Recently, a new suture was designed to minimize laxity in order to preserve consistent tissue approximation while improving footprint compression after tendon repair. The aims of this study were: (1) to compare the biomechanical competence of two different high strength sutures in terms of slippage and failure load, (2) to investigate the influence of both knots number and different media (air, saline and fat) on the holding capacity of the knots. Alternating surgical knots of two different high-strength sutures (group1: FibreWire; group2: DynaCord; n = 105) were tied on two roller bearings with 50N tightening force. Biomechanical testing was performed in each medium applying ramped monotonic tension to failure defined in terms of either knot slippage or suture rupture. For each group and medium, seven specimens with either 3, 4, 5, 6, or 7 knots each were tested, evaluating their knot slippage and ultimate load to failure. The minimum number of knots preventing slippage failure and thus resulting in suture rupture was determined in each group and medium, and taken as a criterium for better performance when comparing the groups. In each group and medium failure occurred via suture rupture in all specimens for the following minimum knot numbers: group1: air – 7, saline – 7, fat – 7; group2: air – 6; saline – 4; fat – 5. The direct comparison between the groups when using 7 knots demonstrated significantly larger slippage in group1 (6.5 ± 2.2 mm) versus group2 (3.5 ± 0.4 mm) in saline (p < 0.01) but not in the other media (p ≥0.52). Ultimate load was comparable between the two groups for all three media (p ≥ 0.06). The lower number of required knots providing sufficient repair stability, smaller slippage levels and identical suture strength, combined with the known laxity alleviation effect demonstrate advantages of DynaCord versus FibreWire

Summary Statement. The purpose is to evaluate the effects of internet usage on new patient referral patterns to identify optimal patient recruitment and communication. Overall, social networking and internet may be an effective way for surgeons to recruit a wider patient population. Introduction. Prior studies in other medical specialties have shown that social networking and internet usage has become an increasingly important means of patient communication and referral. However, this information is lacking in the orthopaedics literature. In this study, we evaluate the means by which new patients arrive at orthopaedic clinics in a major academic center. The purpose is to evaluate the effects of internet or social media usage on new patient referral patterns to identify avenues to optimise patient recruitment and communication. Patients and Methods. New patients were recruited in a major academic orthopaedic clinic to complete a 15-item questionnaire with demographic information, social media use/networking and referral method. Data was collected for all orthopaedic sub-specialties and analyzed accordingly. Statistical analysis was performed. Results. Of the 752 responses, there were 66% female and 34% male responses. Responses were obtained from hand (142), sports medicine (303), foot and ankle (129), joints/tumor (95) and trauma (83) services. Overall, 51% report using social networking sites such as Facebook or Twitter. Of the patients that report not using social network sites, 92% are over the age of 40. Joints/tumor patients most commonly had seen another orthopaedic surgeon prior to their visit (59%) and had prior surgery (42%). Most patients traveled under 60 miles and were referred by their primary care physicians. Between 18–26% of all patients used a physician review website before consultation. The majority of the patients prefer communicating with their physician via the phone(68%) compared to email(32%). Independent associations found that sports medicine patients tend to be higher social networking users (35.9%) relative to other services (9.8–17.9%) and was statistically higher when compared to the joints/tumor service (P<.0001). The multivariate logistic regression model showed that the sports service was generally more likely to have social networking users with the exception of the foot/ankle service), however these differences were not statistically significant. The biggest indicator predicting social media usage in the orthopaedic population was age. The older the patient population, the less likely patients will use social networking sites. Non-doctorate patients were more likely to be social media users compared to doctorate level individuals, but was not statistically significant. Patients that lived from 120 to 180 miles from the hospital were significantly more likely to be social media users, as were patients that did research on their condition prior to their new patient appointment. Discussion and Conclusion. Orthopaedic patients who use social media are more likely to be younger, research their condition prior to their appointment and undergo an average day's travel (120–180 miles) to see a physician. Up to 26% of all patients have seen or used a physician review site prior to their visit. Despite the increased social media usage, most orthopaedic patients still prefer telephone communication with their physicians. Overall, social networking and internet may be an effective way for surgeons to recruit a wider patient population. In an increasingly competitive market, surgeons with younger patient populations (Sports Medicine) will need to utilise social networking and the internet to capture new patient referrals

