Differential proteomics profile of microcapillary networks in response to sound pattern-driven local cell density enhancement.

Spatial cell organization and biofabrication of microcapillary networks in vitro has a great potential in tissue engineering and regenerative medicine. This study explores the impact of local cell density enhancement achieved through an innovative sound-based patterning on microcapillary networks formation and their proteomic profile. Human umbilical vein endothelial cells (HUVEC) and human pericytes from placenta (hPC-PL) were mixed in a fibrin suspension. The mild effect of sound-induced hydrodynamic forces condensed cells into architected geometries showing good fidelity to the numerical simulation of the physical process. Local cell density increased significantly within the patterned areas and the capillary-like structures formed following the cell density gradient. Over five days, these patterns were well-maintained, resulting in concentric circles and honeycomb-like structures. Proteomic analysis of the pre-condensed cells cultured for 5 days, revealed over 900 differentially expressed proteins when cells were preassembled through mild-hydrodynamic forces. Gene ontology (GO) enrichment analysis identified cellular components, molecular functions, and biological processes that were up- and down-regulated, providing insights regarding molecular processes influenced by the local density enhancement. Furthermore, we employed Ingenuity Pathway Analysis (IPA) to identify altered pathways and predict upstream regulators. Notably, VEGF-A emerged as one of the most prominent upstream regulators. Accordingly, this study initiates the unraveling of the changes in microcapillary networks at both molecular and proteins level induced by cell condensation obtained through sound patterning. The findings provide valuable insights for further investigation into sound patterning as a biofabrication technique for creating more complex microcapillary networks and advancing in vitro models.

Osteochondral explants for diarthrodial joint diseases: bridging the gap between bench and bedside.

Diarthrodial joint diseases, affecting hundreds of millions of people worldwide, mainly include osteoarthritis and cartilage injuries. No consensus on joint disease models has been achieved so far owing to the complex aetiologies, pathophysiological mechanisms and heterogeneity of disorders. The disease models established using isolated chondrocytes or small animals have the weaknesses of lacking native extracellular matrix and inter-species differences in anatomical and biomechanical cartilage properties. Osteochondral explants (OCEs) from large-animal or human joints present characteristics of native articular cartilage, showing promising potential for application in research on joint diseases. The present review focuses on OCEs and highlights the OCE sources, harvesting techniques, culture systems, applications and future developments. The OCE-centred ex vivo system has the potential to develop into preclinical models mimicking human joint diseases to help elucidate disease mechanisms, prompt therapeutic strategies and facilitate the clinical translation of findings in basic research.

Development and characterization of a predictive microCT-based non-union model in Fischer F344 rats.

INTRODUCTION: Non-unions remain a clinical problem and are characterised by the failure to heal after a defined period of time. Current preclinical non-union models apply a wide variety of techniques to diminish intrinsic healing potential deviating from the clinical situation. The aim of this study was to develop and characterise a non-union model in rats using internal plate fixation without the need for additional healing insults, whereby bone healing can be longitudinally assessed using microCT. It was hypothesized that healing/non-unions can be accurately predicted at early time points by microCT. MATERIALS AND METHODS: Female, skeletally mature Fischer F344 rats received a 2 mm or 1 mm femoral osteotomy, stabilized with either a 2 mm thick plate or a 1.25 mm thick plate. Healing was monitored by microCT over 14 weeks and histological analysis at euthanasia. The mechanical environment was characterised using finite element (FE) modelling and biomechanical testing. RESULTS: The majority of animals receiving the 2 mm thick plate displayed poor healing responses in both the 2 mm and 1 mm defect size groups. Bone and cartilage formation were markedly improved using the 1.25 mm thick plate. MicroCT could accurately predict bone forming capacity at early time points (3-4 weeks). CONCLUSIONS: The 2 mm thick plating system confers poor healing responses in female Fischer F344 rats, comparable to atrophic non-unions. By reducing plate thickness to increase interfragmentary strain within the defect site healing is improved, leading to borderline healing situations or increased abundance of cartilage tissue present in the defect site with ultimate failure to bridge the defect (hypertrophic non-union). Furthermore, microCT can reliably identify delayed/non-healing animals within 4 weeks, thereby allowing their selective targeting for the testing of novel, clinically relevant treatment strategies in different clinical situations aimed at restoring impaired bone healing.

