Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Aim. Despite the expanding research focusing on bacterial biofilm formation, specific histochemical biofilm stains have not been developed for light microscopy. Therefore, pathologists are often not aware of the presence of biofilm formation when examining slides for diagnosing bacterial infections, including orthopaedic infections. The aim of the present study was to develop a combined histochemical and immunohistochemical biofilm stain for simultaneous visualization of Staphylococcus aureus bacteria and extracellular matrix in different colours using light microscopy. Methods. Infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with the biofilm forming S. aureus strain S54F9. The infection time was 5 and 15 days, respectively. First, 25 common histochemical protocols were used in order to find stains that could identify extracellular biofilm matrix. Hereafter, the histochemical protocols for Alcian Blue pH3, Luna and Methyl-pyronin green were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. Finally, the three new combined protocols were applied to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. For all combined protocols applied on all types of tissue (porcine and human) the number of double stained bacterial aggregates were counted. On the same sections the percentage of extracellular matrix of representative bacterial aggregates was calculated by image analysis. Results. Simultaneous visualization of bacterial cells and extracellular matrix in different colours was detected in both porcine and human tissue sections with all three combined protocols. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain i.e. if it was Alcian blue pH3 (colouring polysaccharides), Luna or Methyl green-pyronin (both colouring extracellular DNA), respectively. In the porcine models, 10 percent of the bacterial aggregates in a 10× magnification field revealed both the extracellular matrix and bacteria simultaneously in two different colours. For the human case, this was seen in 90 percent of the bacterial aggregates. The percentage of extracellular matrix of representative bacterial aggregates was 60 and 20 percent in the human and porcine tissues, respectively. Conclusions. The amount of S. aureus biofilm extracellular matrix increased with infection time. A combination of histochemical and immunohistochemical staining is a practical method for identification and evaluation of S. aureus biofilm in orthopaedic infections

Tissue engineering by self-assembly is a technique that consists of growing cells on surfaces made of thermoresponsive polymers, that allow the production of contiguous cell sheets by simply lowering the temperature below the polymer's low critical solution temperature. In this approach cell-cell junctions and deposited extracellular matrix (ECM) remain intact, which provides a better cell localisation at the site of injury. However, these systems lack the possibility to fabricate multi-layered and three-dimensional cell sheets that would better recapitulate native tissues. Moreover, the fabrication of ECM-rich cell sheets would be highly desirable. This limitation could be overcome by inducing macromolecular crowding (MMC) conditions. Herein we venture to fabricate electrospun thermoresponsive nanofibres to sustain the growth and detachment of ECM-rich tissue substitutes in the presence of a MMC microenvironment. A copolymer of 85% poly-N-isopropylacrylamide and 15% N-tert-butylacrylamide (pNIPAAm/NTBA) were used for all experiments. To create aligned nanofibers, the polymer was electrospun and collected on a mandrel rotating at 2000 rpm. Human adipose derived stem cells (hADSC) were treated with media containing macromolecular crowders to enhance matrix deposition. Cell viability and morphology were assessed, and immunocytochemistry was conducted in order to estimate matrix deposition and composition. Adipogenic, osteogenic and chondrogenic assays were performed both with and without the presence of MMC. Non-invasive cell detachment was enabled by decreasing the temperature of culture to 10 °C for 20 minutes. The electrospinning process resulted in the production of pNIPAm/NTBA fibres in the diameter range from 1 to 2 µm and an overall alignment of 80%. Cell viability, proliferation and metabolic activity revealed that hADSCs were able to grow on the thermoresponsive scaffold. The cells were able to detach as an intact cell sheet in presence of MMC. Moreover, it was demonstated that MMC, by a volume extrusion effect, enhances Collagen type I deposition, which is one of the main components of the ECM. Histological analysis revealed that in the presence of MMC the cells were able to self-assembled into three dimensional multi-layers. The cells were able to differentiate towards the osteogenic and adipogenic lineage in the presence of MMC. Interestingly we were able to fabricate three-dimensional chondrogenic cell sheet both with and without MMC. Collectively the pNIPAm/NTBA thermoresponsive fibres were able to sustain the growth and the detachment of ECM-rich multi-layered cell sheets. The pNIPAm/NTBA fibres were able to successfully sustain growth and detachment of ECM-rich tissue equivalents. We believe that replacement, repair and restoration of tissue function can be accomplished best using cells that create their own tissue-specific extracellular matrix with a precision and stoichiometric efficiency still unmatched by man-made devices