Tendon injuries are common and current therapies often are unsuccessful. Cell-based therapy using mesenchymal stem cells (MSCs) seems to be the most promising approach to heal tendon. Moreover, providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices (GMP). Adipose-derived stem cells (n=4) were cultured in 6-well plates coated with type-I collagen in a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL). At passage 4, ASCs were induced to tendon lineage for 14 days using 100ng/ml CTGF, 10ng/ml TGFβ3, 50ng/ml BMP12 and 50µg/ml ascorbic acid in the SF (SF-TENO) or in the hPL (hPL-TENO) medium. Cells cultured without any supplements are used as control. Morphological appearance, cell viability and FACS were performed in undifferentiated cells to evaluate the xenogenic-free culture conditions; the gene and protein expression were performed by RT-PCR and immunofluorescence to evaluate to expression of stem cell- and tendon-related markers upon cell differentiation. SF-CTRL and hPL-CTRL showed similar viability and MSC's surface proteins and expressed the stemness markers NANOG, OCT4 and Ki67. Moreover, both SF-TENO and hPL-TENO expressed significant higher levels of SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 genes already at 3d (p<0.05) respect to CTRLs. Scleraxis and collagen were also detected in both SF-TENO and hPL-TENO at protein level in higher amount than CTRLs. In conclusion, ASCs exposed to CTGF, BMP12, TGFb3 and AA in both serum and xenogenic-free media possess similar tenogenic differentiation ability moving forward the GMP-compliant approaches for the clinical use of ASCs

Titanium alloys are one of the most used for orthopaedic implants and the fabrication of them by 3D printing technology is a raising technology, which could effectively resolve existing challenges. Surface modification of Ti surfaces is often necessary to improve biocorrosion resistance, especially in inflammatory conditions. Such modification can be made by coatings based on hydrogels, like alginate (Alg) - a naturally occurring anionic polymer. The properties of the hydrogel can be further enhanced with calcium phosphates like octacalcium phosphate (OCP) as a precursor of biologically formed hydroxyapatite. Formed Alg-OCP matrices have a high potential in wound healing, delivery of bioactive agents etc. but their effect on 3D printed Ti alloys performance was not well known.

In this work, Alg-OCP coated 3D printed samples were studied with electrochemical measurements and revealed significant variations of corrosion resistance vs. composition of the coating. The potentiodynamic polarization test showed that the Alg-OCP-coated samples had lower corrosion current density than simple Alg-coated samples. Electrochemical impedance spectroscopy indicated that OCP incorporated hydrogels had also a high value of the Bode modulus and phase angle. Hence Alg-OCP hydrogels could be highly beneficial in protecting 3D printed Ti alloys especially when the host conditions for the implant placement are inflammatory.

AcThis work was supported by the European Union Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Actions GA860462 (PREMUROSA). The authors also acknowledge the access to the infrastructure and expertise of the BBCE – Baltic Biomaterials Centre of Excellence (European Union Horizon 2020 programme under GA857287).

Low back pain resulting from Interertebral disc (IVD) degeneration is a serious worldwide problem, with poor treatment options available. Notochordal (NC) cells, are a promising therapeutic cell source with anti-catabolic and regenerative effect. However, their behaviour in the harsh degenerate environment is unknown. Porcine NC cells (pNCs), and Human NP cells from degenerate IVDs were cultured in alginate beads to maintain phenotype. Cells were cultured alone or in combination, or co-stimulated with notochordal cell condition media (NCCM), in media to mimic the healthy and degenerate disc environment, together with controls for up to 1 week. Following culture viability, qPCR and proteomic analysis using Digiwest was performed. A small increase in pNC cell death was observed in degenerated media compared to standard and healthy media, with a further decrease seen when cultured with IL-1β. Whilst no significant differences were seen in phenotypic marker expression in pNCs cultured in any media at gene level (ACAN, KRT8, KRT18, FOXA2, COL1A1 and Brachyury). Preliminary Digiwest analysis showed increased protein production for Cytokeratin 18, src and phosphorylated PKC but a decrease in fibronectin in degenerated media compared to standard media. Human NP cells cultured with NCCM, showed a decrease in IL-8 production compared to human NP cells alone when cultured in healthy media. However, gene expression analysis (ACAN, VEGF, MMP3 and IL-1β) demonstrated no significant difference between NP only and NP+NCCM groups. Studying the behaviour of the NCs in in vitro conditions that mimic the in vivo healthy or degenerate niche will help us to better understand their potential for therapeutic approaches. The potential use of NC cell sources for regenerative therapies can then be translated to investigate the potential use of iPSCs differentiated into NC cells as a regenerative cell source

