Infrapatellar fat pad adipose-derived stem cells co-cultured with articular chondrocytes from osteoarthritis patients exhibit increased chondrogenic gene expression.

AIM: The variable results in clinical trials of adipose tissue-derived stem cells (ASCs) for chondral defects may be due to the different ex vivo culture conditions of the ASCs which are implanted to treat the lesions. We sought to determine the optimal in vitro chondrocyte co-culture condition that promotes infrapatellar fat pad-derived (IFPD) ASC chondrogenic gene expression in a novel co-culture combination. METHODS: In our study, we utilized an in vitro autologous co-culture of IFPD ASCs and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic human knee joints at ASC-to-chondrocyte seeding log ratios of 1:1, 10:1, and 100:1. Gene expression following in vitro co-culture was quantified by RT-qPCR with a panel comprising COL1A1, COL2A1, COL10A1, L-SOX5, SOX6, SOX9, ACAN, HSPG2, and COMP for chondrogenic gene expression. RESULTS: The chondrogenic gene expression profiles from co-cultures were greater than would be expected from an expression profile modeled from chondrocyte and ASC-only monocultures. Additionally, chondrogenic gene expression decreased with increasing ASC-to-chondrocyte seeding ratios. CONCLUSIONS: These findings provide insight into the mechanisms underlying clinical ASC therapies and signifies that IFPD ASCs pre-conditioned by chondrocyte co-culture may have improved chondrogenic potential for cartilage repair. This model can help further understand IFPD ASCs in chondral and osteochondral repair and the chondrogenic pathways involved. Video Abstract.

Human osteoblasts obtained from distinct periarticular sites demonstrate differences in biological function in vitro.

AIMS: Accumulated evidence indicates that local cell origins may ingrain differences in the phenotypic activity of human osteoblasts. We hypothesized that these differences may also exist in osteoblasts harvested from the same bone type at periarticular sites, including those adjacent to the fixation sites for total joint implant components. METHODS: Human osteoblasts were obtained from the acetabulum and femoral neck of seven patients undergoing total hip arthroplasty (THA) and from the femoral and tibial cuts of six patients undergoing total knee arthroplasty (TKA). Osteoblasts were extracted from the usually discarded bone via enzyme digestion, characterized by flow cytometry, and cultured to passage three before measurement of metabolic activity, collagen production, alkaline phosphatase (ALP) expression, and mineralization. RESULTS: Osteoblasts from the acetabulum showed lower proliferation (p = 0.034), cumulative collagen release (p < 0.001), and ALP expression (p = 0.009), and produced less mineral (p = 0.006) than those from the femoral neck. Osteoblasts from the tibia produced significantly less collagen (p = 0.021) and showed lower ALP expression than those from the distal femur. CONCLUSION: We have demonstrated for the first time an anatomical regional variation in the biological behaviours of osteoblasts on either side of the hip and knee joint. The lower osteoblast proliferation, matrix production, and mineralization from the acetabulum compared to those from the proximal femur may be reflected in differences in bone formation and implant fixation at these sites. Cite this article: Bone Joint Res 2021;10(9):611-618.

Linking chondrocyte and synovial transcriptional profile to clinical phenotype in osteoarthritis.

OBJECTIVES: To determine how gene expression profiles in osteoarthritis joint tissues relate to patient phenotypes and whether molecular subtypes can be reproducibly captured by a molecular classification algorithm. METHODS: We analysed RNA sequencing data from cartilage and synovium in 113 osteoarthritis patients, applying unsupervised clustering and Multi-Omics Factor Analysis to characterise transcriptional profiles. We tested the association of the molecularly defined patient subgroups with clinical characteristics from electronic health records. RESULTS: We detected two patient subgroups in low-grade cartilage (showing no/minimal degeneration, cartilage normal/softening only), with differences associated with inflammation, extracellular matrix-related and cell adhesion pathways. The high-inflammation subgroup was associated with female sex (OR 4.12, p=0.0024) and prescription of proton pump inhibitors (OR 4.21, p=0.0040). We identified two independent patient subgroupings in osteoarthritis synovium: one related to inflammation and the other to extracellular matrix and cell adhesion processes. A seven-gene classifier including MMP13, APOD, MMP2, MMP1, CYTL1, IL6 and C15orf48 recapitulated the main axis of molecular heterogeneity in low-grade knee osteoarthritis cartilage (correlation ρ=-0.88, p<10-10) and was reproducible in an independent patient cohort (ρ=-0.85, p<10-10). CONCLUSIONS: These data support the reproducible stratification of osteoarthritis patients by molecular subtype and the exploration of new avenues for tailored treatments.

