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Background: While growth factors have the potential to halt degeneration and decrease inflammation in animal models, the literature investigating the effect of dosage on human cells is lacking. Moreover, despite the completion of clinical trials using growth differentiation factor-5 (GDF-5), no results have been publicly released. Aims: The overall objective was to quantitatively assess the effect of three clinically relevant concentrations of GDF-5 (0.25, 1, and 2 mg) as a therapeutic for disc regeneration. Materials and methods: Firstly, this work experimentally determined the effects of GDF-5 concentration on the metabolic and matrix synthesis rates of human nucleus pulposus (NP) cells. Secondly, in silico modeling was employed to predict the subsequent regenerative effect of different GDF-5 treatments (± cells). Results: This study suggests a trend of increased matrix synthesis with 0.25 and 1 mg of GDF-5. However, 2 mg of GDF-5 significantly upregulates oxygen consumption. Despite this, in silico models highlight the potential of growth factors in promoting matrix synthesis compared to cell-only treatments, without significantly perturbing the nutrient microenvironment. Discussion: This work elucidates the potential of GDF-5 on human NP cells. Although the results did not reveal statistical differences across all doses, the variability and response among donors is an interesting finding. It highlights the complexity of human response to biological treatments and reinforces the need for further human research and personalized approaches. Furthermore, this study raises a crucial question about whether these potential biologics are more regenerative in nature or better suited as prophylactic therapies for younger patient groups. Conclusion: Biological agents exhibit unique characteristics and features, demanding tailored development strategies and individualized assessments rather than a one-size-fits-all approach. Therefore, the journey to realizing the full potential of biological therapies is long and costly. Nonetheless, it holds the promise of revolutionizing spinal healthcare and improving the quality of life for patients suffering from discogenic back pain.
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During intervertebral disc (IVD) degeneration, microenvironmental challenges such as decreasing levels of glucose, oxygen, and pH play crucial roles in cell survival and matrix turnover. Antacids, such as Mg(OH)2 and CaCO3, entrapped in microcapsules are capable of neutralizing acidic microenvironments in a controlled fashion and therefore may offer the potential to improve the acidic niche of the degenerated IVD and enhance cell-based regeneration strategies. The objectives of this work were, first, to develop and characterize antacid microcapsules and assess their neutralization capacity in an acidic microenvironment and, second, to combine antacid microcapsules with cellular microcapsules in a hybrid gel system to investigate their neutralization effect as a potential therapeutic in a disc explant model. To achieve this, we screened five different pH- neutralizing agents (Al(OH)3, Mg(OH)2, CaCO3, and HEPES) in terms of their pH neutralization capacities, with Mg(OH)2 or CaCO3 being carried forward for further investigation. Antacid-alginate microcapsules were formed at different concentrations using the electrohydrodynamic spraying process and assessed in terms of size, buffering kinetics, cell compatibility, and cytotoxicity. Finally, the combination of cellular microcapsules and antacid capsules was examined in a bovine disc explant model under physiological degenerative conditions. Overall, CaCO3 was found to be superior in terms of neutralization capacities, release kinetics, and cellular response. Specifically, CaCO3 elevated the acidic pH to neutral levels and is estimated to be maintained for several weeks based on Ca2+ release. Using a disc explant model, it was demonstrated that CaCO3 microcapsules were capable of increasing the local pH within the core of a hybrid cellular gel system. This work highlights the potential of antacid microcapsules to positively alter the challenging acidic microenvironment conditions typically observed in degenerative disc disease, which may be used in conjunction with cell therapies to augment regeneration.
