The Effects of Chronological Age on the Chondrogenic Potential of Mesenchymal Stromal Cells: A Systematic Review.

Tissue engineering and cell therapy for regenerative medicine have great potential to treat chronic disorders. In musculoskeletal disorders, mesenchymal stromal cells (MSCs) have been identified as a relevant cell type in cell and regenerative strategies due to their multi-lineage potential, although this is likely to be a result of their trophic and immunomodulatory effects on other cells. This PRISMA systematic review aims to assess whether the age of the patient influences the chondrogenic potential of MSCs in regenerative therapy. We identified a total of 3027 studies after performing a search of four databases, including Cochrane, Web of Science, Medline, and PubMed. After applying inclusion and exclusion criteria, a total of 14 papers were identified that were reviewed, assessed, and reported. Cell surface characterization and proliferation, as well as the osteogenic, adipogenic, and chondrogenic differentiation, were investigated as part of the analysis of these studies. Most included studies suggest a clear link between aged donor MSCs and diminished clonogenic and proliferative potential. Our study reveals a heterogeneous and conflicting range of outcomes concerning the chondrogenic, osteogenic, and adipogenic potential of MSCs in relation to age. Further investigations on the in vitro effects of chronological age on the chondrogenic potential of MSCs should follow the outcomes of this systematic review, shedding more light on this complex relationship.

Mechanical-Stress-Related Epigenetic Regulation of ZIC1 Transcription Factor in the Etiology of Postmenopausal Osteoporosis.

Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.

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.

Co-localisation of intra-nuclear membrane type-1 matrix metalloproteinase and hypoxia inducible factor-2α in osteosarcoma and prostate carcinoma cells.

Increased membrane type-1 matrix metalloproteinase (MT1-MMP) expression in osteosarcoma is predictive of poor prognosis and directs bone metastasis in prostate carcinoma. MT1-MMP subcellular localisation varies with oxygen tension, and, therefore, the aim of the present study was to assess protein interactions between MT1-MMP and the hypoxia inducible factors (HIF-1α and HIF-2α). MT1-MMP protein expression was investigated across a panel of cancer cell lines, including a positive and negative control. The hypoxia-induced alteration in subcellular location of MT1-MMP, HIF-1α and HIF-2α in the U2OS osteosarcoma cell line was assessed using subcellular fractionation. A proximity ligation assay was utilised to assess protein to protein interactions in the osteosarcoma U2OS and prostate carcinoma PC3 cell lines. U2OS and PC3 cells exhibited a significantly increased intra-nuclear interaction between MT1-MMP and HIF-2α in response to hypoxia. The role of this warrants further investigation as it may unveil novel opportunities to target MT1-MMP, which is of particular significance for osteosarcoma since current treatment options are limited.

Using apheresis-derived cells to augment microdrilling in the treatment of chondral defects in an ovine model.

The treatment of chondral defects using microdrilling often results in a mechanically weak fibrocartilagenous repair, rather than a more robust hyaline cartilage repair. Many different microfracture/microdrilling augmentation techniques have been described, including the use of cellular products to enhance healing. Autologous peripheral blood progenitor cells can be obtained via apheresis after administration of granulocyte colony-stimulating factor (G-CSF) and have been used successfully to augment microdrilling in clinical patients. The objective of this study was to use apheresis-derived mononuclear blood cells to augment microdrilling treatment of a cartilage defect in an ovine model to determine the effect on healing. Forty adult female sheep were used in this study and were divided into a control group (microdrilling alone) and a treatment group (microdrilling, hyaluronic acid, and apheretic product). Outcome measurements included weight-bearing on the operated limb, macroscopic scoring of the joint, histology, and immunohistochemistry. In addition, magnetic resonance imaging was used to attempt to identify SPION-labeled cells from the apheretic product in the operated limbs. The results showed a significant increase in healing as measured by the modified O'Driscoll sore in the treated group. No evidence of homing of SPION-labeled cells to the defect was found and no correlation was found between the response to G-CSF administration or concentration of CD34+  and outcome. A correlation was found between healing and the concentration of white blood cells and peripheral blood mononuclear cell numbers in the apheretic product.

