Thermoresponsive Poly(N,N'-dimethylacrylamide)-Based Diblock Copolymer Worm Gels via RAFT Solution Polymerization: Synthesis, Characterization, and Cell Biology Applications.

RAFT solution polymerization is used to polymerize 2-hydroxypropyl methacrylate (HPMA). The resulting PHPMA precursor is then chain-extended using N,N'-dimethylacrylamide (DMAC) to produce a series of thermoresponsive PHPMA-PDMAC diblock copolymers. Such amphiphilic copolymers can be directly dispersed in ice-cold water and self-assembled at 20 °C to form spheres, worms, or vesicles depending on their copolymer composition. Construction of a pseudo-phase diagram is required to identify the pure worm phase, which corresponds to a rather narrow range of PDMAC DPs. Such worms form soft, free-standing gels in aqueous solution at around ambient temperature. Rheology studies confirm the thermoresponsive nature of such worms, which undergo a reversible worm-to-sphere on cooling below ambient temperature. This morphological transition leads to in situ degelation, and variable temperature 1H NMR studies indicate a higher degree of (partial) hydration for the weakly hydrophobic PHPMA chains at lower temperatures. The trithiocarbonate end-group located at the end of each PDMAC chain can be removed by treatment with excess hydrazine. The resulting terminal secondary thiol group can form disulfide bonds via coupling, which produces PHPMA-PDMAC-PHPMA triblock copolymer chains. Alternatively, this reactive thiol group can be used for conjugation reactions. A PHPMA141-PDMAC36 worm gel was used to store human mesenchymal stem cells (MSCs) for up to three weeks at 37 °C. MSCs retrieved from this gel subsequently underwent proliferation and maintained their ability to differentiate into osteoblastic cells.

Elastomeric Porous Poly(glycerol sebacate) Methacrylate (PGSm) Microspheres as 3D Scaffolds for Chondrocyte Culture and Cartilage Tissue Engineering.

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.

It's time for a minimum synoptic operation template in patients undergoing laparoscopic cholecystectomy: a systematic review.

BACKGROUND: Despite the call to enhance accuracy and value of operation records few international recommended minimal standards for operative notes documentation have been described. This study undertook a systematic review of existing operative reporting systems for laparoscopic cholecystectomy (LC) to fashion a comprehensive, synoptic operative reporting template for the future. METHODS: A search for all relevant articles was conducted using PubMed version of Medline, Scopus and Web of Science databases in June 2021, for publications from January 1st 2011 to October 25th 2021, using the keywords: laparoscopic cholecystectomy AND operation notes OR operative notes OR proforma OR documentation OR report OR narrative OR audio-visual OR synoptic OR digital. Two reviewers (NOC, GMC) independently assessed each published study using a MINORS score of ≥ 16 for comparative and ≥ 10 for non-comparative for inclusion. This systematic review followed PRISMA guidelines and was registered with PROSPERO. Synoptic operative templates from published data were assimilated into one "ideal" laparoscopic operative report template following international input from the World Society of Emergency Surgery board. RESULTS: A total of 3567 articles were reviewed. Following MINORS grading 25 studies were selected spanning 14 countries and 4 continents. Twenty-two studies were prospective. A holistic overview of the operative procedure documentation was reported in 6/25 studies and a further 19 papers dealt with selective surgical aspects of LC. A unique synoptic LC operative reporting template was developed and translated into Chinese/Mandarin, French and Arabic. CONCLUSION: This systematic review identified a paucity of publications dealing with operative reporting of LC. The proposed new template may be integrated digitally with hospitals' medical systems and include additional narrative text and audio-visual data. The template may help define new OR (operating room) recording standards and impact on care for patients undergoing LC.

Stimulation of Metabolic Activity and Cell Differentiation in Osteoblastic and Human Mesenchymal Stem Cells by a Nanohydroxyapatite Paste Bone Graft Substitute.

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.

* Substituted Borosilicate Glasses with Improved Osteogenic Capacity for Bone Tissue Engineering.

Borosilicate bioactive glasses (BBGs) have shown the capacity to promote higher formation of new bone when compared with silicate bioactive glasses. Herein, we assessed the capacity of BBGs to induce osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) as a function of their substituted divalent cations (Mg2+, Ca2+, Sr2+). To this purpose, we synthesized BBG particles by melt quenching. The cell viability, proliferation, and morphology (i.e., PrestoBlue®, PicoGreen®, and DAPI and Phalloidin stainings, respectively), as well as protein expression (i.e., alkaline phosphatase, ALP; osteopontin, OP; and osteocalcin, OC), of BM-MSCs in contact with BBGs were evaluated for 21 days. We observed an enhanced expression of bone-specific proteins (ALP, OP, and OC) and high mineralization of BM-MSCs under BBG-Mg and BBG-Sr-conditioned osteogenic media for concentrations of 20 and 50 mg/mL with low cytotoxic effects. Moreover, BBG-Sr, at a concentration of 50 mg/mL, was able to increase the mineralization and expression of the same bone-specific proteins even under basal medium conditions. These results indicated that the proposed BBGs improved osteogenic differentiation of BM-MSCs, therefore showing their potential as relevant biomaterials for bone tissue regeneration, not only by bonding to bone tissue but also by stimulating new bone formation.