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Background: Current guidelines recommend several direct oral anticoagulant agents (DOACs) equally for managing cancer-associated venous thromboembolism (VTE). Objectives: The aim of this study was to assess the efficacy and safety of DOACs in patients with active cancer. Methods: Literature searches were conducted in PubMed, Embase, and Cochrane Central in November 2022. Randomized controlled trials investigating anticoagulation strategies (vitamin K antagonists, parenteral anticoagulation [eg, low-molecular weight heparin], and DOACs) for VTE in patients with active cancer were identified for network meta-analysis. The outcomes included recurrent VTE, recurrent pulmonary embolism, recurrent deep venous thrombosis, major bleeding, clinically relevant nonmajor bleeding (CRNMB), and a composite outcome of major bleeding or CRNMB. Pooled HRs and 95% CIs were estimated using either the HR or relative risk provided from each study. Random-effects models were used for all the analyses. Results: Seventeen randomized controlled trials involving 6,623 patients with active cancer were included. No significant differences were found among the DOACs for efficacy outcomes (recurrent VTE, pulmonary embolism, and deep venous thrombosis). In terms of major bleeding, apixaban was similarly safe compared with dabigatran and rivaroxaban but was associated with a decreased risk compared with edoxaban (HR: 0.38; 95% CI: 0.15-0.93). Regarding CRNMB, edoxaban was similarly safe compared with apixaban but was associated with a decreased risk compared with rivaroxaban (HR: 0.31; 95% CI: 0.10-0.91). Compared with parenteral anticoagulation, apixaban was associated with a reduced risk for recurrent VTE (HR: 0.60; 95% CI: 0.38-0.93) without increasing bleeding, edoxaban was associated with an increased risk for major bleeding or CRNMB (HR: 1.35; 95% CI: 1.02-1.79), and rivaroxaban was associated with an increased risk for CRNMB (HR: 3.76; 95% CI: 1.43-9.88). Conclusions: DOACs demonstrate comparable efficacy but exhibit different safety profiles. Apixaban may confer an antithrombotic benefit without an increased risk for bleeding, distinguishing it from other contemporary anticoagulation strategies in patients with active cancer and VTE.
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INTRODUCTION: Pancreas ductal adenocarcinoma (PDAC) is a frequently lethal malignancy that poses unique therapeutic challenges. The current mainstay of therapy for metastatic PDAC (mPDAC) is cytotoxic chemotherapy. NALIRIFOX (liposomal irinotecan, fluorouracil, leucovorin, oxaliplatin) is an emerging standard of care in the metastatic setting. An evolving understanding of PDAC pathogenesis is driving a shift toward targeted therapy. Olaparib, a poly-ADP-ribose polymerase (PARP) inhibitor, has regulatory approval for maintenance therapy in BRCA-mutated mPDAC along with other targeted agents receiving disease-agnostic approvals including for PDAC with rare fusions and mismatch repair deficiency. Ongoing research continues to identify and evaluate an expanding array of targeted therapies for PDAC. AREAS COVERED: This review provides a brief overview of standard therapies for PDAC and an emphasis on current and emerging targeted therapies. EXPERT OPINION: There is notable potential for targeted therapies for KRAS-mutated PDAC with opportunity for meaningful benefit for a sizable portion of patients with this disease. Further, emerging approaches are focused on novel immune, tumor microenvironment, and synthetic lethality strategies.
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Antinéoplasiques , Carcinome du canal pancréatique , Tumeurs du pancréas , Humains , Tumeurs du pancréas/traitement médicamenteux , Tumeurs du pancréas/génétique , Antinéoplasiques/effets indésirables , Carcinome du canal pancréatique/traitement médicamenteux , Carcinome du canal pancréatique/génétique , Fluorouracil/usage thérapeutique , Oxaliplatine , Microenvironnement tumoralRÉSUMÉ
Meniscus-related injuries are a common orthopedic challenge with an increasing incidence in the population. While the preservation of viable meniscal tissue is the preferred approach in repair strategies, complex or total traumatic lesions may require alternative therapeutic approaches such as meniscal reconstruction using allografts or engineered equivalents. Although clinical studies suggest promising outcomes with the use of acellular implants, further development is needed to improve their biological and mechanical requirements. Decellularized extracellular matrix (dECM) derived from menisci is a promising biomaterial for meniscus tissue engineering due to its recapitulation of the native tissue environment and the maintenance of tissue-specific cues. However, the associated mechanical limitations of dECM-derived scaffolds frequently impedes their adoption, requiring additional reinforcement or combining with stiffer biomaterials to increase their load-bearing properties. In this study, decellularized extracellular matrix was extracted and its fibrillation was controlled by adjusting both pH and salt concentrations to fabricate mechanically functional meniscal tissue equivalents. The effect of collagen fibrillation on the mechanical properties of the dECM constructs was assessed, and porcine-derived fibrochondrocytes were used to evaluate in vitro biocompatibility. It was also possible to fabricate meniscus-shaped implants by casting of the dECM and to render the implants suitable for off-the-shelf use by adopting a freeze-drying preservation method. Suture pull-out tests were also performed to assess the feasibility of using existing surgical methods to fix such implants within a damaged meniscus. This study highlights the potential of utilizing ECM-derived materials for meniscal tissue substitutes that closely mimic the mechanical and biological properties of native tissue.