Assessing the impact of a 'bundle of care' approach to Staphylococcus aureus bacteraemia in a tertiary hospital.

BACKGROUND: Staphylococcus aureus bacteraemia is associated with significant morbidity and mortality. There is evidence that standardised care bundle implementation may improve the rates of appropriate investigations and improve overall management. A S. aureus bacteraemia care bundle was introduced at Christchurch Hospital, New Zealand in early 2014. We assessed the impact of the intervention on the management and outcome of S. aureus bacteraemia. METHODS: A cohort study of cases of S. aureus bacteraemia was conducted following standardised care bundle introduction. Prospective enrolment of post-intervention patients occurred from 1st January 2014 to 30th June 2015, with retrospective review of pre-intervention cases from 1st January 2009 to 31st December 2013. RESULTS: In the pre-intervention period 447 patients had at least one episode of S. aureus bacteraemia compared to 151 patients in the post-intervention period. The two groups were similar by gender, ethnicity, and age. Significant increases in Infectious Diseases consultation rate (86.6% vs 94.8%; p=0.009), echocardiography (76.3% vs 96.3%; p<0.001), urine culture (74.0% vs 91.9%; p<0.001), follow up blood cultures (44.2% vs 83.0%; p<0.001), and at least 2 weeks of parenteral therapy (83.5% vs 92.9%; p=0.014) were observed after introduction of the bundle. There were no significant differences in rates 30-day mortality (18.6% vs. 20.5%; p=0.596), but there was a reduction in episodes of relapsed infection in the post-intervention cohort (7.4% vs 1.3%; p=0.004). CONCLUSION: An integrated care bundle for the management of S. aureus bacteraemia resulted in increased use of quality of care indicators and infectious diseases review and improved patient outcome.

Lessons Learned From a Randomized Controlled Trial of Short-Course Intravenous Antibiotic Therapy for Erysipelas and Cellulitis of the Lower Limb (Switch Trial).

BACKGROUND: The diagnosis of cellulitis is made clinically without a gold standard diagnostic test, and cellulitis has many disease mimics. There is currently no consensus for optimal antimicrobial treatment duration or method of antimicrobial delivery. METHODS: This was a randomized controlled open-label multicenter trial to determine the safety and efficacy of 24 hours of intravenous (IV) therapy compared with ≥72 hours of IV therapy, both followed by oral therapy to a maximum of 7-10 days' duration for the treatment of lower limb cellulitis. RESULTS: Over 40 months, 80 patients were recruited. Thirty-nine patients were assigned to 24 hours of IV antibiotics and 41 to ≥72 hours of IV antibiotics. The mean duration (range) of IV antibiotics in the 24-hour group was 25.5 (17-40) hours, and in the ≥72-hour group it was 78 (41.5-210) hours. Three patients in the 24-hour arm and 4 patients in the ≥72-hour arm were excluded from the analysis due to withdrawal from the trial. Analysis of the remaining patients revealed that 6 patients (4 in the intervention arm and 2 in the control arm) did not achieve an adequate response to therapy. Only 1 patient experienced self-limiting adverse effects of treatment. CONCLUSIONS: The noninferiority of short-course IV therapy cannot be determined from this trial. Challenges included resource limitations for recruitment, misdiagnosis, participant withdrawal, and subjective responses to therapy based on visual assessment by treating clinicians. Further studies are needed to determine if short-course IV therapy is a suitable treatment option. AUSTRALIA COUNCIL OF CLINICAL TRIALS REGISTRY NO: ACTRN12613001366741.

Albumin-Enriched Fibrin Hydrogel Embedded in Active Ferromagnetic Networks Improves Osteoblast Differentiation and Vascular Self-Organisation.