A molecular quantitative trait locus map for osteoarthritis.

Osteoarthritis causes pain and functional disability for over 500 million people worldwide. To develop disease-stratifying tools and modifying therapies, we need a better understanding of the molecular basis of the disease in relevant tissue and cell types. Here, we study primary cartilage and synovium from 115 patients with osteoarthritis to construct a deep molecular signature map of the disease. By integrating genetics with transcriptomics and proteomics, we discover molecular trait loci in each tissue type and omics level, identify likely effector genes for osteoarthritis-associated genetic signals and highlight high-value targets for drug development and repurposing. These findings provide insights into disease aetiopathology, and offer translational opportunities in response to the global clinical challenge of osteoarthritis.

Stimulation of Human Osteoblast Differentiation in Magneto-Mechanically Actuated Ferromagnetic Fiber Networks.

There is currently an interest in "active" implantable biomedical devices that include mechanical stimulation as an integral part of their design. This paper reports the experimental use of a porous scaffold made of interconnected networks of slender ferromagnetic fibers that can be actuated in vivo by an external magnetic field applying strains to in-growing cells. Such scaffolds have been previously characterized in terms of their mechanical and cellular responses. In this study, it is shown that the shape changes induced in the scaffolds can be used to promote osteogenesis in vitro. In particular, immunofluorescence, gene and protein analyses reveal that the actuated networks exhibit higher mineralization and extracellular matrix production, and express higher levels of osteocalcin, alkaline phosphatase, collagen type 1α1, runt-related transcription factor 2 and bone morphogenetic protein 2 than the static controls at the 3-week time point. The results suggest that the cells filling the inter-fiber spaces are able to sense and react to the magneto-mechanically induced strains facilitating osteogenic differentiation and maturation. This work provides evidence in support of using this approach to stimulate bone ingrowth around a device implanted in bone and can pave the way for further applications in bone tissue engineering.

Poly(ADP-Ribose) Links the DNA Damage Response and Biomineralization.

Biomineralization of the extracellular matrix is an essential, regulated process. Inappropriate mineralization of bone and the vasculature has devastating effects on patient health, yet an integrated understanding of the chemical and cell biological processes that lead to mineral nucleation remains elusive. Here, we report that biomineralization of bone and the vasculature is associated with extracellular poly(ADP-ribose) synthesized by poly(ADP-ribose) polymerases in response to oxidative and/or DNA damage. We use ultrastructural methods to show poly(ADP-ribose) can form both calcified spherical particles, reminiscent of those found in vascular calcification, and biomimetically calcified collagen fibrils similar to bone. Importantly, inhibition of poly(ADP-ribose) biosynthesis in vitro and in vivo inhibits biomineralization, suggesting a therapeutic route for the treatment of vascular calcifications. We conclude that poly(ADP-ribose) plays a central chemical role in both pathological and physiological extracellular matrix calcification.

Widespread epigenomic, transcriptomic and proteomic differences between hip osteophytic and articular chondrocytes in osteoarthritis.