Evaluation of the influence of platelet-rich plasma (PRP), platelet lysate (PL) and mechanical loading on chondrogenesis in vitro.

The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-ß). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-ß1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-ß and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-ß1 was quantified in the PL group. Latent TGF-ß1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-ß1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.

Sodium hyaluronate supplemented culture medium combined with joint-simulating mechanical loading improves chondrogenic differentiation of human mesenchymal stem cells.

In vitro models aim to recapitulate the in vivo situation. To more closely mimic the knee joint environment, current in vitro models need improvements to reflect the complexity of the native tissue. High molecular weight hyaluronan (hMwt HA) is one of the most abundant bioactive macromolecules in healthy synovial fluid, while shear and dynamic compression are two joint-relevant mechanical forces. The present study aimed at investigating the concomitant effect of joint-simulating mechanical loading (JSML) and hMwt HA-supplemented culture medium on the chondrogenic differentiation of primary human bone-marrow-derived mesenchymal stem cells (hBM-MSCs). hBM-MSC chondrogenesis was investigated over 28 d at the gene expression level and total DNA, sulphated glycosaminoglycan, TGF-ß1 production and safranin O staining were evaluated. The concomitant effect of hMwt HA culture medium and JSML significantly increased cartilage-like matrix deposition and sulphated glycosaminoglycan synthesis, especially during early chondrogenesis. A stabilisation of the hBM-MSC-derived chondrocyte phenotype was observed through the reduced upregulation of the hypertrophic marker collagen X and an increase in the chondrogenic collagen type II/X ratio. A combination of JSML and hMwt HA medium better reflects the complexity of the in vivo synovial joint environment. Thus, JSML and hMwt HA medium will be two important features for joint-related culture models to more accurately predict the in vivo outcome, therefore reducing the need for animal studies. Reducing in vitro artefacts would enable a more reliable prescreening of potential cartilage repair therapies.

The waves that make the pattern: a review on acoustic manipulation in biomedical research.

Novel approaches, combining technology, biomaterial design, and cutting-edge cell culture, have been increasingly considered to advance the field of tissue engineering and regenerative medicine. Within this context, acoustic manipulation to remotely control spatial cellular organization within a carrier matrix has arisen as a particularly promising method during the last decade. Acoustic or sound-induced manipulation takes advantage of hydrodynamic forces exerted on systems of particles within a liquid medium by standing waves. Inorganic or organic particles, cells, or organoids assemble within the nodes of the standing wave, creating distinct patterns in response to the applied frequency and amplitude. Acoustic manipulation has advanced from micro- or nanoparticle arrangement in 2D to the assembly of multiple cell types or organoids into highly complex in vitro tissues. In this review, we discuss the past research achievements in the field of acoustic manipulation with particular emphasis on biomedical application. We survey microfluidic, open chamber, and high throughput devices for their applicability to arrange non-living and living units in buffer or hydrogels. We also investigate the challenges arising from different methods, and their prospects to gain a deeper understanding of in vitro tissue formation and application in the field of biomedical engineering.

Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model.

The repair of focal cartilage defects remains one of the foremost issues in the field of orthopaedics. Chondral defects may arise from a variety of joint pathologies and left untreated, will likely progress to osteoarthritis. Current repair techniques, such as microfracture, result in short-term clinical improvements but have poor long-term outcomes. Emerging scaffold-based repair strategies have reported superior outcomes compared to microfracture and motivate the development of new biomaterials for this purpose. In this study, unique composite implants consisting of a base porous reinforcing component (woven poly(ε-caprolactone)) infiltrated with 1 of 2 hydrogels (self-assembling peptide or thermo-gelling hyaluronan) or bone marrow aspirate were evaluated. The objective was to evaluate cartilage repair with composite scaffold treatment compared to the current standard of care (microfracture) in a translationally relevant large animal model, the Yucatan minipig. While many cartilage-repair studies have shown some success in vivo, most are short term and not clinically relevant. Informed by promising 6-week findings, a 12-month study was carried out and those results are presented here. To aid in comparisons across platforms, several structural and functionally relevant outcome measures were performed. Despite positive early findings, the long-term results indicated less than optimal structural and mechanical results with respect to cartilage repair, with all treatment groups performing worse than the standard of care. This study is important in that it brings much needed attention to the importance of performing translationally relevant long-term studies in an appropriate animal model when developing new clinical cartilage repair approaches.

