Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds

open access articleTissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)- N0-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen

Bone density of the femoral neck following Birmingham hip resurfacing: A 2-year prospective study in 27 hips

Background Resurfacing is a popular alternative to a standard hip replacement in young arthritic patients. Despite bone preservation around the femoral component, there is little information regarding the bone quality

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

Controlled spatial and conformational display of immobilised bone morphogenetic protein-2 and osteopontin signalling motifs regulates osteoblast adhesion and differentiation in vitro

<p>Abstract</p> <p>Background</p> <p>The interfacial molecular mechanisms that regulate mammalian cell growth and differentiation have important implications for biotechnology (production of cells and cell products) and medicine (tissue engineering, prosthetic implants, cancer and developmental biology). We demonstrate here that engineered protein motifs can be robustly displayed to mammalian cells <it>in vitro </it>in a highly controlled manner using a soluble protein scaffold designed to self assemble on a gold surface.</p> <p>Results</p> <p>A protein was engineered to contain a C-terminal cysteine that would allow chemisorption to gold, followed by 12 amino acids that form a water soluble coil that could switch to a hydrophobic helix in the presence of alkane thiols. Bioactive motifs from either bone morphogenetic protein-2 or osteopontin were added to this scaffold protein and when assembled on a gold surface assessed for their ability to influence cell function. Data demonstrate that osteoblast adhesion and short-term responsiveness to bone morphogenetic protein-2 is dependent on the surface density of a cell adhesive motif derived from osteopontin. Furthermore an immobilised cell interaction motif from bone morphogenetic protein supported bone formation <it>in vitro </it>over 28 days (in the complete absence of other osteogenic supplements). In addition, two-dimensional patterning of this ligand using a soft lithography approach resulted in the spatial control of osteogenesis.</p> <p>Conclusion</p> <p>These data describe an approach that allows the influence of immobilised protein ligands on cell behaviour to be dissected at the molecular level. This approach presents a durable surface that allows both short (hours or days) and long term (weeks) effects on cell activity to be assessed. This widely applicable approach can provide mechanistic insight into the contribution of immobilised ligands in the control of cell activity.</p

Targeted protein delivery: carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds.

Tissue engineering response may be tailored via controlled, sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional (3D) ice-templated collagen scaffolds. However, the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored. Here, we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide) microparticles. We probe the effects of subsequent N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride crosslinking on protein release, using microparticles with different internal protein distributions. Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug. The scaffolds display a homogeneous microparticle distribution, and a reduction in pore size and percolation diameter with increased microparticle addition, although these values did not fall below those reported as necessary for cell invasion. The protein distribution within the microparticles, near the surface or more deeply located within the microparticles, was important in determining the release profile and effect of crosslinking, as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold. Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release. Protein located within the bulk of the microparticles, was protected from the crosslinking reaction and no delay in the overall release profile was seen.This work was supported by the European Research Council [ERC Advanced Grant 320598 3D-E] and was also funded by a grant from the Medical Research Council, Arthritis Research UK, Reumafonds and the UKRM

Association of subchondral bone texture on magnetic resonance imaging with radiographic knee osteoarthritis progression: data from the Osteoarthritis Initiative Bone Ancillary Study.

OBJECTIVES: To assess whether initial or 12-18-month change in magnetic resonance imaging (MRI) subchondral bone texture is predictive of radiographic knee osteoarthritis (OA) progression over 36 months. METHODS: This was a nested case-control study including 122 knees/122 participants in the Osteoarthritis Initiative (OAI) Bone Ancillary Study, who underwent MRI optimised for subchondral bone assessment at either the 30- or 36-month and 48-month OAI visits. Case knees (n = 61) had radiographic OA progression between the 36- and 72-month OAI visits, defined as ≥ 0.7 mm minimum medial tibiofemoral radiographic joint space (minJSW) loss. Control knees (n = 61) without radiographic OA progression were matched (1:1) to cases for age, sex, body mass index and initial medial minJSW. Texture analysis was performed on the medial femoral and tibial subchondral bone. We assessed the association of texture features with radiographic progression by creating a composite texture score using penalised logistic regression and calculating odds ratios. We evaluated the predictive performance of texture features for predicting radiographic progression using c-statistics. RESULTS: Initial (odds ratio [95% confidence interval] = 2.13 [1.41-3.40]) and 12- 18-month change (3.76 [2.04-7.82]) texture scores were significantly associated with radiographic OA progression. Combinations of texture features were significant predictors of radiographic progression using initial (c-statistic [95% confidence interval] = 0.65 [0.64-0.65], p = 0.003) and 12-18-month change (0.68 [0.68-0.68], p < 0.001) data. CONCLUSIONS: Initial and 12-18-month changes in MRI subchondral bone texture score were significantly associated with radiographic progression at 36 months, with better predictive performance for 12-18-month change in texture. These results suggest that texture analysis may be a useful biomarker of subchondral bone in OA. KEY POINTS: • Subchondral bone MRI texture analysis is a promising knee osteoarthritis imaging biomarker. • In this study, subchondral bone texture was associated with knee osteoarthritis progression. • This demonstrates predictive and concurrent validity of MRI subchondral bone texture analysis. • This method may be useful in clinical trials with interventions targeting bone

Insights into patient preferences for elective surgery during the COVID-19 pandemic.

