluid was investigated. The effect of hyaluronic acid content, crosslinkers and immersion time on mineralization behaviour and interface properties as well as the metabolic activity of different cultured cells were also determined. The bioactivity of the poly[2-ethyl(2-pyrrolidone) methacrylate] and hyaluronic acid hydrogels along with cell viability data indicated their potential application in bone tissue engineering.

Fibroblasts are the major cellular component of connective tissue and experience mechanical perturbations due to matrix remodelling and interstitial fluid movement. Transforming growth factor β1 (TGF-β1) can promote differentiation of fibroblasts in vitro to a contractile myofibroblastic phenotype characterised by the presence of α-smooth muscle actin (α-SMA) rich stress fibres. To study the role of mechanical stimulation in this process, we examined the response of primary human fibroblasts to physiological levels of fluid movement and its influence on fibroblast differentiation and responses to TGF-β1. We reported that in both oral and dermal fibroblasts, physiological levels of fluid flow induced widespread changes in gene expression compared to static cultures, including up-regulation of genes associated with TGFβ signalling and endocytosis. TGF-β1, activin A and markers of myofibroblast differentiation including α-SMA and collagen IA1 were also increased by flow but surprisingly the combination of flow and exogenous TGF-β1 resulted in reduced differentiation. Our findings suggest this may result from enhanced internalisation of caveolin and TGF-β receptor II. These findings suggest that a) low levels of fluid flow induce myofibroblast differentiation and b) fluid flow antagonises the fibroblast response to pro-differentiation signals such as TGF-β1. We propose that this may be a novel mechanism by which mechanical forces buffer responses to chemical signals in vivo, maintaining a context-specific fibroblast phenotype. J. Cell. Biochem. 118: 878-890, 2017. © 2016 Wiley Periodicals, Inc.

Three novel borosilicate bioactive glasses (BBGs) of general formula of 0.05Na2O·0.35x·0.20B2O3·0.40SiO2 (molar ratio, where x = MgO or CaO or SrO) were prepared and used to investigate the effect of crystallization on their properties including cytotoxicity. The three postmelt compositions were determined using X-ray fluorescence spectroscopy, and crystallization events were studied using differential thermal analysis and X-ray diffraction. This information was used to determine heat treatments to prepare glass-ceramics by controlled crystallization. X-ray diffraction analysis and Fourier transform infrared spectroscopy showed that, after higher heat treatment temperatures (800–900 °C), borosilicate bioactive glass-ceramics (BBGCs) contained mainly borate and silicate crystalline phases. Specifically, BBG-Mg, BBG-Ca, and BBG-Sr glass-ceramics detected the presence of magnesium silicate-Mg2(SiO3)2 and magnesium borate-Mg2B2O5; wollastonite-2M-CaSiO3 and calcium borate-Ca(BO2)2; and strontium silicate-SrSiO3 and strontium borate-Sr2B2O5, respectively. In vitro cytotoxicity tests were performed using the mouse fibroblast cell line (L929). Glass and glass ceramic at concentrations lower than 50 mg/mL did not exhibit any level of cytotoxicity when compared with the control. However, quantitative evaluation indicated that greater cell growth occurred in the presence of materials with crystalline phases. Control of BBGs crystallization may therefore be used to influence the biocompatibility of these glass-ceramic systems., Portuguese Foundation for Science and Technology (Ph.D. Grant BD/73162/2010), European Union’s Seventh Framework Programme (FP7/2007-2013) under Grant No. REGPOT-CT2012-31633-POLARIS and UK EPSRC Centre for Innovative Manufacturing of Medical Devices-MeDe Innovation (EPSRC Grant EP/K029592/1)

A key feature of osteoarthritis and rheumatoid arthritis is the loss of articular cartilage. Cartilage breakdown is mediated by complex interactions of proinflammatory cytokines, such as IL-1, inflammatory mediators, including nitric oxide and prostaglandin E2, and proteases, including matrix metalloproteinases and aggrecanases, such as ADAMTS-4 and -5. Cannabinoids have been shown to reduce joint damage in animal models of arthritis. They have also been shown to prevent IL-1-induced matrix breakdown of collagen and proteoglycan, indicating that cannabinoids may mediate chondroprotective effects. Cannabinoids produce their effects via several cannabinoid receptors and it is important to identify the key cannabinoids and their receptors that are involved in chondroprotection. This review aims to outline the current and future prospects of cannabinoids as anti-arthritic therapeutics, in terms of their ability to prevent cartilage breakdown.