Introduction. Autologous fat grafting has favourable potential as a regenerative strategy and is the current gold-standard to repair large contour defects, as needed in breast reconstruction after mastectomy and traumatic soft tissue reconstruction. Clinically, there is a limit on the volume of lipoaspirate which can be utilised to repair a soft-tissue defect. Surgical complications are the result of poor structural fidelity of lipoaspirate and graft resorption as a filling material and are hindered further by poor graft vascularisation. This study aims to develop injectable lipoaspirate-derived adipose tissue grafts with enhanced biologically and clinically-admissible structural and functional properties adopting light photocrosslinking of unmodified lipoaspirate. Methods. Patient-derived lipoaspirate was harvested and crosslinked using novel photoinitiator and exposure to visible light (wavelength 450nm) in surgery, establishing bonds between extracellular matrix (ECM) proteins within the material. The degree of crosslinking was tuned (photoinitiator concentration, light exposure, light intensity) and covalent bond formation measured using mass spectrometry. To predict patient response, SWATH-MS was used to identify differences in patient ECM and crosslinked grafts were implanted in vivo using a subcutaneous mouse model. Functional vessel formation and resorption were quantified using micro-CT and tissue-remodelling was assessed via histology. Results. There was an increase in the relative abundance of covalent bonds present with increasing degree of crosslinking. When injected, crosslinked lipoaspirate had better shape fidelity compared with native lipoaspirate – demonstrated by a smaller fibre diameter. Crosslinked lipoaspirate remained viable over long term culture and resulted in more predictable resorption profiles when implanted in vivo. Conclusions. The crosslinking approach described here is tunable and functional across different patient samples. Improving the structural properties of lipoaspirate through minimal manipulation has clinical utility for the delivery of grafts with higher shape fidelity and therefore increased graft survival when implanted

Osteogenesis Imperfecta (OI) is a heritable bone disorder characterized by bone fragility and often caused by mutations in the Type I collagen-encoding genes COL1A1 and COL1A2. The pathophysiology of OI, particularly at the cellular level, is still not well understood. This contributes to the lack of a cure for this disorder as well as an effective preventive or management options of its complications. In the bone environment, mesenchymal stem cells (MSCs) and osteoblasts (Ob) exert their function, at least partially, through the secretion of extracellular vesicles (EV). EV is a heterogeneous group of nanosized membrane-enclosed vesicles that carry/transfer a cargo of proteins, lipid and nucleic acids from the secreting cell to its target cells. Our objective is to characterize EVs secreted by human control (HC)- and OI-MSCs and their derived Obs, with focus on their protein content. We hypothesize that there will be differences in the protein content of EVs secreted by OI-Obs compared to HC-Ob, which may indicate a deviation from healthy Ob behavior and, thus, a role in OI pathophysiology. MSCs were harvested from the adipose tissue of four COL1A1-OI and two HC patients. They were proliferated in an EV-depleted media, then induced to differentiate to extracellular matrix (ECM)-producing osteoblasts, which then gets mineralized. EVs secreted by MSCs (MSC-EV) and Obs (Ob-EV) were then purified and concentrated. Using liquid chromatography- tandem mass spectrometry, proteomic analysis of the EV groups was done. A total of 384 unique proteins were identified in all EVs, 373 were found in Vesiclepedia indicating a good enrichment of our samples with EV proteins. 67 proteins of the total 384 were exclusively or significantly upregulated (p-value < 0 .05) in OI-Ob-EV and 28 proteins in the HC-Ob-EVs, relative to each other. These two groups of differentially expressed proteins were compared by Gene Ontology (GO) analysis of their cellular compartment, molecular functions and biological processes. We observed that there were differences in the cellular origin of EV-proteins, which may indicate heterogeneity of the isolated EVs. Molecular function and biological process analyses of the HC-Ob-EV proteins showed, as expected, predominantly calcium-related activities such as extracellular matrix (ECM) mineralization. OI-Ob-EV proteins were still predominantly exhibiting ECM organization and formation functions. Annexins A1,2,4,5 and 6 were differentially and significantly upregulated by the HC-Ob-EVs. Fibronectin (FN), Fibulin-1 and −2, and Laminins (α4 & γ1), which are amongst the early non-collagenous proteins to form the ECM, were differentially and significantly upregulated in the OI-Ob-EVs. We concluded that the persistent expression of Fibronectin (FN), Fibulin-1 and −2, and Laminins in OI-Ob-EVs might indicate the presence of an immature ECM that the OI-Obs are trying to organize. ECM mineralization is largely dependent on the presence of an organized mature ECM, and this being compromised in OI bone environment, may be a contributor to the bone fragility seen in these patients. Annexins, which are calcium-binders that are vital for ECM mineralization, were significantly downregulated in the OI-Ob-EVs and this may be a further contributor to ECM mineralization impairment and bone fragility

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. However, in vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate the in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media mimicking the dense extracellular matrix. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking, we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing synthesis and deposition of tissue-specific ECM. Human tendons were kindly provided from University Hospital Galway, after obtaining appropriate licenses, ethical approvals and patient consent. Afterwards, tenocytes were extracted using the migration method. Experiments were conducted at passage three. Optimization of MMC conditions was assessed using 50 to 500 μg/ml carrageenan (Sigma Aldrich, UK). For variable oxygen tension cultures, tenocytes were incubated in a Coy Lab (USA) hypoxia chamber. ECM synthesis and deposition were assessed using SDS-PAGE (BioRad, UK) and immunocytochemistry (ABCAM, UK) analysis. Protein analysis for Scleraxis (ABCAM, UK) was performed using western blot. Gene analysis was conducted using a gene array (Roche, Ireland). Cell morphology was assessed using bright-field microscopy. All experiments were performed at least in triplicate. MINITAB (version 16, Minitab, Inc.) was used for statistical analysis. Two-sample t-test for pairwise comparisons and ANOVA for multiple comparisons were conducted. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased synthesis and deposition of collagen type I, the major component of tendon ECM. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, protein analysis revealed elevated dexpression of the tendon-specific protein Sclearaxis, while a detailed gene analysis revealed upregulation of tendon-related genes and downregulation of trans-differentiation markers again when cells cultured with MMC at 2% oxygen tension. Finally, low oxygen tension and MMC did not affect the metabolic activity, proliferation and viability of human tenocytes. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance. Currently, the addition of substrate aligned topography together with MMC and hypoxia is being investigated in this multifactorial study for the development of an implantable device for tendon regeneration