AIMS: To investigate factors that contribute to patient decisions regarding attendance for arthroplasty during the COVID-19 pandemic. METHODS: A postal questionnaire was distributed to patients on the waiting list for hip or knee arthroplasty in a single tertiary centre within the UK. Patient factors that may have influenced the decision to attend for arthroplasty, global quality of life (QoL) (EuroQol five-dimension three-level (EQ-5D-3L)), and joint-specific QoL (Oxford Hip or Knee Score) were assessed. Patients were asked at which 'COVID-alert' level they would be willing to attend an NHS and a "COVID-light" hospital for arthroplasty. Independent predictors were assessed using multivariate logistic regression. RESULTS: Of 540 distributed questionnaires, 400 (74.1%; 236 awaiting hip arthroplasty, 164 awaiting knee arthroplasty) complete responses were received and included. Less than half (48.2%) were willing to attend for hip or knee arthroplasty while a UK COVID-19 epidemic was in circulation (COVID-alert levels 3 to 5). Patients with worse joint-specific QoL had a preference to proceed with surgery at COVID-alert levels 3 to 5 compared to levels 1 and 2 (hip arthroplasty odds ratio (OR) 1.54 (95% confidence interval (CI) 1.45 to 1.63); knee arthroplasty OR 1.16 (1.07 to 1.26)). The odds of patients with worse joint-specific QoL being willing to attend for surgery at COVID-alert levels 3 to 5 increased further if surgery in a private, "COVID-light" hospital was available (hip arthroplasty OR 3.50 (95% CI 3.26 to 3.71); knee arthroplasty OR 1.41 (95% CI 1.29 to 1.53). CONCLUSION: Patient decisions surrounding elective surgery have been influenced by the global COVID-19 pandemic, highlighting the importance of patient involvement in ensuring optimized provision of elective surgery during these challenging times. Cite this article: Bone Jt Open 2021;2(4):261-270

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

Arthroscopic hip surgery compared with physiotherapy and activity modification for the treatment of symptomatic femoroacetabular impingement: multicentre randomised controlled trial.

OBJECTIVE: To compare arthroscopic hip surgery with physiotherapy and activity modification for improving patient reported outcome measures in patients with symptomatic femoroacetabular impingement (FAI). DESIGN: Two group parallel, assessor blinded, pragmatic randomised controlled trial. SETTING: Secondary and tertiary care centres across seven NHS England sites. PARTICIPANTS: 222 participants aged 18 to 60 years with symptomatic FAI confirmed clinically and with imaging (radiography or magnetic resonance imaging) were randomised (1:1) to receive arthroscopic hip surgery (n=112) or a programme of physiotherapy and activity modification (n=110). Exclusion criteria included previous surgery, completion of a physiotherapy programme targeting FAI within the preceding 12 months, established osteoarthritis (Kellgren-Lawrence grade ≥2), and hip dysplasia (centre-edge angle <20 degrees). INTERVENTIONS: Participants in the physiotherapy group received a goal based programme tailored to individual patient needs, with emphasis on improving core stability and movement control. A maximum of eight physiotherapy sessions were delivered over five months. Participants in the arthroscopic surgery group received surgery to excise the bone that impinged during hip movements, followed by routine postoperative care. MAIN OUTCOME MEASURES: The primary outcome measure was the hip outcome score activities of daily living subscale (HOS ADL) at eight months post-randomisation, with a minimum clinically important difference between groups of 9 points. Secondary outcome measures included additional patient reported outcome measures and clinical assessment. RESULTS: At eight months post-randomisation, data were available for 100 patients in the arthroscopic hip surgery group (89%) and 88 patients in the physiotherapy programme group (80%). Mean HOS ADL was 78.4 (95% confidence interval 74.4 to 82.3) for patients randomised to arthroscopic hip surgery and 69.2 (65.2 to 73.3) for patients randomised to the physiotherapy programme. After adjusting for baseline HOS ADL, age, sex, and study site, the mean HOS ADL was 10.0 points higher (6.4 to 13.6) in the arthroscopic hip surgery group compared with the physiotherapy programme group (P<0.001)). No serious adverse events were reported in either group. CONCLUSIONS: Patients with symptomatic FAI referred to secondary or tertiary care achieve superior outcomes with arthroscopic hip surgery than with physiotherapy and activity modification. TRIAL REGISTRATION: ClinicalTrials.gov NCT01893034.NIHR BRC oxfor

A cholinergic neuroskeletal interface promotes bone formation during postnatal growth and exercise.

The autonomic nervous system is a master regulator of homeostatic processes and stress responses. Sympathetic noradrenergic nerve fibers decrease bone mass, but the role of cholinergic signaling in bone has remained largely unknown. Here, we describe that early postnatally, a subset of sympathetic nerve fibers undergoes an interleukin-6 (IL-6)-induced cholinergic switch upon contacting the bone. A neurotrophic dependency mediated through GDNF-family receptor-α2 (GFRα2) and its ligand, neurturin (NRTN), is established between sympathetic cholinergic fibers and bone-embedded osteocytes, which require cholinergic innervation for their survival and connectivity. Bone-lining osteoprogenitors amplify and propagate cholinergic signals in the bone marrow (BM). Moderate exercise augments trabecular bone partly through an IL-6-dependent expansion of sympathetic cholinergic nerve fibers. Consequently, loss of cholinergic skeletal innervation reduces osteocyte survival and function, causing osteopenia and impaired skeletal adaptation to moderate exercise. These results uncover a cholinergic neuro-osteocyte interface that regulates skeletogenesis and skeletal turnover through bone-anabolic effects