Objective Arthritic synovial fluid (SF) contains mesenchymal stem cells (MSCs), which could simply reflect their shedding from diseased joint structures. This study used the bovine model to explore SF MSCs in health and enumerated them at the earliest stages of human osteoarthritis (OA) in radiographically normal joints. Methods Clonogenicity and multipotentiality of normal bovine SF MSCs were compared with donor-matched bone marrow (BM) MSCs at the single-cell level. The colony-forming unit–fibroblastic assay was used for MSC enumeration. The XTT assay was employed to assess cell proliferation, and flow cytometry was used to investigate the marker phenotype of bovine and human SF MSCs. Results Single MSCs were present in normal bovine SF, and 96% of them were able to expand at least 1 million–fold. These cells were CD271–, multipotential, considerably more clonogenic, and less adipogenic than matched BM MSCs. In both pellet assays and on polyglycolic acid scaffolds, SF clones displayed consistent chondrogenic differentiation, while BM clones were variable. MSCs were present in arthroscopically normal human joints and were increased 7-fold in early OA (P = 0.034). Their numbers correlated with numbers of free microscopic synovial tissue fragments (r = 0.826, P < 0.0001). OA SF had a growth-promoting effect on synovial MSCs. Conclusion This study confirms the presence of MSCs in normal SF and shows their numerical increase in early human OA. SF MSCs are likely to originate from synovium. These findings provide a platform for the exploration of the potential role of SF MSCs in joint homeostasis and for investigation of their utility in novel joint regeneration strategies.

PUBLISHED, The aim of this study was to determine whether or not biochemical markers can be used as surrogate measures for the mechanical quality of tissue engineered cartilage. The biochemical composition of tissue engineered cartilage constructs were altered by varying either (i) the initial cell seeding density of the scaffold (seeding density protocol) or (ii) the length of time the engineered tissue was cultured (culture period protocol). The aggregate or Young?s moduli of the constructs were measured (by confined or unconfined compression respectively), and compared with the composition of the extracellular matrix by quantitative measurement of the glycosaminoglycan (GAG), hydroxyproline, collagen I and collagen II and collagen cross-links. The aggregate modulus correlated positively with both GAG and collagen II content, but not with collagen I content. Young?s modulus correlated positively with GAG, collagen II and collagen I content, and the ratio of mature to immature cross-links, but not with hydroxyproline content. These results suggested that hydroxyproline may be an unreliable indicator of mechanical quality of tissue engineered cartilage, and that a measure of collagen II and GAG content is required to predict the biomechanical quality of tissue engineered cartilage., This work was funded by the European Union (EU) under the SCAFCART project (contract G5RD-CT-1999-00050). APH is funded in part by an endowed chair from the UK Arthritis Research Campaign.

Hydrogel brushes are materials composed of a water-swollen network, which contains polymer chains that are grafted with another polymer. Using a thermally responsive polymer, poly( N -isopropyl acrylamide) (polyNIPAM), as the graft component we are able to maintain the critical solution temperature ( T crit ), independent of the overall composition of the material, at approximately 32°C. The change in swelling at T crit is a function of the amount of polyNIPAM in the system. However, there is a much smaller change in the surface contact angles at T crit . PolyNIPAM-based materials have generated considerable interest, as ‘smart’ substrates for the culture of cells and here, we show the utility of hydrogel brushes in cell culture. Chondrocytes attached to the hydrogel brushes and yielded viable cell cultures. Moreover, the chondrocytes could be released from the hydrogel brushes without the use of proteases by reducing the temperature of the cultures to below T crit to induce a change in the conformation of the polyNIPAM chain at T crit . The importance of the crosslink hydrogel component is illustrated by significant changes in cell attachment/cell viability as the crosslink density is changed.