3D spheroid culture is a bridge between standard 2D cell culture and in vivo research which mimics the physiological microenvironment in scaffold-free conditions. Here, this 3D technique is being investigated as a potential method for engineering bone tissue in vitro. However, spheroid culture can exhibit limitations, such as necrotic core formation due to the restricted access of oxygen and nutrients. It is therefore important to determine if spheroids without a sizeable necrotic core can be produced. This study aims to understand necrotic core formation and cell viability in 3D bone cell spheroids using different seeding densities and media formulations. Differentiated rat osteoblasts (dRObs) were seeded in three different seeding densities (1×10. 4. , 5×10. 4. , 1×10 cells) in 96 well U-bottom cell-repellent plates and in three different media i.e., Growth medium (GM), Mineralisation medium 1 (MM1) and MM2. Spheroids were analysed from day 1 to 28 (N=3, n=2). Cell count and viability was assessed by trypan blue method. One way ANOVA and post-hoc Tukey test was performed to compare cell viability among different media and seeding densities. Histological spheroid sections were stained with hematoxylin and eosin (H&E) to identify any visible necrotic core. Cell number increased from day 1 to 28 in all three seeding densities with a notable decrease in cell viability. 1×10. 4. cells proliferated faster than 5×10. 4. and 1×10. 5. cells and had proportionately similar cell death. The necrotic core area was relatively equivalent between all cell seeding densities. The larger the spheroid size, the larger is the size of the necrotic core. This study has demonstrated that 3D spheroids can be formed from dRobs at a variety of seeding densities with no marked difference in necrotic core formation. Future studies will focus on utilising the bone cell spheroids for engineering scalable scaffold-free bone tissue constructs

Abstract. Objective. The preparation of host degenerate cartilage for repair typically requires cutting and/or scraping to remove the damaged tissue. This can lead to mechanical injury and cartilage cell (chondrocytes) death, potentially limiting the integration of repair material. This study evaluated cell death at the site of cutting injury and determined whether raising the osmotic pressure (hyper-osmolarity) prior to injury could be chondroprotective. Methods. Ex vivo human femoral head cartilage was obtained from 13 patients (5 males and 8 females: 71.8 years old) with Ethical Permission and Patient consent. Cartilage wells were created using 3 or 5mm biopsy punches. Cell death at the wounded edge of the host cartilage and the edge of the extracted explants were assessed by quantifying the percentage of cell death (PCD) and measuring the width of the cell death zone at identified regions of interest (ROI) using the confocal laser scanning microscopy and image analysis software. To assess the chondroprotective effect of hyper-osmolarity, cartilage specimens were incubated in 340 or 600mOsm media, five minutes prior to injury to allow the chondrocytes to respond to the altered osmolarity. Wounded cartilage explants and cartilage wells were then cultured for a further 150 minutes following injury. Results. In 340mOsm media, the PCD around the 3mm cartilage wells was significantly less compared to the corresponding explants (20.05±10.24% vs 35.25±4.86%; P=0.0003). When using the 5mm biopsy punch, the PCD at the wound edges was significantly lower when compared to the 3mm cartilage wells (13.33±7.80% vs 20.05±10.24%; P=0.0121) at the same osmolarity. The width of the cell death zone for the well edges for both 3 and 5mm punches was significantly narrower when compared to their corresponding harvested cartilage explants in 340mOsm media (P<0.0001; P=0.0218, respectively). Exposing cartilage to raised osmolarity (600mOsm) prior to injury significantly reduced the PCD for cartilage wells produced by the 3mm biopsy punches (from 20.05±10.24% to 12.24±6.00%; P=0.0025). In addition, the zone of cell death was marginally reduced at the edges of the 5mm cartilage wells (19.25±15.78mm to 12.72±9.09mm; P=0.0499). Conclusions. The choice of biopsy punch and the osmolarity of the incubation medium prior to cartilage injury markedly affected the extent of chondrocyte death both at the edges of the cartilage wells and the explants. The smaller biopsy punch caused more chondrocyte death in the native cartilage wells compared to the larger punch, but this could be compensated for by the chondroprotective effect of raising the osmotic pressure. In general, there was less cell death at the wounded edges of the cartilage wells, compared to the explants. These results suggest that there is scope for further optimising the cutting implements used to create the cartilage wells and protecting chondrocytes by hyper-osmolarity in order to minimize cell death at cut edges and potentially enhance integration between cartilage repair material and host cartilage. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Abstract. OBJECTIVE. Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA. 1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology. 2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues. 3. , expressed in osteocytes. 4. and known to be downregulated in bone OA mechanical loading. 5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients. 6. HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. METHODS. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test. RESULTS. IPSC-derived nociceptor-like cells display elongated (>5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p<0.001), Sema3B (13-fold, p<0.001), Sema3C (0.4-fold, p<0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls. CONCLUSIONS. Here we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