Physiological Fluid Flow Moderates Fibroblast Responses to TGF-ß1.

Fibroblasts are the major cellular component of connective tissue and experience mechanical perturbations due to matrix remodelling and interstitial fluid movement. Transforming growth factor ß1 (TGF-ß1) 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-ß1. 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 TGFß signalling and endocytosis. TGF-ß1, 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-ß1 resulted in reduced differentiation. Our findings suggest this may result from enhanced internalisation of caveolin and TGF-ß 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-ß1. 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.

Repair of bone defects in vivo using tissue engineered hypertrophic cartilage grafts produced from nasal chondrocytes.

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. 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. 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.

Reinforcement of poly-l-lactic acid electrospun membranes with strontium borosilicate bioactive glasses for bone tissue engineering.

UNLABELLED: Herein, for the first time, we combined poly-l-lactic acid (PLLA) with a strontium borosilicate bioactive glass (BBG-Sr) using electrospinning to fabricate a composite bioactive PLLA membrane loaded with 10% (w/w) of BBG-Sr glass particles (PLLA-BBG-Sr). The composites were characterised by scanning electron microscopy (SEM) and microcomputer tomography (µ-CT), and the results showed that we successfully fabricated smooth and uniform fibres (1-3µm in width) with a homogeneous distribution of BBG-Sr microparticles (<45µm). Degradation studies (in phosphate buffered saline) demonstrated that the incorporation of BBG-Sr glass particles into the PLLA membranes increased their degradability and water uptake with a continuous release of cations. The addition of BBG-Sr glass particles enhanced the membrane's mechanical properties (69% higher Young modulus and 36% higher tensile strength). Furthermore, cellular in vitro evaluation using bone marrow-derived mesenchymal stem cells (BM-MSCs) demonstrated that PLLA-BBG-Sr membranes promoted the osteogenic differentiation of the cells as demonstrated by increased alkaline phosphatase activity and up-regulated osteogenic gene expression (Alpl, Sp7 and Bglap) in relation to PLLA alone. These results strongly suggest that the composite PLLA membranes reinforced with the BBG-Sr glass particles have potential as an effective biomaterial capable of promoting bone regeneration. STATEMENT OF SIGNIFICANCE: PLLA membranes were reinforced with 10% (w/w) of strontium-bioactive borosilicate glass microparticles, and their capacity to induce the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) was evaluated. These membranes presented an increased: degradability, water uptake, Young modulus and tensile strength. We also demonstrated that these membranes are non-cytotoxic and promote the attachment of BM-MSCs. The addition of the glass microparticles into the PLLA membranes promoted the increase of ALP activity (under osteogenic conditions), as well as the BM-MSCs osteogenic differentiation as shown by the upregulation of Alpl, Sp7 and Bglap gene expression. Overall, we demonstrated that the reinforcement of PLLA with glass microparticles results in a biomaterial with the appropriate properties for the regeneration of bone tissue.

Expression of Cannabinoid Receptors in Human Osteoarthritic Cartilage: Implications for Future Therapies.

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

Fabrication of drug-loaded anti-infective guided tissue regeneration membrane with adjustable biodegradation property.

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 osteogenic response of mesenchymal stromal cells to strontium-substituted bioactive glasses.

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.

Preparation and in vivo efficient anti-infection property of GTR/GBR implant made by metronidazole loaded electrospun polycaprolactone nanofiber membrane.

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.

Drug loaded homogeneous electrospun PCL/gelatin hybrid nanofiber structures for anti-infective tissue regeneration membranes.

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(ε-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.

Fabrication and evaluation of electrospun PCL-gelatin micro-/nanofiber membranes for anti-infective GTR implants.

Infection is the major reason for GTR/GBR membrane failure in clinical applications. In this work, we developed GTR/GBR membranes with localized drug delivery function to prevent infection. Hierarchical membranes containing micro- and nano-fibers were fabricated. The effects of the incorporation of gelatin and loading content of metronidazole (MNA) (0, 5, 10, 20, 30, and 40 wt% polymer) on the properties of the electrospun membranes were investigated. The interaction between PCL and MNA was identified by molecular dynamics simulation. MNA was released in a controlled manner, and the antibacterial activity of the released MNA remained. The incorporation of gelatin and MNA improved the hydrophilicity, biocompatibility, and in vitro biodegradation rate of PCL nanofibers. The electrospun 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. Subcutaneous implantation results demonstrated that MNA-loaded membranes evoked a less severe inflammatory response than pure PCL nanofibers. These results demonstrated the potential of MNA-loaded membranes as GTR/GBR membranes with antibacterial and anti-inflammatory functions for biomedical applications.

Cannabinoids: novel therapies for arthritis?

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 E(2), 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.

Squeeze pressure bioreactor: a hydrodynamic bioreactor for noncontact stimulation of cartilage constructs.

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 melt-processable chitosan-polybutylene succinate fibre scaffolds for cartilage tissue engineering.

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.

Genetic modification of chondrocytes using viral vectors.

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.

Lentiviral shRNA knock-down of ADAMTS-5 and -9 restores matrix deposition in 3D chondrocyte culture.

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.