Porous coatings on prosthetic implants encourage implant fixation. Enhanced fixation may be achieved using a magneto-active porous coating that can deform elastically in vivo on the application of an external magnetic field, straining in-growing bone. Such a coating, made of 444 ferritic stainless steel fibres, was previously characterised in terms of its mechanical and cellular responses. In this work, co-cultures of human osteoblasts and endothelial cells were seeded into a novel fibrin-based hydrogel embedded in a 444 ferritic stainless steel fibre network. Albumin was successfully incorporated into fibrin hydrogels improving the specific permeability and the diffusion of fluorescently tagged dextrans without affecting their Young's modulus. The beneficial effect of albumin was demonstrated by the upregulation of osteogenic and angiogenic gene expression. Furthermore, mineralisation, extracellular matrix production, and formation of vessel-like structures were enhanced in albumin-enriched fibrin hydrogels compared to fibrin hydrogels. Collectively, the results indicate that the albumin-enriched fibrin hydrogel is a promising bio-matrix for bone tissue engineering and orthopaedic applications.

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.

Engineered mosaic protein polymers; a simple route to multifunctional biomaterials.

BACKGROUND: Engineered living materials (ELMs) are an exciting new frontier, where living organisms create highly functional materials. In particular, protein ELMs have the advantage that their properties can be manipulated via simple molecular biology. Caf1 is a protein ELM that is especially attractive as a biomaterial on account of its unique combination of properties: bacterial cells export it as a massive, modular, non-covalent polymer which is resistant to thermal and chemical degradation and free from animal material. Moreover, it is biologically inert, allowing the bioactivity of each 15 kDa monomeric Caf1 subunit to be specifically engineered by mutagenesis and co-expressed in the same Escherichia coli cell to produce a mixture of bioactive Caf1 subunits. RESULTS: Here, we show by gel electrophoresis and transmission electron microscopy that the bacterial cells combine these subunits into true mosaic heteropolymers. By combining two separate bioactive motifs in a single mosaic polymer we demonstrate its utility by stimulating the early stages of bone formation by primary human bone marrow stromal cells. Finally, using a synthetic biology approach, we engineer a mosaic of three components, demonstrating that Caf1 complexity depends solely upon the variety of monomers available. CONCLUSIONS: These results demonstrate the utility of engineered Caf1 mosaic polymers as a simple route towards the production of multifunctional biomaterials that will be useful in biomedical applications such as 3D tissue culture and wound healing. Additionally, in situ Caf1 producing cells could create complex bacterial communities for biotechnology.

miR-324-5p is up regulated in end-stage osteoarthritis and regulates Indian Hedgehog signalling by differing mechanisms in human and mouse.

The Hedgehog (Hh) signalling pathway plays important roles during embryonic development and in adult tissue homeostasis, for example cartilage, where its deregulation can lead to osteoarthritis (OA). microRNAs (miRNAs) are important regulators of gene expression, and have been implicated in the regulation of signalling pathways, including Hh, thereby impacting upon development and disease. Our aim was to identify the function of miRNAs whose expression is altered in OA cartilage. Here we identified an increase in miR-324-5p expression in OA cartilage and hypothesised that, as in glioma, miR-324-5p would regulate Hh signalling. We determined that miR-324-5p regulates osteogenesis in human mesenchymal stem cells (MSCs) and in mouse C3H10T1/2 cells. Luciferase reporter assays demonstrated that miR-324-5p directly regulated established targets GLI1 and SMO in human but not in mouse, suggesting species-dependent mechanism of Hh pathway regulation. Stable Isotope Labelling with Amino acids in Cell culture (SILAC), mass spectrometry and whole genome transcriptome analysis identified Glypican 1 (Gpc1) as a novel miR-324-5p target in mouse, which was confirmed by real-time RT-PCR, immunoblotting and 3'UTR-luciferase reporters. Knockdown of Gpc1 reduced Hh pathway activity, and phenocopied the effect of miR-324-5p on osteogenesis, indicating that miR-324-5p regulates Hh signalling in mouse via direct targeting of Gpc1. Finally, we showed that human GPC1 is not a direct target of miR-324-5p. Importantly, as well as identifying novel regulation of Indian Hedgehog (Ihh) signalling, this study demonstrates how a miRNA can show conserved pathway regulation in two species but by distinct mechanisms and highlights important differences between human diseases and mouse models.