The enthesis is a specialised zonal tissue interface between tendon and bone, essential for adequate force transmission and composed by four distinct zones, namely tendon, fibrocartilage, mineralized fibrocartilage and bone. Following injuries and surgical repair, the enthesis is often not reestablished and so far, traditionally used tissue substitutes have lacked to reproduce the complexity of the native tissue. In this work, we hypothesised that a collagen-based three-layer scaffold that mimic the composition of the enthesis, in combination with bioactive molecules, will enhance the functional regeneration of the enthesis. A three-layer sponge composed of a tendon-like layer (collagen I), a cartilage-like layer (collagen II) and a bone-like layer (collagen I and hydroxyapatite) was fabricated by an iterative layering freeze-drying technique. Scaffold porosity and structural continuity at the interfaces were assessed through SEM analysis. Bone-marrow derived stem cells (BMSCs) were seeded by syringe vacuum assisted technique on the scaffold. Scaffolds were cultured in basal media for 3 days before switching to differentiation media (chondrogenic, tenogenic and osteogenic). BMSCs metabolic activity, proliferation and viability were assessed by alamarBlue, PicoGreen and Live/Dead assays. At D21 the scaffolds were fixed, cryosectioned and Alizarin Red and Alcian Blue stainings were performed in order to evaluate BMSC differentiation towards osteogenic and chondrogenic lineage. The presence of collagen I and tenascin in the scaffolds was evaluated by immunofluorescence staining at D21 in order to assess tenogenic differentiation of BMSCs. Subsequently, the cartilage-like layer was functionalized with IGF-1, seeded with BMSCs and cultured in basal media up to D21. Structural continuity at the interfaces of the scaffolds was confirmed by SEM and scaffold porosity was assessed as >98%. The scaffolds supported cell proliferation and infiltration homogeneously throughout all the layers up to D21. Osteogenic differentiation of BMSC selectively in the bone-like layer was confirmed by Alizarin red staining in scaffolds cultured in basal and osteogenic media. Alcian blue staining revealed the presence of proteoglycans selectively in the cartilage-like layer in scaffolds cultured in chondrogenic media but not in basal media. Increased expression of the tenogenic markers collagen I and tenascin were observed in the tendon-like layer of scaffolds cultured in tenogenic but not in basal media for 21 days. The presence of IGF-1 increased osteogenic and chondrogenic differentiation of BMSCs, whereas no difference was observed for tenogenic differentiation. In conclusion, a 3-layer collagen sponge was successfully fabricated with distinct but integrated layers; the different collagen composition of the non-functionalized 3-layer sponge was able to regulate BMSC differentiation in a localized manner within the scaffold. The scaffold functionalization with IGF-1 accelerated chondrogenic and osteogenic BMSC differentiation. Overall, functionalization of the 3-layer scaffolds holds promising potential in enthesis regeneration