Giant cell tumors of bone (GCTs) are locally aggressive tumors with recurrence potential that represent up to 10% of primary tumors of the bone. GCTs pathogenesis is driven by neoplastic mononuclear stromal cells that overexpress receptor activator of nuclear factor kappa-B/ligand (RANKL). Treatment with specific anti-RANKL antibody (denosumab) was recently introduced, used either as a neo-adjuvant in resectable tumors or as a stand-alone treatment in unresectable tumors. While denosumab has been increasingly used, a percentage of patients do not improve after treatment. Here, we aim to determine molecular and histological patterns that would help predicting GCTs response to denosumab to improve personalized treatment. Nine pre-treatment biopsies of patients with spinal GCT were collected at 2 centres. In 4 patients denosumab was used as a neo-adjuvant, 3 as a stand-alone and 2 received denosumab as adjuvant treatment. Clinical data was extracted retrospectively. Total mRNA was extracted by using a formalin-fixed paraffin-embedded extraction kit and we determined the transcript profile of 730 immune-oncology related genes by using the Pan Cancer Immune Profiling panel (Nanostring). The gene expression was compared between patients with good and poor response to Denosumab treatment by using the nSolver Analysis Software (Nanostring). Immunohistochemistry was performed in the tissue slides to characterize cell populations and immune response in CGTs. Two out of 9 patients showed poor clinical response with tumor progression and metastasis. Our analysis using unsupervised hierarchical clustering determined differences in gene expression between poor responders and good responders before denosumab treatment. Poor responding lesions are characterized by increased expression of inflammatory cytokines as IL8, IL1, interferon a and g, among a myriad of cytokines and chemokines (CCL25, IL5, IL26, IL25, IL13, CCL20, IL24, IL22, etc.), while good responders are characterized by elevated expression of platelets (CD31 and PECAM), coagulation (CD74, F13A1), and complement classic pathway (C1QB, C1R, C1QBP, C1S, C2) markers, together with extracellular matrix proteins (COL3A1, FN1,. Interestingly the T-cell response is also different between groups. Poor responding lesions have increased Th1 and Th2 component, but good responders have an increased Th17 component. Interestingly, the checkpoint inhibitor of the immune response PD1 (PDCD1) is increased ~10 fold in poor responders. This preliminary study using a novel experimental approach revealed differences in the immune response in GCTs associated with clinical response to denosumab. The increased activity of checkpoint inhibitor PD1 in poor responders to denosumab treatment may have implications for therapy, raising the potential to investigate immunotherapy as is currently used in other neoplasms. Further validation using a larger independent cohort will be required but these results could potentially identify the patients who would most benefit from denosumab therapy

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone

Large cartilage lesions in younger patients can be treated by fresh osteochondral allograft transplantation, a surgical technique that relies on stable initial fixation and a minimum chondrocyte viability of 70% in the donor tissue to be successful. The Missouri Osteochondral Allograft Preservation System (MOPS) may extend the time when stored osteochondral tissues remain viable. This study aimed to provide an independent evaluation of MOPS storage by evaluating chondrocyte viability, chondrocyte metabolism, and the cartilage extracellular matrix using an ovine model. Femoral condyles from twelve female Arcott sheep (6 years, 70 ± 15 kg) were assigned to storage times of 0 (control), 14, 28, or 56 days. Sheep were assigned to standard of care [SOC, Lactated Ringer's solution, cefazolin (1 g/L), bacitracin (50,000 U/L), 4°C storage] or MOPS [proprietary media, 22-25°C storage]. Samples underwent weekly media changes. Chondrocyte viability was assessed using Calcein AM/Ethidium Homodimer and reported as percent live cells and viable cell density (VCD). Metabolism was evaluated with the Alamar blue assay and reported as Relative Fluorescent Units (RFU)/mg. Electromechanical properties were measured with the Arthro-BST, a device used to non-destructively compress cartilage and calculate a quantitative parameter (QP) that is inversely proportional to stiffness. Proteoglycan content was quantified using the dimethylmethylene blue assay of digested cartilage and distribution visualized by Safranin-O/Fast Green staining of histological sections. A two-way ANOVA and Tukey's post hoc were performed. Compared to controls, MOPS samples had fewer live cells (p=0.0002) and lower VCD (p=0.0004) after 56 days of storage, while SOC samples had fewer live cells (p=0.0004, 28 days; p=0.0002, 56 days) and lower VCD (p=0.0002, 28 days; p=0.0001, 56 days) after both 28 and 56 days (Table 1). At 14 days, the percentage of viable cells in SOC samples were statistically the same as controls but VCD was lower (p=0.0197). Cell metabolism in MOPS samples remained the same over the study duration but SOC had lower RFU/mg after 28 (p=0.0005) and 56 (p=0.0001) days in storage compared to controls. These data show that MOPS maintained viability up to 28 days yet metabolism was sustained for 56 days, suggesting that the conditions provided by MOPS storage allowed fewer cells to achieve the same metabolic levels as fresh cartilage. Electromechanical QP measurements revealed no differences between storage methods at any individual time point. QP data could not be used to interpret changes over time because a mix of medial and lateral condyles were used and they have intrinsically different properties. Proteoglycan content in MOPS samples remained the same over time but SOC was significantly lower after 56 days (p=0.0086) compared to controls. Safranin-O/Fast Green showed proteoglycan diminished gradually beginning at the articular surface and progressing towards bone in SOC samples, while MOPS maintained proteoglycan over the study duration (Figure 1). MOPS exhibited superior viability, metabolic activity and proteoglycan retention compared to SOC, but did not maintain viability for 56 days. Elucidating the effects of prolonged MOPS storage on cartilage properties supports efforts to increase the supply of fresh osteochondral allografts for clinical use. For any figures or tables, please contact the authors directly