Multi-length scale bioprinting towards simulating microenvironmental cues.

It is envisaged that the creation of cellular environments at multiple length scales, that recapitulate in vivo bioactive and structural roles, may hold the key to creating functional, complex tissues in the laboratory. This review considers recent advances in biofabrication and bioprinting techniques across different length scales. Particular focus is placed on 3D printing of hydrogels and fabrication of biomaterial fibres that could extend the feature resolution and material functionality of soft tissue constructs. The outlook from this review discusses how one might create and simulate microenvironmental cues in vitro. A fabrication platform that integrates the competencies of different biofabrication technologies is proposed. Such a multi-process, multiscale fabrication strategy may ultimately translate engineering capability into an accessible life sciences toolkit, fulfilling its potential to deliver in vitro disease models and engineered tissue implants.

Osseointegration of porous apatite-wollastonite and poly(lactic acid) composite structures created using 3D printing techniques.

A novel apatite-wollastonite/poly(lactic acid) (AW/PLA) composite structure, which matches cortical and cancellous bone properties has been produced and evaluated in vitro and in vivo. The composites structure has been produced using an innovative combination of 3D printed polymer and ceramic macrostructures, thermally bonded to create a hybrid composite structure. In vitro cell assays demonstrated that the AW structure alone, PLA structure alone, and AW/PLA composite were all biocompatible, with the AW structure supporting the proliferation and osteogenic differentiation of rat bone marrow stromal cells. Within a rat calvarial defect model the AW material showed excellent osseointegration with the formation of new bone, and vascularisation of the porous AW structure, both when the AW was implanted alone and when it was part of the AW/PLA composite structure. However, the AW/PLA structure showed the largest amount of the newly formed bone in vivo, an effect which is considered to be a result of the presence of the osteoinductive AW structure stimulating bone growth in the larger pores of the adjacent PLA structure. The layered AW/PLA structure showed no signs of delamination in any of the in vitro or in vivo studies, a result which is attributed to good initial bonding between polymer and ceramic, slow resorption rates of the two materials, and excellent osseointegration. It is concluded that macro-scale composites offer an alternative route to the fabrication of bioactive bone implants which can provide a match to both cortical and cancellous bone properties over millimetre length scales.

How cell culture conditions affect the microstructure and nanomechanical properties of extracellular matrix formed by immortalized human mesenchymal stem cells: An experimental and modelling study.

This paper presents an investigation of how different culture media (i.e. basal and osteogenic media) affect the nanomechanical properties and microstructure of the mineralized matrix produced by the human mesenchymal stem cell line Y201, from both an experimental and theoretical approach. A bone nodule (i.e. mineralized matrix) cultured from basal medium shows a more anisotropic microstructure compared to its counterpart cultured from an osteogenic medium. As confirmed by finite element simulations, this anisotropic microstructure explains the bimodal distribution of the corresponding mechanical properties very well. The overall nanomechanical response of the bone nodule from the osteogenic medium is poorer compared to its counterpart from the basal medium. The bone nodules, from both basal and osteogenic media, have shown reverse aging effects in terms of mechanical properties. These are possibly due to the fact that cell proliferation outcompetes the mineralization process.

Novel bioglasses for bone tissue repair and regeneration: Effect of glass design on sintering ability, ion release and biocompatibility.