The number of seven needed knots to provide secure hold of high strength sutures was previously reported. New technologies like tape sutures and sutures with a salt infused silicon core have been developed, potentially reducing the number of needed knots. Study aims: To investigate the influence of (1) throw number and (2) different ambient conditions on knot security in two different high-strength sutures, and (3) to compare their biomechanical competence. Two sutures (SutureTape (FT); n=56 and DynaTape (DT); n=56) were assigned for knot tying. Specimens were exposed to different media during tying, namely air, saline solution, and fat. A monotonic tensile ramp was applied. For each suture and ambient condition, seven specimens with 3 to 7 throws each were tested (n=7), evaluating their slippage and ultimate force to failure. The minimum number of throws preventing suture unraveling was determined in each suture and condition. For each suture type and condition failure occurred via rupturing in all specimens for the following minimum number of throws: FT: dry–6, wet–6, fatty-wet–6; DT: dry–6; wet–4; fatty-wet–5. No significant differences were found comparing ultimate load to rupture of the two groups with minimum number of needed throws in each media. (FT dry-6 vs. DT dry-6; p<0.07); (FT wet-6 vs. DT wet-4; p<0.20); (FT fat-6 vs. DT fat-5; p<0.58). Knot slippage of DT was significantly higher in wet and fatty conditions compared to ST p<0.001 and p<0.004. In fatty-wet conditions DT requires 5 throws to achieve a secure knot. In wet conditions this number can be reduced to 4 throws. FT needs 6 throws to provide a stable knot in all conditions. The biomechanical competence of both sutures in terms of knot slippage and peak force are comparable

Introduction. Tendinopathies represent a significant health burden, causing inflammation, pain, and reducing quality of life. The pivotal role of macrophages (Mφ) characterized by their ability to differentiate into proinflammatory (M1) or anti-inflammatory (M2) phenotypes depending on the microenvironment, has gained significant interest in tissue inflammation research. Additionally, existing literature states that the interplay between tenocytes and immune cells during inflammation involves unidentified soluble factors (SF). This study aimed to investigate the effect of extracellular vesicles (EVs) and SF derived from polarized Mφ on tendon cells to provide deeper insights of their potential therapeutic applications in the context of inflammation. Method. Human monocytes were isolated from blood donor buffy coats and differentiated into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, EVs were isolated from the conditioned media from polarized Mφ and comprehensively characterized. In parallel, the elution media containing SF was collected. Furthermore, the EVs and SF were released independently onto tenocytes from human donors, previously induced with IL-1β to simulate an inflammatory environment. Finally, mRNA levels of tendon-related markers were evaluated by qPCR after the exposure to these EVs and SF. Result. Notably, the study found that the viability of the cells was not affected by the exposure to EVs nor SF, indicating their potential safety for therapeutic use. Moreover, the mRNA content of tendon-derived cells was evaluated following exposure to Mφ-EVs and SF revealing alterations in gene expression. Interestingly, a significant increase in the expression of tenomodulin was observed in tendon cells treated with Mφ-EVs. Conclusion. These results mark a significant advancement in understanding the interplay between Mφ and tenocytes at a molecular level. To fully understand the underlying causes of Mφ-EVs effects, and its potential clinical application in tendon inflammatory diseases, further comprehensive research is required. Acknowledgments. Carlos III Health Institute and the European Social Fund for contract CP21/00136 and project PI22/01686