The tissue-renin-angiotensin-system of the human intervertebral disc.

Symptomatic intervertebral disc (IVD) degeneration accounts for significant socioeconomic burden. Recently, the expression of the tissue renin-angiotensin system (tRAS) in rat and bovine IVD was demonstrated. The major effector of tRAS is angiotensin II (AngII), which participates in proinflammatory pathways. The present study investigated the expression of tRAS in human IVDs, and the correlation between tRAS, inflammation and IVD degeneration. Human IVD tissue was collected during spine surgery and distributed according to principal diagnosis. Gene expression of tRAS components, proinflammatory and catabolic markers in the IVD tissue was assessed. Hydroxyproline (OHP) and glycosaminoglycan (GAG) content in the IVD tissue were determined. Tissue distribution of tRAS components was investigated by immunohistochemistry. Gene expression of tRAS components such as angiotensin-converting enzyme (ACE), Ang II receptor type 2 (AGTR2), angiotensinogen (AGT) and cathepsin D (CTSD) was confirmed in human IVDs. IVD samples that expressed tRAS components (n = 21) revealed significantly higher expression levels of interleukin 6 (IL-6), tumour necrosis factor α (TNF-α), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 4 and 5 compared to tRAS-negative samples (n = 37). Within tRAS-positive samples, AGT, matrix-metalloproteinases 13 and 3, IL-1, IL-6 and IL-8 were more highly expressed in traumatic compared to degenerated IVDs. Total GAG/DNA content of non-tRAS expressing IVD tissue was significantly higher compared to tRAS positive tissue. Immunohistochemistry confirmed the presence of AngII in the human IVD. The present study identified the existence of tRAS in the human IVD and suggested a correlation between tRAS expression, inflammation and ultimately IVD degeneration.

Tissue mimetic hyaluronan bioink containing collagen fibers with controlled orientation modulating cell migration and alignment.

Biofabrication is providing scientists and clinicians the ability to produce engineered tissues with desired shapes and gradients of composition and biological cues. Typical resolutions achieved with extrusion-based bioprinting are at the macroscopic level. However, for capturing the fibrillar nature of the extracellular matrix (ECM), it is necessary to arrange ECM components at smaller scales, down to the micron and the molecular level. Herein, we introduce a bioink containing the tyramine derivative of hyaluronan (HA; henceforth known as THA) and collagen (Col) type 1. In this bioink, similar to connective tissues, Col is present in the fibrillar form, and HA functions as a viscoelastic space filler. THA was enzymatically cross-linked under mild conditions allowing simultaneous Col fibrillogenesis, thus achieving a homogeneous distribution of Col fibrils within the viscoelastic HA-based matrix. The THA-Col composite displayed synergistic properties in terms of storage modulus and shear thinning, translating into good printability. Shear-induced alignment of the Col fibrils along the printing direction was achieved and quantified via immunofluorescence and second-harmonic generation. Cell-free and cell-laden constructs were printed and characterized, analyzing the influence of the controlled microscopic anisotropy on human bone marrow-derived mesenchymal stromal cell (hMSC) migration. Anisotropic HA-Col showed cell-instructive properties modulating hMSC adhesion, morphology, and migration from micropellets stimulated by the presence and the orientation of Col fibers. Actin filament staining showed that hMSCs embedded in aligned constructs displayed increased cytoskeleton alignment along the fibril direction. Based on gene expression of cartilage/bone markers and ECM production, hMSCs embedded in the isotropic bioink displayed chondrogenic differentiation comparable with standard pellet culture by means of proteoglycan production (safranin O staining and proteoglycan quantification). The possibility of printing matrix components with control over microscopic alignment brings biofabrication one step closer to capturing the complexity of native tissues.

Functional cell phenotype induction with TGF-ß1 and collagen-polyurethane scaffold for annulus fibrosus rupture repair.