Aim. Staphylococcus epidermidis (S. epidermidis) is one of the main pathogens responsible for bone and joint infections especially those involving prosthetic materials (PJI). Although less virulent than S. aureus, S. epidermidis is involved in chronic infections notably due to its ability to form biofilm. Moreover, it is frequently multiresistant to antibiotics. In this context, the development of additional or alternative antibacterial therapies targeting the biofilm is a priority. Method. The aim of this study was to evaluate in vitro the activity of phage lysin exebacase (CF-301) against biofilms formed by 19 S. epidermidis clinical strains responsible for PJI. We determined the remaining viable bacteria inside the biofilm (counting after serial dilution and plating) and the biomass (bacteria and extracellular matrix, using crystal violet staining) after 24h of exposition to exebacase at different concentrations, alone (0.05; 0.5; 5; 50 and 150 mg/L) or in combination (5, 50 and 150 mg/L) with antibiotics commonly used to treat multi-resistant S. epidermidis PJI (rifampin (1 mg/L), vancomycin (10mg/L) and daptomycin (10mg/L)). In this study, synergy was defined as a significantly higher effect of the association in comparison to the sum of the effect of each molecule. Results. Exebacase showed a dose-dependent reduction of biomass, ranging from 11 % at 0.5 mg/L to 66 % at 150 mg/L. Exebacase showed a significant bactericidal activity at 50 and 150 mg/l, with a mean decrease of the inoculum of 0.94 and 1.7 log, respectively. In addition, synergistic effects were observed in association with i) rifampin (1 mg/L) showing a mean decrease up to 84% of the biomass and 3.5 log CFU at 150 mg/L of exebacase, ii) vancomycin (10 mg/L) showing a mean decrease up to 81% of the biomass and 2.82 log CFU at 150 mg/L of exebacase, iii) and daptomycin (10 mg/L) showing a mean decrease up to 85% of the biomass and 3.1 log CFU at 150 mg/L of exebacase. Conclusions. Exebacase showed, in vitro, synergistic activity with antibiotics against S. epidermidis biofilms. It is a promising adjuvant therapy to rifampin, vancomycin and daptomycin in the context of PJI. Further studies are needed, in vitro to understand the mechanism of action on S. epidermidis biofilm and the heterogeneity of strain behaviour and in vivo to confirm the present data

Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, with cumulative treatment costs for all prosthetic joint infections estimated to be ∼ $1 billion per annum (UK and North America). Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Low intensity pulsed ultrasound is already licensed for clinical use in fracture management and thus could be translated quickly into a clinical treatment. Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged with gentamicin +/− low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 180 minutes and 20 minutes, respectively. The primary outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. Assessment of cellular metabolism was conducted using a liquid-chromatography-mass spectrometry, as well as a triphenyltetrazolium chloride assay coupled with spectrophotometry. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was reduced to 64mg/L. Metabolic activity of biofilm-associated S. aureus was increased by 25% following ultrasound therapy (p < 0 .0001), with identification of key biosynthetic pathways activated by non-lethal dispersal. Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin, bringing the MBEC of gentamicin to within clinically achievable concentrations. The mechanism of action was due to partial disruption of the extracellular matrix which led to an increase of nutrient availability and oxygen tension within the biofilm. This metabolic stimulus was responsible for the reversal of gentamicin tolerance in the biofilm-associated S. aureus

Biologic supplementation and augmentation techniques have become popular in recent years. There has been considerable media attention regarding professional athletes and celebrities who have traveled around the world to receive treatments using proprietary cocktails of platelet rich plasma, bone marrow aspirate concentrates, extracellular matrix, adipose-derived stem cells, human as well as xenograft derived collagen implants and protein supplements among other components. Unfortunately, the medical evidence regarding these treatments has often been inconsistent, inadequate with respect to levels of evidence with a dearth of mid- and long-term data to guide our treatments. This presentation will review the data available regarding the treatment of rotator cuff tendinosis, partial thickness rotator cuff tears, full thickness rotator cuff tears and osteoarthritis of the glenohumeral joint. Unfortunately, there are more questions than answers regarding the use of biologics