Tendon injuries are a common problem that can significantly impact an individual's quality of life. While traditional surgical methods have been used to address this issue, Extracellular Vesicles (EVs) have emerged as a promising approach to promote tendon repair and regeneration mechanisms, as they deliver specific biological signals to neighbouring cells. In this study, we extracted human Tendon Progenitor Stem cells (hTPSCs) from surgery explants and isolated their EVs from perfused and static media. hTPSCs were isolated from tendon surgery biopsy (Review Board prot./SCCE n.151, 29/10/2020) and cultured in both static and dynamic conditions, using a perfusion bioreactor (1ml/min). When cells reached 80% confluence, they were switched into a serum-free medium for 24 hours for EVs-production. Conditioned media was ultra-centrifuged for 90min (100000g). The recovered pellet was then characterized by size and concentration (Nanosight NS300), Zeta potential (Mastersizer S), morphology (SEM and TEM) and protein quantification. hTPSCs stemness and multipotency were confirmed through CD73, CD90, and CD105 expression and confirmation of quad-lineage (adipo-osteo-chondro-teno) differentiation. After 7 days, hTPSCs were ready for EVs-production. Ultracentrifugation revealed the presence of particles with a concentration of 7×107 particles/mL consistent across both cultures. Further characterization indicated that EVs collected from perfused conditions displayed an elevated vesicle mean size (mean 143±6.5 nm) in comparison to static conditions (mean 112±7.4 nm). Consistent with, but not in proportion with, the above protein content was measured at 20 ng/ml (dynamic) and 7 ng/mL (static) indicating a nearly 3-fold increase in concentration associated with a ~22% increase in particle size. Proposed data showed that sub-200 diameter vesicles were successfully collected from multipotent hTPSCs starvation, and the vesicle size and protein concentration were compatible with established EV literature; furthermore, dynamic culture conditions seemed more suitable for EVs-production. Further characterization will be required to better understand, EVs-compositions and their role in tendon regenerative events

Pericytes are contractile, motile cells that surround the capillary. Recent studies have shown that pericytes promoted joint fibrosis and induced subchondral bone angiogenesis, indicating the role of pericytes in osteoarthritis (OA). However, whether pericytes are involved in regulating inflammatory and catabolic response, as well as fibrotic repair of cartilage is still unclear. Here we used 2D and 3D models to investigate the communication of pericytes and chondrocytes under inflammatory osteoarthritis conditions. CD34-CD146+ pericytes were isolated and sorted from human bone marrow. Human OA chondrocytes were isolated from OA joints. In 2D studies, monolayer cultured chondrocytes were treated +/- pericyte conditioned media, +/- 1ng/ml IL1β for 24h. In 3D studies, pericytes and chondrocytes were cultured within fibrin gel in 3D polyurethane scaffolds, separately or combined for 7 days, followed by treatment of +/- IL1β for another 7 days (Fig 2A). The inflammatory response, catabolic activity and expression of fibrosis markers of chondrocytes and pericytes were measured by ELISA and/or q-rtPCR. Pericytes had weak inflammatory, catabolic and fibrotic response to IL1β (data not shown). The 2D study showed that pericyte conditioned media promoted inflammation, catabolism and fibrosis markers of chondrocytes, in the absence of IL1β treatment (Figure 1). However, study in 3D showed that coculture of chondrocytes and pericytes reduced the inflammatory and catabolic response of chondrocytes to IL1β and induced fibrosis markers in chondrocytes (Figure 2). Pericytes are involved in regulating inflammatory response, catabolic response and fibrosis of chondrocytes. The opposite results from 2D and 3D experiments indicate the variety of the regulatory role of pericytes in the interaction with chondrocytes within different culture models. The underlying mechanism is under evaluation with on-going studies. Acknowledgements. This study was funded by SINPAIN project, from European Union's Horizon Europe research and innovation programme under Grant Agreement NO. 101057778. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them. For any figures or tables, please contact the authors directly