Appropriate cell sources, bioactive factors and biomaterials for generation of functional and integrated annulus fibrosus (AF) tissue analogues are still an unmet need. In the present study, the AF cell markers, collagen type I, cluster of differentiation 146 (CD146), mohawk (MKX) and smooth muscle protein 22α (SM22α) were found to be suitable indicators of functional AF cell induction. In vitro 2D culture of human AF cells showed that transforming growth factor ß1 (TGF-ß1) upregulated the expression of the functional AF markers and increased cell contractility, indicating that TGF-ß1-pre-treated AF cells were an appropriate cell source for AF tissue regeneration. Furthermore, a tissue engineered construct, composed of polyurethane (PU) scaffold with a TGF-ß1-supplemented collagen type I hydrogel and human AF cells, was evaluated with in vitro 3D culture and ex vivo preclinical bioreactor-loaded organ culture models. The collagen type I hydrogel helped maintaining the AF functional phenotype. TGF-ß1 supplement within the collagen I hydrogel further promoted cell proliferation and matrix production of AF cells within in vitro 3D culture. In the ex vivo IVD organ culture model with physiologically relevant mechanical loading, TGF-ß1 supplement in the transplanted constructs induced the functional AF cell phenotype and enhanced collagen matrix synthesis. In conclusion, TGF-ß1-containing collagen-PU constructs can induce the functional cell phenotype of human AF cells in vitro and in situ. This combined cellular, biomaterial and bioactive agent therapy has a great potential for AF tissue regeneration and rupture repair.

Evaluation of biomimetic hyaluronic-based hydrogels with enhanced endogenous cell recruitment and cartilage matrix formation.

Biomaterials play a pivotal role in cell-free cartilage repair approaches, where cells must migrate through the scaffold, fill the defect, and then proliferate and differentiate facilitating tissue remodeling. Here we used multiple assays to test the influence of chemokines and growth factors on cell migration and cartilage repair in two different hyaluronan (HA)-based hydrogels. We first investigated bone marrow Mesenchymal Stromal Cells (BMSC) migration in vitro, in response to different concentrations of platelet-derived growth factor-BB (PDGF-BB), chemokine ligand 5 (CCL5/RANTES) and stromal cell-derived factor 1 (SDF-1), using a 3D spheroid-based assay. PDGF-BB was selected as most favourable chemotactic agent, and MSC migration was assessed in the context of physical impediment to cell recruitment by testing Fibrin-HA and HA-Tyramine hydrogels of different cross-linking densities. Supplementation of PDGF-BB stimulated progressive migration of MSC through the gels over time. We then investigated in situ cell migration into the hydrogels with and without PDGF-BB, using a cartilage-bone explant model implanted subcutaneously in athymic mice. In vivo studies show that when placed into an osteochondral defect, both hydrogels supported endogenous cell infiltration and provided an amenable microenvironment for cartilage production. These processes were best supported in Fibrin-HA hydrogel in the absence of PDGF-BB. This study used an advanced preclinical testing platform to select an appropriate microenvironment provided by implanted hydrogels, demonstrating that HA-based hydrogels can promote the initial and critical step of endogenous cell recruitment and circumvent some of the clinical challenges in cartilage tissue repair. STATEMENT OF SIGNIFICANCE: The challenge of articular cartilage repair arises from its complex structure and architecture, which confers the unique mechanical behavior of the extracellular matrix. The aim of our research is to identify biomaterials for implants that can support migration of endogenous stem and progenitor cell populations from cartilage and bone tissue, in order to permanently replace damaged cartilage with the original hyaline structure. Here, we present an in vitro 3D spheroid-based migration assay and an osteochondral defect model, which provide the opportunity to assess biomaterials and biomolecules, and to get stronger experimental evidence of the not well-characterized dynamic process of endogenous cells colonization in an osteochondral defect. Furthermore, the delicate step of early cell migration into biomaterials towards functional tissue engineering is reproduced. These tests can be used for pre-clinical testing of newly developed material designs in the field of scaffold engineering.

CD146/MCAM distinguishes stem cell subpopulations with distinct migration and regenerative potential in degenerative intervertebral discs.