Purpose. Adenosine triphosphate (ATP) has been implicated as an autocrine/paracrine signal in the mechanotransduction pathway of chondrocytes. In this study, human chondrocytes in a 3D agarose scaffold were cultured with exogenous ATP in varying doses to determine its effect on extracellular matrix synthesis by the cells. Further experiments determined basal ATP release, ATP degradation and expression of P2Y1 and P2Y2 purinoreceptors by the cultured cell constructs. Method. Human chondrocytes were obtained by enzymatic digestion of cartilage samples obtained at the time of total joint arthroplasty. The chondrocytes were cultured in a 3D agarose scaffold using standard tissue culture techniques. Various concentrations of exogenous ATP were added to the cultures, along with the radioisotopes to assess matrix synthesis. The cultures were harvested after a 24 hr incubation and radioisotope incorporation was determined by scintillation counting to determine proteoglycan ([35S]-sulfate) and collagen ([3H]-proline) synthesis, respectively. DNA content was determined by the Hoescht 33258 binding assay, and the proteoglycan and collagen synthesis were normalized to DNA content. Basal ATP release and degradation of exogenous ATP were determined by luciferase assay and luminometry. Expression of P2Y1 and P2Y2 purinoreceptors were determined by flow cytometry. Results. Cartilage was obtained and cultured from 22 patients. We identified responders (16/22) and non-responders (6/22) to ATP stimulation. Patients demographics, co-morbidities and medications were reviewed and no correlating characteristics were identified. The average increase in [3H]-proline incorporation was 242% the control (range 115%–388%, p<0.02) and the average increase in [35S]-sulfate incorporation was 238% (range 124%–711%, p<0.02). The expression of P2Y1 and P2Y2 receptors varied widely between individuals, with a range of 11–76% expression and of 3–67% expression for P2Y1 and P2Y2 receptors, respectively. Almost all cells expressing P2Y2 receptors also expressed P2Y1 receptors, and 4/8 patients also had significant cell populations expressing P2Y1 but not P2Y2 receptors (range of 4–17% of cells). Of the 8 patients studied, only 1 patient had measurable ATP within the culture media. ATP degradation within the culture media was measured, with the measured ATP half-life and elimination rate constants were determined. The ATP elimination rate constant values showed good correlation to P2Y1 receptor expression (R=0.99). Conclusion. P2Y1 and P2Y2 receptors were expressed on a significant proportion of chondrocytes from patients with osteoarthritis and there was a significant correlation of the expression of these receptors to the ATP elimination rate constants. The addition of exogenous ATP increased both the proteoglycan and collagen synthesis of the developing cartilage constructs in a subset of patients and appears to be a promising technique to improve extracellular matrix production in these constructs

The Masquelet or induced membrane technique (IMT) is a two-stage surgical procedure used for the treatment of segmental bone defects. In this technique, the defect is first filled with a polymethyl methacrylate (PMMA) spacer, which triggers the formation of a membrane that will encapsulate the defect. During the second surgery, the spacer is carefully removed and replaced by autologous bone graft while preserving the membrane. This membrane is vascularized, contains growth factors, and provides mechanical stability to the graft, all of which are assumed to prevent graft resorption and promote bone healing. The technique is gaining in popularity and several variations have been introduced in the clinical practice. For instance, orthopaedic surgeons now often include antibiotics in the spacer to treat or prevent infection. However, the consequences of this approach on the properties of the induce membrane are not fully understood. Accordingly, in a small animal model, this study aimed to determine the impact on the induced membrane of impregnating spacers with antibiotics frequently used in the IMT. We surgically created a five-mm segmental defect in the right femur of 25 adult male Sprague Dawley rats. The bone was stabilized with a plate and screws before filling the defect with a PMMA spacer. Animals were divided into five equal groups according to the type and dose of antibiotics impregnated in the spacer: A) no antibiotic (control), B) low-dose tobramycin (1.2 g/40 g of PMMA), C) low-dose vancomycin (1 g/40 g of PMMA), D) high-dose tobramycin (3.6 g/40 g of PMMA), E) high-dose vancomycin (3 g/40 g of PMMA). The animals were euthanized three weeks after surgery and the induced membranes were collected and divided for analysis. We assessed the expression of selected genes (Alpl, Ctgf, Runx2, Tgfb1, Vegfa) within the membrane by quantitative real-time PCR. Moreover, frozen sections of the specimens were used to quantify vascularity by immunohistochemistry (CD31 antigen), proliferative cells by immunofluorescence (Ki-67 antigen), and membrane thickness. Microscopic images of the entire tissue sections were taken and analyzed using FIJI software. Finally, we measured the concentration of vascular endothelial growth factor (VEGF) in the membranes by ELISA. No significant difference was found among the groups regarding the expression of genes related to osteogenesis (Alpl, Runx2), angiogenesis (Vegfa), or synthesis of extracellular matrix (Ctgf, Tgfb1) (n = four or five). Similarly, the density of proliferative cells and blood vessels within the membrane, as well as the membrane thickness, did not vary substantially between the control, low-dose, or high-dose antibiotic groups (n = four or five). The concentration of VEGF was also not significantly influenced by the treatment received (n = four or five). The addition of tobramycin or vancomycin to the spacer, at the defined low and high doses, does not significantly alter the bioactive characteristics of the membrane. These results suggest that orthopaedic surgeons could use antibiotic-impregnated spacers for the IMT without compromising the induced membrane and potentially bone healing