For guided tissue regeneration (GTR) membrane, synchronization of the membrane biodegradation rate and tissue regeneration rate is important. Besides, the major reason for GTR membrane failure in clinical application is infection which can be prevented by loading anti-bacterial drug. To realize the consistency in membrane degradation rate and tissue regeneration rate of the anti-infective membrane, we developed metronidazole-loaded electrospun poly(ɛ-caprolactone)-gelatin nanofiber membranes with different poly(ɛ-caprolactone)/gelatin ratios (95:5, 90:10, 80:20, 70:30, 60:40, and 50:50). Homogeneous nanofibers were successfully fabricated. The mechanical strength of the membranes increased with the poly(ɛ-caprolactone) content, while the hydrophilicity decreased. The controlled and sustained release of metronidazole from all the membranes prevented the colonization of anaerobic bacteria. At all poly(ɛ-caprolactone)/gelatin ratios, all the membranes presented good biocompatibility while the increase of gelatin content resulted in enhanced cell adhesion and proliferation. Subcutaneous implantation in rabbits for 8 months demonstrated that all the membranes showed good biocompatibility without infection. Both in vitro and in vivo results showed that the biodegradation rate of the membranes was accelerated with the increase of gelatin content. The biodegradation rate and biocompatibility of the membranes can be adjusted by changing the PCL/gelatin ratio. The optimal membrane can be chosen based on the patient and tissue type to realize the synchronization of membrane degradation with tissue regeneration for the best treatment effect.

The effects of devitrification of an ionomer glass with a molar composition 4.5SiO(2).3Al(2)O(3).1.5P(2)O(5).3CaO.2CaF(2) on cement formation and in vitro biocompatibility were investigated. Differential thermal analysis was used to study the phase evolution in the glass, and to determine the heat treatments for production of glass-ceramics. X-ray diffraction patterns from glass frit heat-treated at 750 degrees C for 2h contained peaks corresponding to apatite (JCPDS 15-876), whereas for samples heat-treated at 950 degrees C for 2h apatite and mullite (JCPDS 15-776) were the major phases detected. Transmission electron microscopy (TEM) confirmed that apatite and apatite-mullite phases were present after heat treatments at 750 degrees C and 950 degrees C respectively. Glass and glass-ceramics were ground to prepare

Infection is the major reason for guided tissue regeneration/guided bone regeneration (GTR/GBR) membrane failure in clinical application. In this work, we developed GTR/GBR membranes with localized drug delivery function to prevent infection by electrospinning of poly(e-caprolactone) (PCL) and gelatin blended with metronidazole (MNA). Acetic acid (HAc) was introduced to improve the miscibility of PCL and gelatin to fabricate homogeneous hybrid nanofiber membranes. The effects of the addition of HAc and the MNA content (0, 1, 5, 10, 20, 30, and 40 wt.% of polymer) on the properties of the membranes were investigated. The membranes showed good mechanical properties, appropriate biodegradation rate and barrier function. The controlled and sustained release of MNA from the membranes significantly prevented the colonization of anaerobic bacteria. Cells could adhere to and proliferate on the membranes without cytotoxicity until the MNA content reached 30%. Subcutaneous implantation in rabbits for 8 months demonstrated that MNA-loaded membranes evoked a less severe inflammatory response depending on the dose of MNA than bare membranes. The biodegradation time of the membranes was appropriate for tissue regeneration. These results indicated the potential for using MNA-loaded PCL/gelatin electrospun membranes as anti-infective GTR/GBR membranes to optimize clinical application of GTR/GBR strategies.

We read with interest an article by Ando et al. [1] entitled “Clonal analysis of synovial fluid stem cells to characterize and identify stable mesenchymal stromal cell/mesenchymal progenitor cell phenotypes in a porcine model: a cell source with enhanced commitment to the chondrogenic lineage.” The authors isolated clonal populations of mesenchymal stromal cells (MSCs) from pig synovial fluid (SF) and demonstrated good chondrogenic potential of 18 isolated clones. The authors concluded that the inherent chondrogenic phenotype of SF MSCs may be optimal for cartilage repair. Interestingly, the data of Ando et al. closely follow and support original work in the field of SF MSCs, which was unfortunately overlooked by this report. In 2000, Marinova-Mutafchieva et al. [2] showed the presence of CD44-positive, highly proliferative fibroblastic cells in arthritic SF, and, 4 years later, we demonstrated their tri-potentiality toward osteogenesis, adipogenesis and chondrogenesis at the single-cell level [3]. On average, single-cellederived SFMSCs had high levels of chondrogenicity and low levels of osteogenicity [3]. Furthermore, in this article, we showed increased numbers of MSCs in the SF of patients with osteoarthritis (OA) compared with rheumatoid arthritis; this was later explored by Tokyo [4] and Calgary groups [5] who furnished further evidence that OA may be associated with endogenous repair processes leading to an expansion of MSCs in the joint space. In our subsequent report (2008), we explored the nature of SF MSCs in health with the use of a bovine model and in early human OA [6]. We showed that in the bovine, clonally derived SF MSCs had a more