Objectives: To identify molecular differences between chondrocytes from osteophytic and articular cartilage tissue from OA patients. Methods: We investigated genes and pathways by combining genome-wide DNA methylation, RNA sequencing and quantitative proteomics in isolated primary chondrocytes from the cartilaginous layer of osteophytes and matched areas of low- and high-grade articular cartilage across nine patients with OA undergoing hip replacement surgery. Results: Chondrocytes from osteophytic cartilage showed widespread differences to low-grade articular cartilage chondrocytes. These differences were similar to, but more pronounced than, differences between chondrocytes from osteophytic and high-grade articular cartilage, and more pronounced than differences between high- and low-grade articular cartilage. We identified 56 genes with significant differences between osteophytic chondrocytes and low-grade articular cartilage chondrocytes on all three omics levels. Several of these genes have known roles in OA, including ALDH1A2 and cartilage oligomeric matrix protein, which have functional genetic variants associated with OA from genome-wide association studies. An integrative gene ontology enrichment analysis showed that differences between osteophytic and low-grade articular cartilage chondrocytes are associated with extracellular matrix organization, skeletal system development, platelet aggregation and regulation of ERK1 and ERK2 cascade. Conclusion: We present a first comprehensive view of the molecular landscape of chondrocytes from osteophytic cartilage as compared with articular cartilage chondrocytes from the same joints in OA. We found robust changes at genes relevant to chondrocyte function, providing insight into biological processes involved in osteophyte development and thus OA progression.

Correction to: The effect of cationically-modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo.

The article "The effect of cationically modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo", written by Jonathan M. Lawton, Mariam Habib, Bingkui Ma, Roger A. Brooks, Serena M. Best, Andrew L. Lewis, Neil Rushton and William Bonfield, was originally published Online First without open access. After publication in volume 28, issue 9, page 144 it was noticed that the copyright was wrong in the PDF version of the article. The copyright of the article should read as "

Evaluation of shared genetic aetiology between osteoarthritis and bone mineral density identifies SMAD3 as a novel osteoarthritis risk locus.

Osteoarthritis (OA) is a common complex disease with high public health burden and no curative therapy. High bone mineral density (BMD) is associated with an increased risk of developing OA, suggesting a shared underlying biology. Here, we performed the first systematic overlap analysis of OA and BMD on a genome wide scale. We used summary statistics from the GEFOS consortium for lumbar spine (n = 31,800) and femoral neck (n = 32,961) BMD, and from the arcOGEN consortium for three OA phenotypes (hip, ncases=3,498; knee, ncases=3,266; hip and/or knee, ncases=7,410; ncontrols=11,009). Performing LD score regression we found a significant genetic correlation between the combined OA phenotype (hip and/or knee) and lumbar spine BMD (rg=0.18, P = 2.23 × 10-2), which may be driven by the presence of spinal osteophytes. We identified 143 variants with evidence for cross-phenotype association which we took forward for replication in independent large-scale OA datasets, and subsequent meta-analysis with arcOGEN for a total sample size of up to 23,425 cases and 236,814 controls. We found robustly replicating evidence for association with OA at rs12901071 (OR 1.08 95% CI 1.05-1.11, Pmeta=3.12 × 10-10), an intronic variant in the SMAD3 gene, which is known to play a role in bone remodeling and cartilage maintenance. We were able to confirm expression of SMAD3 in intact and degraded cartilage of the knee and hip. Our findings provide the first systematic evaluation of pleiotropy between OA and BMD, highlight genes with biological relevance to both traits, and establish a robust new OA genetic risk locus at SMAD3.

The effect of cationically-modified phosphorylcholine polymers on human osteoblasts in vitro and their effect on bone formation in vivo.

The effect of introducing cationic charge into phosphorylcholine (PC)-based polymers has been investigated in this study with a view to using these materials as coatings to improve bone formation and osseointegration at the bone-implant interface. PC-based polymers, which have been used in a variety of medical devices to improve biocompatibility, are associated with low protein adsorption resulting in reduced complement activation, inflammatory response and cell adhesion. However, in some applications, such as orthopaedics, good integration between the implant and bone is needed to allow the distribution of loading stresses and a bioactive response is required. It has previously been shown that the incorporation of cationic charge into PC-based polymers may increase protein adsorption that stimulates subsequent cell adhesion. In this paper, the effect of cationic charge in PC-based polymers on human osteoblasts (HObs) in vitro and the effect of these polymers on bone formation in the rat tibia was assessed. Increasing PC positive surface charge increased HOb cell adhesion and stimulated increased cell differentiation and the production of calcium phosphate deposits. However, when implanted in bone these materials were at best biotolerant, stimulating the production of fibrous tissue and areas of loosely associated matrix (LAM) around the implant. Their development, as formulated in this study, as bone interfacing implant coatings is therefore not warranted.

Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two -omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three -omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.

Effect of Rotation on Scaffold Motion and Cell Growth in Rotating Bioreactors.

Efficient use of different bioreactor designs to improve cell growth in three-dimensional scaffolds requires an understanding of their mechanism of action. To address this for rotating wall vessel bioreactors, fluid and scaffold motion were investigated experimentally at different rotation speeds and vessel fill volumes. Low cost bioreactors with single and dual axis rotation were developed to investigate the effect of these systems on human osteoblast proliferation in free floating and constrained collagen-glycosaminoglycan porous scaffolds. A range of scaffold motions (free fall, periodic oscillation, and orbital motion) were observed at the rotation speeds and vessel fluid/air ratios used, with 85% fluid fill and an outer vessel wall velocity of ∼14 mm s-1 producing a scaffold in a free fall state. The cell proliferation results showed that after 14 and 21 days of culture, this combination of fluid fill and speed of rotation produced significantly greater cell numbers in the scaffolds than when lower or higher rotation speeds (p < 0.002) or when the chamber was 60% or 100% full (p < 0.01). The fluid flow and scaffold motion experiments show that biaxial rotation would not improve the mass transfer of medium into the scaffold as the second axis of rotation can only transition the scaffold toward oscillatory or orbital motion and, hence, reduce mass transport to the scaffold. The cell culture results confirmed that there was no benefit to the second axis of rotation with no significant difference in cell proliferation either when the scaffolds were free floating or constrained (p > 0.05).

Multi-casting approach for vascular networks in cellularized hydrogels.

Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. We report a new method for producing a hierarchical, three-dimensional (3D) and perfusable vasculature in a large, cellularized fibrin hydrogel. Bifurcating channels, varying in size from 1 mm to 200-250 µm, are formed using a novel process in which we convert a 3D printed thermoplastic material into a gelatin network template, by way of an intermediate alginate hydrogel. This enables a CAD-based model design, which is highly customizable, reproducible, and which can yield highly complex architectures, to be made into a removable material, which can be used in cellular environments. Our approach yields constructs with a uniform and high density of cells in the bulk, made from bioactive collagen and fibrin hydrogels. Using standard cell staining and immuno-histochemistry techniques, we showed good cell seeding and the presence of tight junctions between channel endothelial cells, and high cell viability and cell spreading in the bulk hydrogel.

Solid state NMR of isotope labelled murine fur: a powerful tool to study atomic level keratin structure and treatment effects.

We have prepared mouse fur extensively 13C,15N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. 13C double quantum-single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and ß-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.

The effect of particle size on the in vivo degradation of poly(d,l-lactide-co-glycolide)/α-tricalcium phosphate micro- and nanocomposites.