Osteoarthritis (OA) is a multifactorial debilitating disease that affects over four million Canadians. Although the mechanism(s) of OA onset is unclear, the biological outcome is cartilage degradation. Cartilage degradation is typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II) – partly due to the up-regulation of catabolic enzymes - aggrecanases a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). There is currently no treatment that will prevent or repair joint damage, and current medications are aimed mostly at pain management. When pain becomes unmanageable arthroplastic surgery is often performed. Interest has developed over the presence of calcium crystals in the synovial fluid of OA patients, as they have been shown to activate synovial fibroblasts inducing the expression of catabolic agents. We recently discovered elevated levels of free calcium in the synovial fluid of OA patients and raised the question on its role in cartilage degeneration. Articular cartilage was isolated from 5 donors undergoing total hip replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase and expanded in DMEM supplemented with 10% heat-inactivated FBS. OA and normal human articular chondrocytes (PromoCell, Heidelberg, Germany) were transferred to 6-well plates in culture medium containing various concentrations of calcium (0.5, 1, 2.5, and 5 mM CaCl2), and IL-1β. Cartilage explants were prepared from the same donors and included cartilage with the cortical bone approximately 1 cm2 in dimension. Bovine articular cartilage explants (10 months) were used as a control. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. Immunohistochemistry was performed on cartilage explants to measure expression of Col X, MMP-13, and alkaline phosphatase. The sulfated glycosaminoglycan (GAG, predominantly aggrecan) content of cartilage was analyzed using the 1,9-dimethylmethylene blue (DMMB) dye-binding assay, and aggregan fragmentation was determined by Western blotting using antibody targeted to its G1 domain. Western blotting was also performed on cell lysate from both OA and normal chondrocytes to measure aggrecan, Col II, MMP-3 and −13, ADAMTS-4 and −5. Ca2+ significantly decreased the proteoglycan content of the cartilage explants as determined by the DMMB assay. The presence of aggrecan and Col II also decreased as a function of calcium, in both the human OA and bovine cartilage explants. When normal and OA chondrocytes were cultured in medium supplemented with increasing concentrations of calcium (0.5–5 mM Ca2+), aggrecan and Col II expression decreased dose-dependently. Surprisingly, increasing Ca2+ did not induce the release of MMP-3, and −13, or ADAMTS-4 and-5 in conditioned media from OA and normal chondrocytes. Interestingly, inhibition of the extracellular calcium-sensing receptor CaSR) reversed the effects of calcium on matrix protein synthesis. We provide evidence that Ca2+ may play a direct role in cartilage degradation by regulating the expression of aggrecan and Col II through activation of CaSR

Osteoarthritis (OA) is a debilitating disease and the most common joint disorder worldwide. Although the development of OA is considered multifactorial, the mechanisms underlying its initiation and progression remain unclear. A prominent feature in OA is cartilage degradation typified by the progressive loss of extracellular matrix components - aggrecan and type II collagen (Col II). Cartilage homeostasis is maintained by the anabolic and catabolic activities of chondrocytes. Prolonged exposure to stressors such as mechanical loading and inflammatory cytokines can alter the phonotype of chondrocytes favoring cartilage catabolism, and occurs through decreased matrix protein synthesis and upregulation of catabolic enzymes such as aggrecanases (ADAMTS-) 4 and 5 and matrix metalloproteinases (MMPs). More recently, the endoplasmic reticulum (ER) stress response has been implicated in OA. The ER-stress response protects the cell from misfolded proteins however, excessive activation of this system can lead to chondrocyte apoptosis. Acute exposure of chondrocytes to IL-1β has been demonstrated to upregulate ER-stress markers (GADD153 and GRP78), however, it is unclear whether the ER-stress response plays a role on chronic IL-1β exposure. The purpose of this study was to determine whether modulating the ER stress response with tauroursodeoxycholic acid (TUDCA) in human OA chondrocytes during prolonged IL-1β exposure can alter its catabolic effects. Articular cartilage was isolated from donors undergoing total hip or knee replacement. Chondrocytes were recovered from the cartilage of each femoral head or knee by sequential digestion with Pronase followed by Collagenase, and expanded in DMEM-low glucose supplemented with 10% FBS. Chondrocytes were expanded in flasks for one passage before being prepared for micropellet culture. Chondrocyte pellets were cultured in regular growth medium (Control), medium supplemented with IL-1β [10 ng/mL], TUDCA [100 uM] or IL-1β + TUDCA for 12 days. Medium was replaced every three days. Cartilage explants were prepared from the donors undergoing knee replacement, and included cartilage with the cortical bone approximately 1 cm2 in dimension. Explants were cultured in the above mentioned media, however, the incubation period was extended to 21 days. RNA was extracted using Geneaid RNA Mini Kit for Tissue followed by cDNA synthesis. QPCR was performed using Cyber Green mastermix and primers for the following genes: ACAN (aggreacan), COL1A1, COL2A1, COL10A1, ADAMTS-4, ADAMTS-5, MMP-3, and MMP-13, on an ABI 7500 fast qPCR system. Although IL-1β did not significantly decrease the expression of matrix proteins, it did increase the expression of ADAMTS-4, −5, and MMP3 and −13 when compared to controls (Kruskal-Wallis, p < 0 .05, n=3). TUDCA treatment alone did not significantly increase the expression of catabolic enzymes but it did increase the expression of collagen type II. When IL-1β was coincubated with TUDCA, the expression of ADAMTS-4, ADAMTS-5, and MMP-13 significantly decreased by ∼40-fold, ∼10-fold, and ∼3-fold, respectfully. We provide evidence that the catabolic activities of IL-1β on human cartilage can be abrogated through modulation of the ER stress response