Poly(2-ethyl-(2-pyrrolidone)methacrylate)-hyaluronic acid hydrogels based on the free radical polymerization of 2-ethyl-(2-pyrrolidone)methacrylate combined with hyaluronic acid, using N,N′-methylenebisacrylamide or triethylene glycol dimethacrylate, as cross-linking agents, were considered for tissue engineering applications. Bovine articular chondrocytes were seeded onto the poly(2-ethyl-(2-pyrrolidone)methacrylate)-hyaluronic acid hydrogels, under orbital agitation, for a total of 40 days. The engineered cell-constructs were characterized according to cell proliferation, morphology and distribution as well as the biochemical composition of the tissue formed. The chondrocytes were found to be attached and presented a typical spherical morphology. Cells were able to proliferate and synthesize a hyaline-like matrix rich in glycosaminoglycans and collagen type II which were mainly located on the superficial area. Increased content of individual components poly(2-ethyl-(2-pyrrolidone)methacrylate) and hyaluronic acid, in triethylene glycol dimethacrylate-cross-linked networks led to enhanced cell distribution and total glycosaminoglycans content, supporting their potential application for the repair of cartilaginous tissues.

Low-friction surfaces are critical for efficient joint articulation. The tribological properties of articular cartilage have been studied extensively in native tissue and joints. Despite their importance, very few studies have examined the frictional properties of tissue-engineered cartilage. We have therefore reviewed the relationship between composition, structure and friction in tissue-engineered cartilage.

Cytokines, including TNF alpha and IL-l beta, are central to the chronic inflammatory process and tissue damage that characterises diseases such as rheumatoid arthritis. The mechanisms responsible for long-term generation of these molecules are poorly understood. We have previously demonstrated impaired activity of Na, K-ATPase, a key enzyme regulating intracellular cation levels, on rheumatoid mononuclear cells. Mimicking this 'defect' on normal mononuclear cells with ouabain has been shown to induce TNF alpha and, in particular, IL-l beta production, whereas IL-6 synthesis was suppressed. A similar pattern of cytokine generation was noted when mononuclear cells were treated with the sodium ionophore, monensin. Induction of cytokine production was related to up-regulation of the appropriate mRNA, although enhanced secretion of processed IL-l beta was also observed. The mechanism underlying these cellular responses appears to involve sodium/calcium exchange across the cell membrane. Impaired Na,K-ATPase activity might promote pro-inflammatory cytokine secretion in patients with rheumatoid arthritis.

Summary Objective Interleukin-1β (IL-1β) is involved in the up-regulation of matrix metalloproteinases (MMPs) leading to cartilage degradation. Cannabinoids are anti-inflammatory and reduce joint damage in animal models of arthritis. This study aimed to determine a mechanism whereby the synthetic cannabinoid WIN-55,212-2 mesylate (WIN-55) may inhibit cartilage degradation. Methods Effects of WIN-55 were studied on IL-1β stimulated production of MMP-3 and -13 and their inhibitors TIMP-1 and -2 in human chondrocytes. Chondrocytes were obtained from articular cartilage of patients undergoing total knee replacement. Chondrocytes were grown in monolayer and 3D alginate bead cultures. Real-time polymerase chain reaction (PCR) was used to determine the gene expression of MMP-3, -13, TIMP-1 and -2 and Enzyme Linked Immunosorbent Assay (ELISA) to measure the amount of MMP-3 and MMP-13 protein released into media. Immunocytochemistry was used to investigate the expression of cannabinoid receptors in chondrocyte cultures. Results Treatment with WIN-55 alone or in combination with IL-1β, decreased or abolished MMP-3, -13, TIMP-1 and -2 gene expression in human chondrocyte monolayer and alginate bead cultures in both a concentration and time dependent manner. WIN-55 treatment alone, and in combination with IL-1β, reduced MMP-3 and -13 protein production by chondrocytes cultured in alginate beads. Immunocytochemistry demonstrated the expression of cannabinoid receptors in chondrocyte cultures. Conclusion Cannabinoid WIN-55 can reduce both basal and IL-1β stimulated gene and protein expression of MMP-3 and -13. However WIN-55 also decreased basal levels of TIMP-1 and -2 mRNA. These actions of WIN-55 suggest a mechanism by which cannabinoids may act to prevent cartilage breakdown in arthritis.