OBJECTIVE: This study aimed to characterize the mesenchymal stem cell (MSC) subpopulation migrating towards a degenerated intervertebral disc (IVD) and to assess its regenerative potential. DESIGN: Based on initial screening for migration towards C-C motif chemokine ligand 5 (CCL5), the migration potential of CD146+ and CD146- mesenchymal stem cells (MSCs) was evaluated in vitro and in a degenerated organ culture model (degeneration by high-frequency loading in a bioreactor). Discogenic differentiation potential of CD146+ and CD146- MSCs was investigated by in vitro pellet culture assay with supplementation of growth and differentiation factor-6 (GDF6). Furthermore, trypsin degenerated IVDs were treated by either homing or injection of CD146+ or CD146- MSCs and glycosaminoglycan synthesis was evaluated by Sulphur 35 incorporation after 35 days of culture. RESULTS: Surface expression of CD146 led to a higher number of migrated MSCs both in vitro and in organ culture. CD146+ and CD146- pellets responded with a similar up-regulation of anabolic markers. A higher production of sulfated glycosaminoglycans (sGAG)/DNA was observed for CD146+ pellets, while in organ cultures, sGAG synthesis rate was higher for IVDs treated with CD146- MSCs by either homing or injection. CONCLUSIONS: The CD146+ MSC subpopulation held greater migration potential towards degenerative IVDs, while the CD146- cells induced a stronger regenerative response in the resident IVD cells. These findings were independent of the application route (injection vs migration). From a translational point of view, our data suggests that CD146+ MSCs may be suitable for re-population, while CD146- MSCs may represent the primary choice for stimulation of endogenous IVD cells.

Mechanically stimulated osteochondral organ culture for evaluation of biomaterials in cartilage repair studies.

Surgical procedures such as microfracture or autologous chondrocyte implantation have been used to treat articular cartilage lesions; however, repair often fails in terms of matrix organization and mechanical behaviour. Advanced biomaterials and tissue engineered constructs have been developed to improve cartilage repair; nevertheless, their clinical translation has been hampered by the lack of reliable in vitro models suitable for pre-clinical screening of new implants and compounds. In this study, an osteochondral defect model in a bioreactor that mimics the multi-axial motion of an articulating joint, was developed. Osteochondral explants were obtained from bovine stifle joints, and cartilage defects of 4 mm diameter were created. The explants were used as an interface against a ceramic ball applying dynamic compressive and shear loading. Osteochondral defects were filled with chondrocytes-seeded fibrin-polyurethane constructs and subjected to mechanical stimulation. Cartilage viability, proteoglycan accumulation and gene expression of seeded chondrocytes were compared to free swelling controls. Cells within both cartilage and bone remained viable throughout the 10-day culture period. Loading did not wear the cartilage, as indicated by histological evaluation and glycosaminoglycan release. The gene expression of seeded chondrocytes indicated a chondrogenic response to the mechanical stimulation. Proteoglycan 4 and cartilage oligomeric matrix protein were markedly increased, while mRNA ratios of collagen type II to type I and aggrecan to versican were also enhanced. This mechanically stimulated osteochondral defect culture model provides a viable microenvironment and will be a useful pre-clinical tool to screen new biomaterials and biological regenerative therapies under relevant complex mechanical stimuli. STATEMENT OF SIGNIFICANCE: Articular cartilage lesions have a poor healing capacity and reflect one of the most challenging problems in orthopedic clinical practice. The aim of current research is to develop a testing system to assess biomaterials for implants, that can permanently replace damaged cartilage with the original hyaline structure and can withstand the mechanical forces long term. Here, we present an osteochondral ex vivo culture model within a cartilage bioreactor, which mimics the complex motion of an articulating joint in vivo. The implementation of mechanical forces is essential for pre-clinical testing of novel technologies in the field of cartilage repair, biomaterial engineering and regenerative medicine. Our model provides a unique opportunity to investigate healing of articular cartilage defects in a physiological joint-like environment.

3D bioprinting of a hyaluronan bioink through enzymatic-and visible light-crosslinking.

Extrusion-based three-dimensional bioprinting relies on bioinks engineered to combine viscoelastic properties for extrusion and shape retention, and biological properties for cytocompatibility and tissue regeneration. To satisfy these conflicting requirements, bioinks often utilize either complex mixtures or complex modifications of biopolymers. In this paper we introduce and characterize a bioink exploiting a dual crosslinking mechanism, where an enzymatic reaction forms a soft gel suitable for cell encapsulation and extrusion, while a visible light photo-crosslinking allows shape retention of the printed construct. The influence of cell density and cell type on the rheological and printability properties was assessed correlating the printing outcomes with the damping factor, a rheological characteristic independent of the printing system. Stem cells, chondrocytes and fibroblasts were encapsulated, and their viability was assessed up to 14 days with live/dead, alamar blue and trypan blue assays. Additionally, the impact of the printing parameters on cell viability was investigated. Owing to its straightforward preparation, low modification, presence of two independent crosslinking mechanisms for tuning shear-thinning independently of the final shape fixation, the use of visible green instead of UV light, the possibility of encapsulating and sustaining the viability of different cell types, the hyaluronan bioink here presented is a valid biofabrication tool for producing 3D printed tissue-engineered constructs.