An established rabbit model was used to preliminarily investigate the effect of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets on tendon-bone interface healing. Bone, fibrocartilage and tendon tissue were harvested from the rabbits and sectioned into a book-type scaffold. The scaffolds were decellularized and their characterization was presented. BMSCs were isolated and co-cultured with the scaffolds to verify their cytocompatibility. BMSCs sheets were fabricated and inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS complex. The complex was implated in the right knee of rabbits which operated standard partial patellectomy for TBI regeneration using Imaging, histological and biomechanical examinations. The bone, fibrocartilage and tendon tissue were sectioned into a book-type scaffold before decellularization. Then we decellularized the above tissue and mostly preserved their microstructure and composition of the natural extracellular matrix, including collagen and proteoglycan. After the physicochemical and biological properties of the book-type ATS were evaluated, autologous BMSCs sheets were inserted into the book page of the scaffold to construct an autologous BMSCs-sheets/book-type ATS implants for TBI regeneration. In addition, the ATS has the advantages of non-toxicity, suitable for cell adhesion and growth as well as low immunogenicity while co-cultured with the BMSCs. At the same time, different scaffolds has the ability to induce the osteogenic, chondrogenic and tenogenic differentiation of BMSCs by immunofluorescence, reverse transcription-polymerase chain reaction and western blot analysis. To determine the efficacy of the tissue-engineered implants for TBI regeneration, we transplanted it into a rabbit patella-patellar tendon (PPT) injury model, and the rabbits were sacrificed at postoperative week 8 or 16 for the radiological, histological, and mechanical evaluation. Radiologically, Synchrotron radiation micro-computed tomography (SR-μCT) showed that BMSCs/ATS group significantly increased bone area, BV/TV, trabecular thickness and trabecular number at the healing interface as compared with other groups at postoperative week 8 or 16. Histologically, the BMSCs/ATS group showed more woven bone, and a more robust fibrocartilaginous junction with a characteristic matrix rich in proteoglycans was seen at the PPT healing interface in comparison with other groups after 8 weeks. At week 16, the healing interface in 3 groups displayed better remodeling with respect to postoperative week 8. Healing and remodeling at the PPT junction were almost complete, with a resemblance to a healthy BTI consisting of the characteristic 4 zones in all groups. At last, we used biomechanical test as functional parameters to evaluate the quality of tendon-bone healing. Biomechanical testing indicated that BMSCs/ATS group showed significantly higher failure load and stiffness than other groups at postoperative week 8 and 16. The complex composed of acellular triphase, namely bone-cartilage-tendon, scaffold (ATS) sandwiched with autologous bone mesenchymal stem cells (BMSCs) sheets can simulate the gradient structure of tendon-bone interface, inducing stem cell directional differentiation, so as to promote patella-patellar tendon interface healing effectively after injury

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of culture time. Although the characteristic anisotropy of tendon tissue is commonly replicated in scaffolds, relevant physical cues such as tendon rigidity or mechanical loading are often neglected. The objective of this study is to use tendons' main extracellular matrix component, collagen type I, to create scaffolds with an anisotropic surface topography and controlled rigidity, in an effort to engineer functional tendon tissue equivalents, with native organization and strength. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 μm) or planar polydimethylsiloxane (PDMS) molds and dried in a laminar flow hood to obtain 5 mg/ml collagen films. Surface topography and elastic modulus of the final scaffolds were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were isolated from adult tendon tissue and cultured on micro-grooved/planar scaffolds for 4, 7 and 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP at low pH and temperature. The elastic modulus of the scaffolds cross-linked with the highest concentration of 4SP matched the physiological values reported in developing tendons (∼15 kPa). Around eighty percent of the human tendon cells cultured on the cross-linked collagen films aligned in the direction of the anisotropy for 10 days in culture. At 4 days, tenoyctes cultured on micro-grooved substrates presented a significant higher nuclei aspect ratio than tenocytes cultured on planar substrates for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively in the rigid micro-grooved scaffolds after 7 days in culture. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities. Gene expression analysis and a replicate study with hBMSCs will be carried out to support the first results and to further identify the optimal biophysical conditions for tenogenic cell lineage commitment. This potentially leads to the design of smart implants that not only restore immediate tendon functionality but also provide microscopic cues that drive cellular synthesis of organized tissue-specific matrix