Eight novel silicate, phosphate and borate glass compositions (coded as NCLx, where x = 1 to 8), containing different oxides (i.e. MgO, MnO2, Al2O3, CaF2, Fe2O3, ZnO, CuO, Cr2O3) were designed and evaluated alongside apatite-wollastonite (used as comparison material), as potential biomaterials for bone tissue repair and regeneration. Glass frits of all the formulations were processed to have particle sizes under 53 µm, with their morphology and dimensions subsequently investigated by scanning electron microscopy (SEM). In order to establish the nature of the raw glass powders, X-ray diffraction (XRD) analysis was also performed. The sintering ability of the novel materials was determined by using hot stage microscopy (HSM). Ionic release potential was assessed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Finally, the cytotoxic effect of the novel glass powders was evaluated for different glass concentrations via a colorimetric assay, on which basis three formulations are considered promising biomaterials.

Clinical learning environments: place, artefacts and rhythm.

CONTEXT: Health care practitioners learn through experience in clinical environments in which supervision is a key component, but how that learning occurs outside the supervision relationship remains largely unknown. This study explores the environmental factors that inform and support workplace learning within a clinical environment. METHODS: An observational study drawing on ethnographic methods was undertaken in a general medicine ward. Observers paid attention to interactions among staff members that involved potential teaching and learning moments that occurred and were visible in the course of routine work. General purpose thematic analysis of field notes was undertaken. RESULTS: A total of 376 observations were undertaken and documented. The findings suggest that place (location of interaction), rhythm (regularity of activities occurring in the ward) and artefacts (objects and equipment) were strong influences on the interactions and exchanges that occurred. Each of these themes had inherent tensions that could promote or inhibit engagement and therefore learning opportunities. Although many learning opportunities were available, not all were taken up or recognised by the participants. CONCLUSIONS: We describe and make explicit how the natural environment of a medical ward and flow of work through patient care contribute to the learning architecture, and how this creates or inhibits opportunities for learning. Awareness of learning opportunities was often tacit and not explicit for either supervisor or learner. We identify strategies through which tensions inherent within space, artefacts and the rhythms of work can be resolved and learning opportunities maximised.

Three-dimensional printing of porous load-bearing bioceramic scaffolds.

This article reports on the use of the binder jetting three-dimensional printing process combined with sintering to process bioceramic materials to form micro- and macroporous three-dimensional structures. Three different glass-ceramic formulations, apatite-wollastonite and two silicate-based glasses, have been processed using this route to create porous structures which have Young's modulus equivalent to cortical bone and average bending strengths in the range 24-36 MPa. It is demonstrated that a range of macroporous geometries can be created with accuracies of ±0.25 mm over length scales up to 40 mm. Hot-stage microscopy is a valuable tool in the definition of processing parameters for the sintering step of the process. Overall, it is concluded that binder jetting followed by sintering offers a versatile process for the manufacture of load-bearing bioceramic components for bone replacement applications.

Impact of Collagen/Heparin Multilayers for Regulating Bone Cellular Functions.

Bone cell interaction with extracellular matrix (ECM) microenvironment is of critical importance when engineering surface interfaces for bone regeneration. In this work layer-by-layer films of type I collagen (coll), the major constituent of bone ECM, and heparin (hep), a glycosaminoglycan, were assembled on poly(l-lactic acid) (PLLA) substrates to evaluate the impact of the biomacromolecular coating on cell activity. The surface modification of PLLA demonstrated that the hep/coll multilayer is stable after 10 bilayers (confirmed by contact angle, infrared spectroscopy, and morphological analysis). This simple approach provided novel information on the effect of heparin on type I collagen hierarchical organization and subsequent cell response of osteoblast-like (MC3T3-E1) and human bone marrow-derived mesenchymal stem cells (hMSCs). Interestingly, the number of deposited heparin layers (1 or 10) appeared to play an important role in the self-assembly of collagen into fibrils, stabilizing the fibrous collagen layer, and potentially impacting hMSCs activity.