Introduction and Objective. Mesenchymal stem cells (MSC) are attractive candidates for bone regeneration approaches. Benefits of MSC therapy are mainly attributed to paracrine effects via soluble factors, exerting both immunoregulatory and regenerative actions. Encapsulation of MSC in hydrogels prepared with extracellular matrix (ECM) proteins has been proposed as a strategy to enhance their survival and potentiate their function after implantation. Functional activity of MSC can be regulated by the physical and mechanical properties of their microenvironment. In this work, we investigated whether matrix stiffness can modulate the crosstalk between MSC encapsulated in collagen hydrogels with macrophages and osteoblasts. Materials and Method. Collagen hydrogels with a final collagen concentration of 1.5, 3 and 6 mg/mL loaded with human MSC were prepared. Viscoelastic properties of hydrogels were measured in a controlled stress rheometer. Cell distribution into the hydrogels was examined using confocal microscopy and the levels of the immunomodulatory factors interleukin-6 (IL-6) and prostaglandin E. 2. (PGE. 2. ) released by MSC were quantified by immunoassays. To determine the effect of matrix stiffness on the immunomodulatory potential of MSC, human macrophages obtained from healthy blood were cultured in media conditioned by MSC in hydrogels. The involvement of IL-6 and PGE. 2. in MSC-mediated immunomodulation was investigated employing neutralizing antibodies. Finally, the influence of soluble factors released by MSC in hydrogels on bone-forming cells was studied using osteoblasts obtained from trabecular bone explants from patients with osteonecrosis of the femoral head during total hip arthroplasty. Results. MSC loaded in hydrogels containing varying concentrations (1.5, 3 and 6 mg/mL) of collagen were viable. Rheology measurements determined that the hydrogel stiffness increased with increasing collagen concentration. Encapsulation of MSC into hydrogels barely affected their storage modulus values. MSC acquired a three-dimensional (3D) arrangement in all hydrogels and showed a more elongated shape in hydrogels with higher stiffness. The secretion of IL-6 and PGE. 2. by MSC in hydrogels increased with increasing matrix stiffness. Media conditioned by MSC encapsulated in stiffer hydrogels decreased TNF-α levels secreted by macrophages to a higher extent than media conditioned by MSC in softer hydrogels. This effect was partially mediated by PGE. 2. Finally, our preliminary results indicated that factors released by MSC in hydrogels regulated osteoblast-mediated mineralisation and this effect was dependent on hydrogel stiffness. Conclusions. Our data indicate that matrix stiffness of collagen hydrogels regulates the production of soluble factors by MSC and their paracrine actions on macrophages and osteoblasts

The extracellular matrix (ECM) is the non-cellular structural support that provides cells with a network of biochemical and biomechanical factors for cellular processes. The ECM regulates cell function, differentiation and homeostasis. Here, we present a proteomics characterization of three commonly used additive manufactured polymers: polylactic acid (PLA), polyactive (PEOT/PBT) and polycaprolactone (PCL). We cultured human mesenchymal stromal cells (hMSCs) and make them undergo chondrogenic and osteogenic differentiation on 3D printed PCL, PEOT/PBT and PLA scaffolds. hMSCs were cultured in basal, chondrogenic and osteogenic media (200000 cells/scaffold) and analyzed after 35 days of culture. Differentiation was proved through biochemical assays, immunofluorescence and histology. The protein content was explored using label free liquid chromatography mass spectrometry (LC-MS), which revealed upregulated proteins and their related pathways. A higher difference was found among different media compared to the scaffold type through principal component analysis (PCA). Interestingly, in all three materials, chondrogenesis was characterized by a lower but more diverse amount of proteins. PCL induced ECM production in both differentiation media, but it led to more apoptosis and GAG degradation in the chondrogenic medium compared to the osteogenic one. During chondrogenesis in PEOT/PBT and PLA, cell differentiation resulted in the activation of stress response cascades, collagen formation and ECM remodelling. On the other hand, in osteogenesis, PCL enhanced insulin-like growth factor pathway and fibrin clot related pathways

Aspiration arthrography using an iodinated contrast medium is a useful tool for the investigation of septic or aseptic loosening of arthroplasties and of septic arthritis. Previously, the contrast media have been thought to cause false negative results in cultures when present in aspirated samples of synovial fluid, probably because free iodine is bactericidal, but reports have been inconclusive. We examined the influence of the older, high osmolar contrast agents and the low osmolar media used currently on the growth of ten different micro-organisms capable of causing deep infection around a prosthesis. Five media were tested, using a disc diffusion technique and a time-killing curve method in which high and low inocula of micro-organisms were incubated in undiluted media. The only bactericidal effects were found with low inocula of Escherichia coli and Pseudomonas aeruginosa in ioxithalamate, one of the older ionic media. The low and iso-osmolar iodinated contrast media used currently do not impede culture. Future study must assess other causes of false negative cultures of synovial fluid and new developments in enhancing microbial recovery from aspirated samples