Introduction: The tissue dynamics of a growing tumor differ from normal tissue, with increased matrix stiffness and mechanical pressure causing alterations in interstitial fluid movement in the tumor microenvironment (TME). This change in fluid flow is known to affect the ability of chemotherapeutic drugs to penetrate tumors, but the effect of flow on the phenotype of cells of the tumor microenvironment has not been studied. The predominant cell type of the TME, the fibroblast, is known to become activated to a pro-tumorigenic phenotype upon exposure to factors such as TGF-β1 released by cancer cells, immune cells and also by mechanical forces. The effect of fluid flow on the fibroblast phenotype, however, remains undetermined. Objectives: We hypothesize that stromal fibroblasts are activated by interstitial fluid flow and thereby promote tumor growth. We aim to investigate this by studying the markers of fibroblast activation under mechanically relevant interstitial fluid flow conditions. Methods: Normal oral (NOF) and dermal (NHDF) fibroblasts were plated on collagen coated Thermonox cover slips, transferred into Quasi-vivo bioreactors and subjected to flow (150 μl/min) for 24 h. Expression of fibroblast activation markers, α-SMA and collagen IA1, were assessed by qPCR and microarray gene expression analysis was performed using RNA harvested from the cells. α-SMA and phospho-SMAD3 protein expression was studied using immunofluorescence and western blot. Results: Media flow altered gene expression in oral and skin fibroblasts, increasing expression of α-SMA by 5.2 fold (NHDF) and 2.6 fold (NOF) and collagen IA by 2.9 fold (NHDF) and 3.2 fold (NOF) when compared to cells in static culture. TGF-β1 stimulation in the presence of flow decreased expression of these markers, but increased them in static culture. In dermal fibroblasts, flow stimulated α-SMA protein expression and translocation of phosphorylated SMAD3 to the cell nucleus and enhanced the expression of caveolin-1 alpha, which regulates the TGF-β1 pathway. Dermal fibroblasts exposed to flow revealed significant variations in expression of 16348 genes that may regulate the TGF-β1 pathway. Conclusion: Flow activates fibroblasts to a similar extent as TGF-β1 stimulated static cultures. Concurrent TGF-β1 treatment with flow, however, does not cause fibroblast activation. It is possible that TGF-β1 stimulation of fibroblasts under flow leads to caveolin-mediated endocytosis of the receptor or negative regulation of SMAD intracellular signaling pathways resulting in decreased expression of activation markers. Regardless of their origin (oral or dermal), fibroblasts respond to even modest flow conditions. Our findings suggest caution should be used when extrapolating findings from static models to in vivo conditions. It further prompts exploration of novel anti-tumor strategies that alter the physical properties of the tumor microenvironment. Citation Format: Sadhvi Nithiananthan, Aileen Crawford, Daniel W. Lambert, Simon Whawell. Modeling interstitial fluid movement in the tumor microenvironment: A role in fibroblast activation? [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B35.

A novel squeeze pressure bioreactor for noncontact hydrodynamic stimulation of cartilage is described. The bioreactor is based on a small piston that moves up and down, perpendicular to a tissue construct, in a fluid-filled chamber. Fluid displaced by the piston generates a pressure wave and shear stress as it moves across the sample, simulating the dynamic environment of a mobile joint. The fluid dynamics inside the squeeze pressure bioreactor was modeled using analytical and computational methods to simulate the mechanical stimuli imposed on a construct. In particular, the pressure, velocity field, and wall shear stress generated on the surface of the construct were analyzed using the theory of hydrodynamic lubrication, which describes the flow of an incompressible fluid between two surfaces in relative motion. Both the models and in-situ pressure measurements in the bioreactor demonstrate that controlled cyclic stresses of up to 10 kPa can be applied to tissue constructs. Initial tests on three-dimensional scaffolds seeded with chondrocytes show that glycosaminoglycan production is increased with regard to controls after 24 and 48 h of cyclic noncontact stimulation in the bioreactor.