Adolescent idiopathic scoliosis (AIS) is a poorly understood progressive curvature of the spine. The 3-dimmensionnal spinal deformation brings abnormal biomechanical stresses on the load-bearing organs. We have recently reported for the first time the presence of facet joint cartilage degeneration comparable to age-related osteoarthritis in scoliotic adolescents. To better understand the degenerative mechanisms and explore new therapeutic possibilities, we focused on Toll-like receptors (TLRs) which are germline-encoded pattern recognition receptors that recognize pathogens and endogenous proteins such as fragmented extracellular matrix components (alarmins) present in intervertebral discs (IVD) and articular cartilage. Once activated, they regulate the production pro-inflammatory cytokines, proteases and neurotrophins which can lead to matrix catabolism, inflammation and potentially pain. These mechanisms have however not been studied in the context of AIS or facet joints. Facet joints of AIS patients undergoing corrective surgery and of cadaveric donors (non-scoliotic) were collected from consenting patients or organ donors with ethical approval. Cartilage biopsies and chondrocytes were isolated using 3mm biopsy punches and collagenase type 2 digestion respectively. qPCR was used to assess gene expression of the degenerative factors (MMP3, MMP13, IL-1ß, IL-6, IL-8) The biopsies were cut into two equal halves, one was treated for 4 days with a TLR2 agonist (Pam2CSK4, Invivogen) in serum-free chondrocyte media while the other one was cultured in media alone. MMP3, MMP13, IL-6 and IL-8 ELISAs and DMMB assays were performed on the biopsy cultured media. The ex vivo cartilage was then fixed, cryosectionned and also stained with SafraninO-Fast Green dyes. Baseline gene expression levels of TLR1,−2,−4,−6 were all upregulated in scoliotic chondodryctes compared to non-scoliotic. Pearson correlation analysis revealed that all TLR1,−2,−4,−6 gene expression correlated strongly and significantly with degenerative markers (MMP3, MMP13, IL-6, IL-8) in scoliotic chondrocytes but not in non-scoliotic. (Figure 1) When monolayer facet joint chondrocytes were activated with Pam2CSk4, there was a significant upregulation in previously described degenerative markers, TLR2 and NGF, a potent neurotrophin. These findings were strengthened by protein secretion analysis of select markers such as MMP-3, −13, IL-6 and IL-8 which were all upregulated after TLR2 activation. The scoliotic biopsies which were treated with Pam2CSK4 had a significant loss of proteoglycan content as shown by histology, was reflected in the proteoglycan content found in the media by DMMB. TLR gene expression levels were upregulated and correlated with proteases and pro-inflammatory cytokines in degenerating scoliotic cartilage, suggesting they promote cartilage degradation, especially considering the lack of correlations in non-scoliotic healthy cartilage. Furthermore, when TLRs are activated by Pam2CSK4 it triggers the release of the same proteases and pro-inflammatory cytokines in our ex vivo experiment. All this exacerbates the loss of proteoglycan in the cartilage ex vivo model after four days of insult with a TLR2 specific agonist. These results suggest that TLRs are an important pathway partaking in the cartilage degeneration of scoliotic facet joints and potentially all cartilage beyond our scope. Future studies aim at blocking TLRs to alleviate proteolysis and inflammation. For any figures or tables, please contact the authors directly

The zonal organization of articular cartilage is crucial in providing the tissue with mechanical properties to withstand compression and shearing force. Current treatments available for articular cartilage injury are not able to restore the hierarchically organized architecture of the tissue. Implantation of zonal chondrocyte as a multilayer tissue construct could overcome the limitation of current treatments. However, it is impeded by the lack of efficient zonal chondrocyte isolation protocol and dedifferentiation of chondrocytes during expansion on tissue culture plate (TCP). This study aims to develop a protocol to produce an adequate number of high-quality zonal chondrocytes for clinical application via size-based zonal chondrocyte separation using inertial spiral microchannel device and expansion under dynamic microcarrier culture. Full thickness (FT) chondrocytes isolated from porcine femoral condyle cartilage were subjected to two serial of size-based sorting into three subpopulations of different cell sizes, namely small (S1), medium (S2), and large (S3) chondrocytes. Zonal phenotype of the three subpopulations was characterised. To verify the benefit of stratified zonal chondrocyte implantation in the articular cartilage regeneration, a bilayer hydrogel construct composed of S1 chondrocytes overlaying a mixture of S2 and S3 (S2S3) chondrocytes was delivered to the rat osteochondral defect model. For chondrocyte expansion, two dynamic microcarrier cultures, sort-before-expansion and sort-after-expansion, which involved expansion after or before zonal cells sorting, were studied to identify the best sort-expansion strategy. Size-sorted zonal chondrocytes showed zone-specific characteristics in qRT-PCR with a high level of PRG4 expression in S1 and high level of aggrecan, Type II and IX collagen expression in S2 and S3. Cartilage reformation capability of sorted zonal chondrocytes in three-dimensional fibrin hydrogel showed a similar trend in qRT-PCR, histology, extracellular matrix protein quantification and mechanical compression test, indicating the zonal characteristics of S1, S2 and S3 as superficial (SZ), middle (MZ) and deep (DZ) zone chondrocytes, respectively. Implantation of bilayered zonal chondrocytes resulted in better cartilage tissue regeneration in a rat osteochondral defect model than FT control group, with predominantly Type II hyaline cartilage tissue and significantly lower Type I collagen. Dynamic microcarrier expansion of sorted zonal chondrocytes was able to retain the zonal cell size difference that correlate to zonal phenotype, while maintaining the rounded chondrocyte morphology and F-actin distribution similar to that in mature articular cartilage. With the better retention of zonal cell size and zonal phenotype relation on microcarrier, zonal cells separation was achievable in the sort-after-expansion strategy with cells expanded on microcarrier, in comparison to cells expanded on TCP. Inertial spiral microchannel device provides a label-free and high throughput method to separate zonal chondrocytes based on cell size. Stratified implantation of zonal chondrocytes has the potential to improve articular cartilage regeneration. Dynamic microcarrier culture allows for size-based zonal chondrocyte separation to be performed on expanded chondrocytes, thus overcoming the challenge of limited tissue availability from the patients. Our novel zonal chondrocyte isolation and expansion protocol provide a translatable strategy for stratified zonal chondrocyte implantation that could improve articular cartilage regeneration of critical size defects