This paper reports the effect of particle size within a resorbable composite on the in vivo degradation rate and host response. Resorbable composites based on poly(d,l-lactide-co-glycolide) (PLGA) reinforced with tricalcium phosphate (TCP) have shown suitable degradation, biological and mechanical properties for bone repair. Composites with nano-sized TCP particles degrade more homogenously in vitro than equivalent composites with micro-sized particles. In this study, PLGA and PLGA/TCP composites containing micro- or nano-sized α-TCP particles were implanted into an ovine distal femoral condyle defect and harvested at 6, 12, 18 and 24weeks. An intimate interface was observed between the new bone tissue and degrading implants. Visual scoring of histological images and semi-automated segmentation of X-ray images were used to quantify implant degradation and the growth of new bone tissue in the implant site. Bone growth into the implant site occurred at a similar rate for both composites and the PLGA control. However, the in vivo degradation rate of the nanocomposite was slower than that of the microcomposite and consequently more closely matched the rate of bone growth. For the first 6weeks, the rate of in vivo degradation matched that of in vitro degradation, but lagged significantly at longer time points. These results point to the potential use of ceramic particle size in controlling composite degradation whilst maintaining good bone formation. STATEMENT OF SIGNIFICANCE: This paper concerns degradable composites for orthopaedic application. The effect of particle size on implant degradation in vivo is not yet well characterised and these results give the first opportunity to directly compare in vitro and in vivo degradation rates for composites with micro- and nano-sized particles. This type of data is vital for the validation of models of composite degradation behaviour, which will lead to the design and manufacture of composites with a tailored, predictable degradation profile. The trainable segmentation tool can be used for future studies where X-rays of partially degraded implants (which have complicated greyscales and morphologies) need to be quantified without bias.

Adsorption of Amorphous Silica Nanoparticles onto Hydroxyapatite Surfaces Differentially Alters Surfaces Properties and Adhesion of Human Osteoblast Cells.

Silicon (Si) is suggested to be an important/essential nutrient for bone and connective tissue health. Silicon-substituted hydroxyapatite (Si-HA) has silicate ions incorporated into its lattice structure and was developed to improve attachment to bone and increase new bone formation. Here we investigated the direct adsorption of silicate species onto an HA coated surface as a cost effective method of incorporating silicon on to HA surfaces for improved implant osseointegration, and determined changes in surface characteristics and osteoblast cell adhesion. Plasma-sprayed HA-coated stainless steel discs were incubated in silica dispersions of different concentrations (0-42 mM Si), at neutral pH for 12 h. Adsorbed Si was confirmed by XPS analysis and quantified by ICP-OES analysis following release from the HA surface. Changes in surface characteristics were determined by AFM and measurement of surface wettability. Osteoblast cell adhesion was determined by vinculin plaque staining. Maximum Si adsorption to the HA coated disc occurred after incubation in the 6 mM silica dispersion and decreased progressively with higher silica concentrations, while no adsorption was observed with dispersions below 6 mM Si. Comparison of the Si dispersions that produced the highest and lowest Si adsorption to the HA surface, by TEM-based analysis, revealed an abundance of small amorphous nanosilica species (NSP) of ~1.5 nm in diameter in the 6 mM Si dispersion, with much fewer and larger NSP in the 42 mM Si dispersions. 29Si-NMR confirmed that the NSPs in the 6 mM silica dispersion were polymeric and similar in composition to the larger NSPs in the 42 mM Si dispersion, suggesting that the latter were aggregates of the former. Amorphous NSP adsorbed from the 6 mM dispersion on to a HA-coated disc surface increased the surface's water contact angle by 53°, whereas that adsorbed from the 42 mM dispersion decreased the contact angle by 18°, indicating increased and decreased hydrophobicity, respectively. AFM showed an increase in surface roughness of the 6 mM Si treated surface, which correlated well with an increase in number of vinculin plaques. These findings suggest that NSP of the right size (relative to charge) adsorb readily to the HA surface, changing the surface characteristics and, thus, improving osteoblast cell adhesion. This treatment provides a simple way to modify plasma-coated HA surfaces that may enable improved osseointegration of bone implants.

Preparation of highly and generally enriched mammalian tissues for solid state NMR.

An appreciable level of isotope labelling is essential for future NMR structure elucidation of mammalian biomaterials, which are either poorly expressed, or unexpressable, using micro-organisms. We present a detailed protocol for high level (13)C enrichment even in slow turnover murine biomaterials (fur keratin), using a customized diet supplemented with commercial labelled algal hydrolysate and formulated as a gel to minimize wastage, which female mice consumed during pregnancy and lactation. This procedure produced approximately eightfold higher fur keratin labelling in pups, exposed in utero and throughout life to label, than in adults exposed for the same period, showing both the effectiveness, and necessity, of this approach.