Novel chitosan/polybutylene succinate fibre-based scaffolds (C-PBS) were seeded with bovine articular chondrocytes in order to assess their suitability for cartilage tissue engineering. Chondrocytes were seeded onto C-PBS scaffolds using spinner flasks under dynamic conditions, and cultured under orbital rotation for a total of 6 weeks. Non-woven polyglycolic acid (PGA) felts were used as reference materials. Tissue-engineered constructs were characterized by scanning electron microscopy (SEM), hematoxylin-eosin (H&E), toluidine blue and alcian blue staining, immunolocalization of collagen types I and II, and dimethylmethylene blue (DMB) assay for glycosaminoglycans (GAG) quantification at different time points. SEM showed the chondrocytes' typical morphology, with colonization at the surface and within the pores of the C-PBS scaffolds. These observations were supported by routine histology. Toluidine blue and alcian blue stains, as well as immunohistochemistry for collagen types I and II, provided qualitative information on the composition of the engineered extracellular matrix. More pronounced staining was observed for collagen type II than collagen type I. Similar results were observed with constructs engineered on PGA scaffolds. These also exhibited higher amounts of matrix glycosaminoglycans and presented a central region which contained fewer cells and little matrix, a feature that was not detected with C-PBS constructs., J. T. O. would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for his grant (SFRH/BD17135/2004). The authors would like to thank Dr. Chris Hill for his help with the SEM analysis. This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283).

Aggrecan is one of the two major constituents of articular cartilage, and during diseases such as osteoarthritis (OA) it is subject to degradation by proteolytic enzymes. The primary proteases responsible for aggrecan cleavage are the aggrecanases, identified as members of the ADAMTS family of proteases, which are upregulated in response to inflammatory stimuli. It is uncertain which of the six aggrecanases (ADAMTS-1, -4, -5, -8, -9 and -15) are primarily responsible for the degradation of aggrecan in human cartilage. Here we show that four of the six aggrecanases are expressed in immortalized chondrocyte cell-lines and can be upregulated in response to inflammatory cytokines. Using RNA interference, we demonstrate robust knock-down of ADAMTS-5 and -9 expression in these cells and, by culturing them on three-dimensional (3D) scaffolds, show that reduction in expression of ADAMTS-5 enzyme results in an increase in matrix deposition. These data suggest that the quality of tissue-engineered cartilage matrix might be improved by targeted depletion of aggrecanase expression. Moreover, this work also provides further evidence that ADAMTS-5 may be a therapeutic target in the treatment of arthritic disease.

The use of isolated cells to construct engineered tissues provides the opportunity to genetically modify those cells prior to the formation of tissue. This should make it possible to create transgenic human model tissues that can be used to determine gene function as well as to identify or validate potential therapeutic targets. As proof of principle, we have used RNA interference to selectively suppress the expression of aggrecanase genes in human chondrocytes, in an attempt to determine which of these key enzymes have roles in arthritic cartilage destruction. This combination of gene targeting and tissue engineering we are using should be equally applicable to the identification of gene function in other biological systems.

The use of isolated cells to construct engineered tissues provides the opportunity to genetically modify those cells prior to the formation of tissue. This should make it possible to create transgenic human model tissues that can be used to determine gene function as well as to identify or validate potential therapeutic targets. As proof of principle, we have used RNA interference to selectively suppress the expression of aggrecanase genes in human chondrocytes, in an attempt to determine which of these key enzymes have roles in arthritic cartilage destruction. This combination of gene targeting and tissue engineering we are using should be equally applicable to the identification of gene function in other biological systems.