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Ménisque , Structures d'échafaudage tissulaires , Animaux , Suidae , Structures d'échafaudage tissulaires/composition chimique , Matrice extracellulaire décellularisée , Matrice extracellulaire/composition chimique , Ingénierie tissulaire/méthodes , Ménisque/composition chimique , Matériaux biocompatibles , Concentration en ions d'hydrogèneRÉSUMÉ
Background: A significant hurdle for potential cell-based therapies is the subsequent survival and regenerative capacity of implanted cells. While many exciting developments have demonstrated promise preclinically, cell-based therapies for intervertebral disc (IVD) degeneration fail to translate equivalent clinical efficacy. Aims: This work aims to ascertain the clinical relevance of both a small and large animal model by experimentally investigating and comparing these animal models to human from the perspective of anatomical scale and their cellular metabolic and regenerative potential. Materials and Methods: First, this work experimentally investigated species-specific geometrical scale, native cell density, nutrient metabolism, and matrix synthesis rates for rat, goat, and human disc cells in a 3D microspheroid configuration. Second, these parameters were employed in silico to elucidate species-specific nutrient microenvironments and predict differences in temporal regeneration between animal models. Results: This work presents in silico models which correlate favorably to preclinical literature in terms of the capabilities of animal regeneration and predict that compromised nutrition is not a significant challenge in small animal discs. On the contrary, it highlights a very fine clinical balance between an adequate cell dose for sufficient repair, through de novo matrix deposition, without exacerbating the human microenvironmental niche. Discussion: Overall, this work aims to provide a path towards understanding the effect of cell injection number on the nutrient microenvironment and the "time to regeneration" between preclinical animal models and the large human IVD. While these findings help to explain failed translation of promising preclinical data and the limited results emerging from clinical trials at present, they also enable the research field and clinicians to manage expectations on cell-based regeneration. Conclusion: Ultimately, this work provides a platform to inform the design of clinical trials, and as computing power and software capabilities increase in the future, it is conceivable that generation of patient-specific models could be used for patient assessment, as well as pre- and intraoperative planning.
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Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.
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Back pain is a global epidemiological and socioeconomic problem often associated with intervertebral disc degeneration; a condition believed to initiate in the nucleus pulposus (NP). There is considerable interest in developing early therapeutic interventions to target the NP and halt degeneration. Rat caudal models of disc degeneration have demonstrated significant utility in the study of disease progression and its impact on tissue structure, composition, and mechanical performance. One significant advantage of the caudal model is the ease of access and high throughput nature. However, considerable variability exists across the literature in terms of experimental setup and parameters. The objective of this article is to aid researchers in the design and development of caudal puncture models by providing details and insight into the most reported experimental parameters. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were employed to screen the existing literature and 80 manuscripts met the inclusion criteria. Disc geometry, surgical approaches, effect of needle gauge size to induce degeneration, therapeutic volume, outcome measures, and associated limitations are considered and discussed, and a range of recommendations based on different research questions are presented.