Mechanical loading of intervertebral disc modulates microglia proliferation, activation, and chemotaxis.

OBJECTIVE: The aim of the study is to assess the effects of the neuroinflammatory microenvironment of a mechanically-induced degenerating intervertebral disc (IVD) on neuroinflammatory like cells such as microglia, in order to comprehend the role of microglial cells in degenerative disc disease. METHODS: Bovine caudal IVDs were kept in culture in an ex vivo bioreactor under high frequency loading and limited nutrition or in free swelling conditions as control samples. Conditioned media (CM) were collected, analysed for cytokine and neurotrophin content and applied to microglial cells for neuroinflammatory activation assessment. RESULTS: Degenerative conditioned medium (D-CM) induced a higher production of interleukin (IL)-8, nerve growth factor (NGF), interferon (IFN)-γ, IL-17 from IVD cells than unloaded control conditioned medium (U-CM). Upon 48 h of co-incubation with microglia, D-CM stimulated microglia proliferation, activation, with increased expression of ionized calcium binding adaptor molecule 1 (IBA1) and CD68, and chemotaxis. Moreover, an increment of nitrite production was observed. Interestingly, D-CM caused an upregulation of IL-1ß, IL-6, tumour necrosis factor α (TNFα), inducible NO synthase (iNOS), IBA1, and vascular endothelial growth factor (VEGF) genes in microglia. Similar results were obtained when microglia were treated with the combination of the measured cytokines. CONCLUSIONS: Our findings show that in IVD degenerative microenvironment, IL-8, NGF, IFN-γ, IL-17 drive activation of microglia in the spinal cord and increase upregulation of neuroinflammatory markers. This, in turn, enhances the inflammatory milieu within IVD tissues and in the peridiscal space, aggravating the cascade of degenerative events. This study provides evidence for an important role of microglia in maintaining IVD neuroinflammatory microenvironment and probably inducing low back pain.

Neutral detergent fibre in piglet diets: digestibility, performance, and deposition of body nutrients

ABSTRACT A total 120 piglets with an average live weight of 7.00 kg, weaned at 21 days, were used to evaluate the effect of neutral detergent fibre levels on the digestibility of nutrients and energy from the diets, productive performance, and the composition and rate of deposition of nutrients and energy in the bodies of piglets in the nursery phase. The animals were distributed according to a randomized-block design into five treatments, which consisted of neutral detergent fibre levels, with six replicates and four animals per plot. A quadratic effect was detected for the digestibility coefficients of nutrients and energy, feed intake and weight gain. The increase in fibre level promoted a linear increase in fat content in the carcass, blood, and body, whereas the energy in the carcass, organs, and body showed an inverse response. The results showed a quadratic effect on the nutrient deposition rate in the carcass, organs and body. In conclusion, the best digestibility of nutrients and energy from the diet is obtained with 10-11.5% neutral detergent fibre, as higher weight gain and greater protein deposition in the body are achieved at neutral detergent fibre levels of 10.6% and 10.3%, respectively.

Cobalt-containing bioactive glasses reduce human mesenchymal stem cell chondrogenic differentiation despite HIF-1α stabilisation.

Bioactive glasses (BGs) are excellent delivery systems for the sustained release of therapeutic ions and have been extensively studied in the context of bone tissue engineering. More recently, due to their osteogenic properties and expanding application to soft tissue repair, BGs have been proposed as promising materials for use at the osteochondral interface. Since hypoxia plays a critical role during cartilage formation, we sought to investigate the influence of BGs releasing the hypoxia-mimicking agent cobalt (CoBGs) on human mesenchymal stem cell (hMSC) chondrogenesis, as a novel approach that may guide future osteochondral scaffold design. The CoBG dissolution products significantly increased the level of hypoxia-inducible factor-1 alpha in hMSCs in a cobalt dose-dependent manner. Continued exposure to the cobalt-containing BG extracts significantly reduced hMSC proliferation and metabolic activity, as well as chondrogenic differentiation. Overall, this study demonstrates that prolonged exposure to cobalt warrants careful consideration for cartilage repair applications.