In vitro osteoclast formation and resorption of silicon-substituted hydroxyapatite ceramics.

Materials that participate in bone remodeling at the implant/tissue interface represent a modern tissue engineering approach with the aim of balancing implant resorption and nascent tissue formation. Silicon-substituted hydroxyapatite (SiHA) ceramics are capable of stimulating new bone formation, but little is known about their interaction with osteoclasts (OC). The effects of soluble silicate and SiHA on OCs were investigated in this study. Soluble silicate below 500 µM did not stimulate cell metabolism at 4 days or alter resorption area at 7 days on calcium phosphate discs. On sintered ceramics, OC numbers were similar on HA, Si0.3 HA (0.5 wt % Si) and Si0.5 HA (1.2 wt % Si) after 21 days in vitro, but actin ring sealing zone morphology on SiHA resembled that commonly found on bone or on carbonate-substituted hydroxyapatite (CHA). Smaller and thicker actin rings on SiHA as compared to HA were probably the result of altered surface chemistry and solubility differences. The more stable sealing zones and increased lattice solubility likely contributed to increased individual pit volumes observed on Si0.5 HA. The delayed formation of OCs on Si0.5 HA (lower numbers at day 14) excludes earlier differentiation as a possible mechanism of increased individual OC pit volumes at later times (day 21). Materials characterization of Si containing biomaterials remains paramount as the Si type and amounts can subsequently impact downstream OC behaviour in a complex manner.

Physical and biological characterization of ferromagnetic fiber networks: effect of fibrin deposition on short-term in vitro responses of human osteoblasts.

Ferromagnetic fiber networks have the potential to deform in vivo imparting therapeutic levels of strain on in-growing periprosthetic bone tissue. 444 Ferritic stainless steel provides a suitable material for this application due to its ability to support cultures of human osteoblasts (HObs) without eliciting undue inflammatory responses from monocytes in vitro. In the present article, a 444 fiber network, containing 17 vol% fibers, has been investigated. The network architecture was obtained by applying a skeletonization algorithm to three-dimensional tomographic reconstructions of the fiber networks. Elastic properties were measured using low-frequency vibration testing, providing globally averaged properties as opposed to mechanical methods that yield only local properties. The optimal region for transduction of strain to cells lies between the ferromagnetic fibers. However, cell attachment, at early time points, occurs primarily on fiber surfaces. Deposition of fibrin, a fibrous protein involved in acute inflammatory responses, can facilitate cell attachment within this optimal region at early time points. The current work compared physiological (3 and 5 g·L(-1)) and supraphysiological fibrinogen concentrations (10 g·L(-1)), using static in vitro seeding of HObs, to determine the effect of fibrin deposition on cell responses during the first week of cell culture. Early cell attachment within the interfiber spaces was observed in all fibrin-containing samples, supported by fibrin nanofibers. Fibrin deposition influenced the seeding, metabolic activity, and early stage differentiation of HObs cultured in the fibrin-containing fiber networks in a concentration-dependant manner. While initial cell attachment for networks with fibrin deposited from low physiological concentrations was similar to control samples without fibrin deposition, significantly higher HObs attached onto high physiological and supraphysiological concentrations. Despite higher cell numbers with supraphysiological concentrations, cell metabolic activities were similar for all fibrinogen concentrations. Further, cells cultured on supraphysiological concentrations exhibited lower cell differentiation as measured by alkaline phosphatase activity at early time points. Overall, the current study suggests that physiological fibrinogen concentrations would be more suitable than supraphysiological concentrations for supporting early cell activity in porous implant coatings.

Collagen labelling with an azide-proline chemical reporter in live cells.

We have developed a strategy for selective imaging of collagen in live foetal ovine osteoblasts. Our approach involves the incorporation of an azide-tagged proline in the biosynthesis of collagen followed by labelling using a strain-promoted [3+2] azide-alkyne cycloaddition reaction.