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Background: It is well established that the unique biochemical microenvironment of the intervertebral disc plays a predominant role in cell viability and biosynthesis. However, unless the effect of microenvironmental conditions is primary to a study objective, in vitro culture parameters that are critical for reproducibility are both varied and not routinely reported. Aims: This work aims to investigate the local microenvironments of commonly used culture configurations, highlighting physiological relevance, potential discrepancies, and elucidating possible heterogeneity across the research field. Materials and Methods: This work uses nutrient-transport in silico models to reflect on the effect of often underappreciated parameters, such as culture geometry and diffusional distance (vessel, media volume, construct size), seeding density, and external boundary conditions on the local microenvironment of two-dimensional (2D) and three-dimensional (3D) in vitro culture systems. Results: We elucidate important discrepancies between the external boundary conditions such as the incubator level or media concentrations and the actual local cellular concentrations. Oxygen concentration and cell seeding density were found to be highly influential parameters and require utmost consideration when utilizing 3D culture systems. Discussion: This work highlights that large variations in the local nutrient microenvironment can easily be established without consideration of several key parameters. Without careful deliberation of the microenvironment within each specific and unique system, there is the potential to confound in vitro results leading to heterogeneous results across the research field in terms of biosynthesis and matrix composition. Conclusion: Overall, this calls for a greater appreciation of key parameters when designing in vitro experiments. Better harmony and standardization of physiologically relevant local microenvironments are needed to push toward reproducibility and successful translation of findings across the research field.
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Articular cartilage defects fail to heal spontaneously, typically progressing to osteoarthritis. Bone marrow stimulation techniques such as microfracture (MFX) are the current surgical standard of care; however MFX typically produces an inferior fibro-cartilaginous tissue which provides only temporary symptomatic relief. Here we implanted solubilised articular cartilage extracellular matrix (ECM) derived scaffolds into critically sized chondral defects in goats, securely anchoring these implants to the joint surface using a 3D-printed fixation device that overcame the need for sutures or glues. In vitro these ECM scaffolds were found to be inherently chondro-inductive, while in vivo they promoted superior articular cartilage regeneration compared to microfracture. In an attempt to further improve the quality of repair, we loaded these scaffolds with a known chemotactic factor, transforming growth factor (TGF)-ß3. In vivo such TGF-ß3 loaded scaffolds promoted superior articular cartilage regeneration. This study demonstrates that ECM derived biomaterials, either alone and particularly when combined with exogenous growth factors, can successfully treat articular cartilage defects in a clinically relevant large animal model.
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Background: Despite exciting advances in regenerative medicine, cell-based strategies for treating degenerative disc disease remain in their infancy. To maximize the potential for successful clinical translation, a more thorough understanding of the in vivo microenvironment is needed to better determine and predict how cell therapies will respond when administered in vivo. Aims: This work aims to reflect on the in vivo nutrient microenvironment of the degenerating IVD through consolidating what has already been measured together with investigative in silico models. Materials and Methods: This work uses in silico modeling, underpinned by more recent experimentally determined parameters of degeneration and nutrient transport from the literature, to re-evaluate the current knowledge in terms of grade-specific stages of degeneration. Results: Through modeling only the metabolically active cell population, this work predicts slightly higher glucose concentrations compared to previous in silico models, while the predicted results show good agreement with previous intradiscal pH and oxygen measurements. Increasing calcification with degeneration limits nutrient transport into the IVD and initiates a build-up of acidity; however, its effect is compensated somewhat by a reduction in diffusional distance due to decreasing disc height. Discussion: This work advances in silico modeling through a strong foundation of experimentally determined grade-specific input parameters. Taken together, pre-existing measurements and predicted results suggest that metabolite concentrations may not be as critically low as commonly believed, with calcification not appearing to have a detrimental effect at stages of degeneration when cell therapies are an appropriate intervention. Conclusion: Overall, our initiative is to provoke greater deliberation and consideration of the nutrient microenvironment when performing in vitro cell culture and cell therapy development. This work highlights urgency for robust experimental glucose measurements in healthy and degenerating IVDs, not only to validate in silico models but to significantly advance the field in fully elucidating the nutrient microenvironment and refining in vitro techniques to accelerate clinical translation.