Biomaterials for articular cartilage tissue engineering: Learning from biology.

Articular cartilage is commonly described as a tissue that is made of up to 80% water, is devoid of blood vessels, nerves, and lymphatics, and is populated by only one cell type, the chondrocyte. At first glance, an easy tissue for clinicians to repair and for scientists to reproduce in a laboratory. Yet, chondral and osteochondral defects currently remain an open challenge in orthopedics and tissue engineering of the musculoskeletal system, without considering osteoarthritis. Why do we fail in repairing and regenerating articular cartilage? Behind its simple and homogenous appearance, articular cartilage hides a heterogeneous composition, a high level of organisation and specific biomechanical properties that, taken together, make articular cartilage a unique material that we are not yet able to repair or reproduce with high fidelity. This review highlights the available therapies for cartilage repair and retraces the research on different biomaterials developed for tissue engineering strategies. Their potential to recreate the structure, including composition and organisation, as well as the function of articular cartilage, intended as cell microenvironment and mechanically competent replacement, is described. A perspective of the limitations of the current research is given in the light of the emerging technologies supporting tissue engineering of articular cartilage. STATEMENT OF SIGNIFICANCE: The mechanical properties of articular tissue reflect its functionally organised composition and the recreation of its structure challenges the success of in vitro and in vivo reproduction of the native cartilage. Tissue engineering and biomaterials science have revolutionised the way scientists approach the challenge of articular cartilage repair and regeneration by introducing the concept of the interdisciplinary approach. The clinical translation of the current approaches are not yet fully successful, but promising results are expected from the emerging and developing new generation technologies.

Neutral detergent fibre in piglet diets: digestibility, performance, and deposition of body nutrients.

A total 120 piglets with an average live weight of 7.00 kg, weaned at 21 days, were used to evaluate the effect of neutral detergent fibre levels on the digestibility of nutrients and energy from the diets, productive performance, and the composition and rate of deposition of nutrients and energy in the bodies of piglets in the nursery phase. The animals were distributed according to a randomized-block design into five treatments, which consisted of neutral detergent fibre levels, with six replicates and four animals per plot. A quadratic effect was detected for the digestibility coefficients of nutrients and energy, feed intake and weight gain. The increase in fibre level promoted a linear increase in fat content in the carcass, blood, and body, whereas the energy in the carcass, organs, and body showed an inverse response. The results showed a quadratic effect on the nutrient deposition rate in the carcass, organs and body. In conclusion, the best digestibility of nutrients and energy from the diet is obtained with 10-11.5% neutral detergent fibre, as higher weight gain and greater protein deposition in the body are achieved at neutral detergent fibre levels of 10.6% and 10.3%, respectively.

Bioreactor mechanically guided 3D mesenchymal stem cell chondrogenesis using a biocompatible novel thermo-reversible methylcellulose-based hydrogel.

Autologous chondrocyte implantation for cartilage repair represents a challenge because strongly limited by chondrocytes' poor expansion capacity in vitro. Mesenchymal stem cells (MSCs) can differentiate into chondrocytes, while mechanical loading has been proposed as alternative strategy to induce chondrogenesis excluding the use of exogenous factors. Moreover, MSC supporting material selection is fundamental to allow for an active interaction with cells. Here, we tested a novel thermo-reversible hydrogel composed of 8% w/v methylcellulose (MC) in a 0.05 M Na2SO4 solution. MC hydrogel was obtained by dispersion technique and its thermo-reversibility, mechanical properties, degradation and swelling were investigated, demonstrating a solution-gelation transition between 34 and 37 °C and a low bulk degradation (<20%) after 1 month. The lack of any hydrogel-derived immunoreaction was demonstrated in vivo by mice subcutaneous implantation. To induce in vitro chondrogenesis, MSCs were seeded into MC solution retained within a porous polyurethane (PU) matrix. PU-MC composites were subjected to a combination of compression and shear forces for 21 days in a custom made bioreactor. Mechanical stimulation led to a significant increase in chondrogenic gene expression, while histological analysis detected sulphated glycosaminoglycans and collagen II only in loaded specimens, confirming MC hydrogel suitability to support load induced MSCs chondrogenesis.