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds’ porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold., M.L. Alves da Silva would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her grant (SFRH/BD/28708/2006), Marie Curie Actions-ALEA JACTA EST (MEST-CT-2004-008104), European NoE EXPERTISSUES (NMP3-CT-2004-500283), IP GENOSTEM (LSHB-CT-2003-503161) and CARTISCAFF (POCTI/SAUIBMA/58982)

Silks have been proposed as potential scaffold materials for tissue engineering, mainly because of their physical properties. They are stable at physiological temperatures, flexible and resist tensile and compressive forces. Bombyx mori (silkworm) cocoon silk has been used as a suture material for over a century, and has proved to be biocompatible once the immunogenic sericin coating is removed. Spider silks have a similar structure to silkworm silk but do not have a sericin coating. This paper provides a general overview on the use of silk protein in biomaterials, with a focus on skeletal tissue engineering. Copyright © 2008 John Wiley & Sons, Ltd.

Introduction The morphology of a substrate on which cells are seeded is known to affect cells in multiple ways including morphology, proliferation differentiation and matrix production. However, it is difficult to characterise the effects of mechanical loading on cells seeded in scaffolds of different morphology but identical chemistry. The aim of this project is to create polyurethane foam and fibre scaffolds from the same linear polyurethane and to analyse the effect of scaffold morphology on mechanical properties, cell viability, and cell morphology. The current work aims to produce scaffolds of sufficient mechanical strength for mechanical stimulation experiments. Materials and Methods Foams were fabricated from a polypropylene oxide polyol and a toluene diisocyanate (TDI) by the direct foaming technique. Fibres were fabricated by dissolving the foam overnight in a 20wt% solution of dimethylacetamide under stirring conditions. The fibres were spun using an electrospinning rig with a 15kV electric field, 30cm working distance, flow rate of 3.5ml/hr and collected on a rotating drum. MC-3T3-E1 osteoblastic cells were seeded onto 2cm diameter, thin discs of each scaffold type at a density of 100 x 10 cells per sample. The seeded scaffolds were then left for 1hr to allow cell attachment before immersion in 3ml of media and culture for 21 days with a media change every 3 days. The distribution of viable cells was analysed by an assay of metabolic activity (MTS). Results Both foam and fibre morphologies are suitable for support of cell growth over medium term culture periods (21 days). Cell viability analysis showed no significant difference between the two morphologies. Microscopy analysis indicated cell attachment and survival within the pores of the foam scaffold with cells adhering to the struts. Both scaffolds can be cut into disk or dumbbell shapes suitable for mechanical stimulation in compression or tension respectively.

Together, the chondrocyte and its pericellular matrix have been collectively termed the chondron. Current opinion is that the pericellular matrix has both protective and signalling functions between chondrocyte and extracellular matrix. Formation of a native chondrocyte pericellular matrix or chondron structure might therefore be advantageous when tissue engineering a functional hyaline cartilage construct. The presence of chondrons has not been previously described in cartilage engineered on a scaffold. In this paper, we describe a modified immunochemical method to detect collagen VI, a key molecular marker for the pericellular matrix, and an investigation of type VI collagen distribution in engineered hyaline cartilage constructs. Cartilage constructs were engineered from adult human or bovine hyaline chondrocytes cultured on sponge or nonwoven fiber based HYAFF 11 scaffolds. Type VI collagen was detected in all constructs, but a distinctive, high-density, chondron-like distribution of collagen VI was present only in constructs exhibiting additional features of hyaline cartilage engineered using nonwoven HYAFF 11. Chondron structures were localized in areas of the extracellular matrix displaying strong collagen II and GAG staining of constructs where type II collagen composed a high percentage (over 65%) of the total collagen.

Adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] was shown to be present in cultured human articular chondrocytes. Optimal conditions of incubation time, protein and substrate concentrations and pH were determined in whole cell lysates. Maximal activity occurred at pH 8.5 with no decrease in activity up to pH 10.0. Adenylate cyclase activity of particulate membrane preparations was enhanced by the addition of crude cytosol preparations. The prostaglandins E1, E2, F1 alpha, F2 alpha, D2, B1, B2, A1 and A2, as well as adrenaline and isoprenaline, stimulated adenylate cyclase derived from either adult or foetal chondrocytes. No significant stimulation was observed in the presence of human calcitonin or glucagon. Bovine parathyroid hormone always significantly stimulated the adenylate cyclase derived from foetal chondrocytes, but not from adult chondrocytes. Preincubation of the chondrocytes in culture with indomethacin and with or without supernatant medium from cultured mononuclear cells increased the responsiveness of the adenylate cyclase to prostaglandin E1.