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While some clinical advances in cartilage repair have occurred, osteochondral (OC) defect repair remains a significant challenge, with current scaffold-based approaches failing to recapitulate the complex, hierarchical structure of native articular cartilage (AC). To address this need, we fabricated bilayered extracellular matrix (ECM)-derived scaffolds with aligned pore architectures. By modifying the freeze-drying kinetics and controlling the direction of heat transfer during freezing, it was possible to produce anisotropic scaffolds with larger pores which supported homogenous cellular infiltration and improved sulfated glycosaminoglycan deposition. Neo-tissue organization in vitro could also be controlled by altering scaffold pore architecture, with collagen fibres aligning parallel to the long-axis of the pores within scaffolds containing aligned pore networks. Furthermore, we used in vitro and in vivo assays to demonstrate that AC and bone ECM derived scaffolds could preferentially direct the differentiation of mesenchymal stromal cells (MSCs) towards either a chondrogenic or osteogenic lineage respectively, enabling the development of bilayered ECM scaffolds capable of spatially supporting unique tissue phenotypes. Finally, we implanted these scaffolds into a large animal model of OC defect repair. After 6 months in vivo, scaffold implantation was found to improve cartilage matrix deposition, with collagen fibres preferentially aligning parallel to the long axis of the scaffold pores, resulting in a repair tissue that structurally and compositionally was more hyaline-like in nature. These results demonstrate how scaffold architecture and composition can be spatially modulated to direct the regeneration of complex interfaces such as the osteochondral unit, enabling their use as cell-free, off-the-shelf implants for joint regeneration. STATEMENT OF SIGNIFICANCE: The architecture of the extracellular matrix, while integral to tissue function, is often neglected in the design and evaluation of regenerative biomaterials. In this study we developed a bilayered scaffold for osteochondral defect repair consisting of tissue-specific extracellular matrix (ECM)-derived biomaterials to spatially direct stem/progenitor cell differentiation, with a tailored pore microarchitecture to promote the development of a repair tissue that recapitulates the hierarchical structure of native AC. The use of this bilayered scaffold resulted in improved tissue repair outcomes in a large animal model, specifically the ability to guide neo-tissue organization and therefore recapitulate key aspects of the zonal structure of native articular cartilage. These bilayer scaffolds have the potential to become a new therapeutic option for osteochondral defect repair.
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Cartilage articulaire , Structures d'échafaudage tissulaires , Animaux , Matériaux biocompatibles/composition chimique , Chondrogenèse , Collagène , Matrice extracellulaire , Ingénierie tissulaire/méthodes , Structures d'échafaudage tissulaires/composition chimiqueRÉSUMÉ
Nerve guidance conduits (NGCs) are sub-optimal for long-distance injuries with inflammation and poor vascularization related to poor axonal repair. This study used a multi-factorial approach to create an optimized biomaterial NGC to address each of these issues. Through stepwise optimization, a collagen-chondroitin-6-sulfate (Coll-CS) biomaterial was functionalized with extracellular matrix (ECM) components; fibronectin, laminin 1 and laminin 2 (FibL1L2) in specific ratios. A snap-cooled freeze-drying process was then developed with optimal pore architecture and alignment to guide axonal bridging. Culture of adult rat dorsal root ganglia on NGCs demonstrated significant improvements in inflammation, neurogenesis and angiogenesis in the specific Fib:L1:L2 ratio of 1:4:1. In clinically relevant, large 15 mm rat sciatic nerve defects, FibL1L2-NGCs demonstrated significant improvements in axonal density and angiogenesis compared to unmodified NGCs with functional equivalence to autografts. Therefore, a multiparameter ECM-driven strategy can significantly improve axonal repair across large defects, without exogenous cells or growth factors.
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Régénération nerveuse , Nerf ischiatique , Animaux , Matériaux biocompatibles , Ganglions sensitifs des nerfs spinaux , Inflammation/génétique , RatsRÉSUMÉ
BACKGROUND: Ex vivo disc organ culture systems have become a valuable tool for the development and pre-clinical testing of potential intervertebral disc (IVD) regeneration strategies. Bovine caudal discs have been widely selected due to their large availability and comparability to human IVDs in terms of size and biochemical composition. However, despite their extensive use, it remains to be elucidated whether their nutrient microenvironment is comparable to human degeneration. AIMS: This work aims to create the first experimentally validated in silico model which can be used to predict and characterize the metabolite concentrations within ex vivo culture systems. MATERIALS & METHODS: Finite element models of cultured discs governed by previously established coupled reaction-diffusion equations were created using COMSOL Multiphysics. Experimental validation was performed by measuring oxygen, glucose and pH levels within discs cultured for 7 days, in a static compression bioreactor. RESULTS: The in silico model was successfully validated through good agreement between the predicted and experimentally measured concentrations. For an ex vivo organ cultured in high glucose medium (4.5 g/L or 25 mM) and normoxia, a larger bovine caudal disc (Cd1-2 to Cd3-4) had a central concentration of ~2.6 %O2, ~8 mM of glucose and a pH value of 6.7, while the smallest caudal discs investigated (Cd6-7 and Cd7-8), had a central concentration of ~6.5 %O2, ~12 mM of glucose and a pH value of 6.9. DISCUSSION: This work advances the knowledge of ex vivo disc culture microenvironments and highlights a critical need for optimization and standardization of culturing conditions. CONCLUSION: Ultimately, for assessment of cell-based therapies and successful clinical translation based on nutritional demands, it is imperative that the critical metabolite values within organ cultures (minimum glucose, oxygen and pH values) are physiologically relevant and comparable to the stages of human degeneration.