Growing evidence has suggested that paracrine mechanisms of Mesenchymal stem cell (MSC) may be involved in the underlying mechanism of MSC after transplantation, and extracellular vesicles (EVs) are an important component of this paracrine role. The aim of this study was to investigate the in vitro osteogenic effects of EVs derived from undifferentiated mesenchymal stem cells and from chemically induced to differentiate into osteogenic cells for 7 days. Further, the osteoinductive potential of EVs for bone regeneration in rat calvarial defects was assessed. We could isolate and characterize EVs from naïve and osteogenic-induced MSCs. Proteomic analysis revealed that EVs contained distinct protein profiles, with Osteo-EVs having more differentially expressed proteins with osteogenic properties. EVs were found to enhance the proliferation and migration of cultured MSC. In addition, the study found that Osteo-EVs/MEM combination scaffolds could enhance greater bone formation after 4 weeks as compared to native MEM loaded with serum-free media. The study suggests that EVs derived from chemically osteogenic-induced MSCs for 7 days can significantly enhance both the osteogenic differentiation activity of cultured hMSCs and the osteoinductivity of MEM scaffolds. The results indicate that Osteo-MSC-secreted nanocarriers-EVs combined with MEM scaffolds can be used for repairing bone defects

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure

Cartilage lacks the ability to self-repair when damaged, which can lead to the development of degenerative joint disease. Despite intensive research in the field of cartilage tissue engineering, there is still no regenerative treatment that consistently promotes the development of hyaline cartilage. Extracellular matrix (ECM) derived hydrogels have shown to support cell adhesion, growth and differentiation [1,2]. In this study, porcine articular cartilage was decellularized, solubilised and subsequently modified into a photo-crosslinkable methacrylated cartilage ECM hydrogel. Bone marrow derived mesenchymal stem/stromal cells (MSCs) were encapsulated into both methacrylated ECM hydrogels (ECM-MA) and gelatin methacryloyl (GelMA) as control hydrogel, and their chondrogenic potential was assessed using biochemical assays and histological analysis. We found that successful decellularization of the cartilage tissue could be achieved while preserving key ECM components, including collagen and glycosaminoglycans. A live-dead assay demonstrated good viability of MSCs withing both GelMA and ECM-MA hydrogels on day 7. Large increases in sGAG accumulation was observed after 21 days of culture in chondrogenic media in both groups. Histological analysis revealed the presence of a more fibrocartilage tissue in the GelMA group, while cells embedded within the ECM-MA showed a round and chondrocytic-like morphology. Both groups stained positively for proteoglycans and collagen, with limited evidence of calcium deposition following Alizarin Red staining. These results show that ECM-MA hydrogels support a hyaline cartilage phenotype and robust cartilaginous matrix production. Future studies will focus on the printability of ECM-MA hydrogels to enable their use as bioinks for the biofabrication of functional tissues

Device-associated bacterial infections are a major and costly clinical challenge. This project aimed to develop a smart new biomaterial for implants that helps to protect against infection and inflammation, promote bone growth, and is biodegradable. Gallium (Ga) doped strontium-phosphate was coated on pure Magnesium (Mg) through a chemical conversion process. Mg was distributed in a graduated manner throughout the strontium-phosphate coating GaSrPO4, with a compact structure and a Ga-rich surface. We tested this sample for its biocompatibility, effects on bone remodeling and antibacterial activities including Staphylococcus aureus, S. epidermidis and E. coli - key strains causing infection and early failure of the surgical implantations in orthopaedics and trauma. Ga was distributed in a gradient way throughout the entire strontium-phosphate coating with a compact structure and a gallium-rich surface. The GaSrPO4 coating protected the underlying Mg from substantial degradation in minimal essential media at physiological conditions over 9 days. The liberated Ga ions from the coatings upon Mg specimens inhibited the growth of bacterial tested. The Ga dopants showed minimal interferences with the SrPO4 based coating, which boosted osteoblasts and undermined osteoclasts in in vitro co-cultures model. The results evidenced this new material may be further translated to preclinical trial in large animal model and towards clinical trial. Acknowledgements: Authors are grateful to the financial support from the Australian Research Council through the Linkage Scheme (ARC LP150100343). The authors acknowledge the facilities, and the scientific and technical assistance of the RMIT University and John Curtin School of Medical Research, Australian National University