1. The addition of 50 000g cytosol preparations of isolated human platelets, cultured rat osteogenic sarcoma or cultured bone cells to particulate preparations of adenylate cyclase, from the same or unrelated tissues, caused marked enhancement of the hormone-stimulated enzyme activities. 2. The degree of enhancement obtained by addition of the cytosol preparations was similar to that observed on addition of GTP. 3. The enhancing activity of the three cytosol types was found to be sensitive to digestion by trypsin and alkaline phosphatase, partially heat-labile and partially inactivated by exposure to charcoal. 4. Gel filtration studies indicated an apparent molecular weight of 20 000–30 000. Further, the 20000-30000-mol.wt. fractions obtained by gel filtration could enhance the adenylate cyclase activity of particulate preparations derived from unrelated cell types. 5. The results suggest a common or similar adenylate-cyclase-enhancing factor or factors, protein in nature, present in the three cytosol types.

Interleukin-1 (IL-1) is the name given to a family of related proteins showing a variety of activities. It was originally shown to be produced by monocytes and macrophages but is now known to be produced by numerous cell types, including synovial cells. From the point of view of arthritis, its most interesting activities are those on connective tissue cells in vitro. These include stimulation of production of prostaglandins, plasminogen activator and metalloproteinases such as collagenase and proteoglycanase. IL-1 is also mitogenic for synoviocytes and bone cells, and can alter rates of production of extracellular matrix constituents. The presence of IL-1 in synovial fluids from rheumatoid and osteoarthritic joints and its actions on connective tissues in vitro suggest that IL-1 may play an important role in the pathogenesis of arthritis. There are several potential cellular sources of IL-1 in the inflamed rheumatoid joint and interactions between these cells, T lymphocytes and plasma cells may continually induce IL-1 so contributing to the chronicity of the disease. The mechanism of action of IL-1 on connective tissue cells is at present uncertain though preliminary studies suggest that IL-1 may induce cellular responses by stimulating phosphoinositide turnover and possibly protein kinase C activity.

1. Homogenates of whole tissues were shown to contain both intracellular and extracellular factors that affected particulate adenylate cyclase activity in vitro. Factors present in the extracellular fluids produced an inhibition of basal, hormone- and fluoride-stimulated enzyme activity but factors present in the cell cytosol increased hormone-stimulated activity with relatively little effect on basal or fluoride-stimulated enzyme activity. 2. The existence of this cytosol factor or factors was investigated using freshly isolated human platelets, freshly isolated rat hepatocytes, and cultured cells derived from rat osteogenic sarcoma, rat calvaria, mouse melanoma, pig aortic endothelium, human articular cartilage chondrocytes and human bronchial carcinoma (BEN) cells. 3. The stimulation of the hormone response by the cytosol factor ranged from 60 to 890% depending on the tissue of origin of the adenylate cyclase. 4. In each case the behaviour of the factor was similar to the action of GTP on that particular adenylate cyclase preparation. 5. No evidence of tissue or species specificity was found, as cytosols stimulated adenylate cyclase from their own and unrelated tissues to the same degree. 6. In the human platelet, the inclusion of the cytosol in the assay of adenylate cyclase increased the rate of enzyme activity in response to stimulation by prostaglandin E1 without affecting the amount of prostaglandin E1 required for half-maximal stimulation or the characteristics of enzyme activation by prostaglandin E.

The effects of prostaglandins (PG's) E 1 , E 2 , I 2 (prostacyclin), 6-keto F 1α and thromboxane (Tx) B 2 were compared in freshly isolated cells from a rat osteogenic sarcoma and in membranes from cultured cells of the same tumour. Cyclic AMP production was measured in cells and adenylate cyclase activity was measured in cell membranes. In both systems PGI 2 was less potent than either of the PGE's, and both TxB 2 and 6-keto PGF 1α were only weak agonists. These effects on bone-derived cells suggest that PGI 2 is unlikely to be a potent bone resorbing agent. Resitance experiments suggested that all the PG's share the same receptor site which appears distinct from the site of action of parathyroid hormone.