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Androgen deprivation therapy or ADT is one of the cornerstones of management of locally advanced or metastatic prostate cancer, alongside radiation therapy. However, despite early response, most advanced prostate cancers progress into an androgen unresponsive or castrate resistant state, which hitherto remains an incurable entity and the second leading cause of cancer-related mortality in men in the US. Recent advances have uncovered multiple complex and intermingled mechanisms underlying this transformation. While most of these mechanisms revolve around androgen receptor (AR) signaling, novel pathways which act independently of the androgen axis are also being discovered. The aim of this article is to review the pathophysiological mechanisms that help bypass the apoptotic effects of ADT to create castrate resistance. The article discusses castrate resistance mechanisms under two categories: 1. Direct AR dependent pathways such as amplification or gain of function mutations in AR, development of functional splice variants, posttranslational regulation, and pro-oncogenic modulation in the expression of coactivators vs corepressors of AR. 2. Ancillary pathways involving RAS/MAP kinase, TGF-beta/SMAD pathway, FGF signaling, JAK/STAT pathway, Wnt-Beta catenin and hedgehog signaling as well as the role of cell adhesion molecules and G-protein coupled receptors. miRNAs are also briefly discussed. Understanding the mechanisms involved in the development and progression of castration-resistant prostate cancer is paramount to the development of targeted agents to overcome these mechanisms. A number of targeted agents are currently in development. As we strive for more personalized treatment across oncology care, treatment regimens will need to be tailored based on the type of CRPC and the underlying mechanism of castration resistance.
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Understanding how the local cellular environment influences cell metabolism, phenotype and matrix synthesis is crucial to engineering functional tissue grafts of a clinically relevant scale. The objective of this study was to investigate how the local oxygen environment within engineered cartilaginous tissues is influenced by factors such as cell source, environmental oxygen tension and the cell seeding density. Furthermore, the subsequent impact of such factors on both the cellular oxygen consumption rate and cartilage matrix synthesis were also examined. Bone marrow derived stem cells (BMSCs), infrapatellar fat pad derived stem cells (FPSCs) and chondrocytes (CCs) were seeded into agarose hydrogels and stimulated with transforming growth factor-ß3 (TGF- ß3). The local oxygen concentration was measured within the center of the constructs, and numerical modeling was employed to predict oxygen gradients and the average oxygen consumption rate within the engineered tissues. The cellular oxygen consumption rate of hydrogel encapsulated CCs remained relatively unchanged with time in culture. In contrast, stem cells were found to possess a relatively high initial oxygen consumption rate, but adopted a less oxidative, more chondrocyte-like oxygen consumption profile following chondrogenic differentiation, resulting in net increases in engineered tissue oxygenation. Furthermore, a greater reduction in oxygen uptake was observed when the oxygen concentration of the external cell culture environment was reduced. In general, cartilage matrix deposition was found to be maximal in regions of low oxygen, but collagen synthesis was inhibited in very low (less than 2%) oxygen regions. These findings suggest that promoting an oxygen consumption profile similar to that of chondrocytes might be considered a key determinant to the success of stem cell-based cartilage tissue engineering strategies.
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Low back pain resulting from intervertebral disc (IVD) degeneration is a significant socioeconomic burden. The main effect of the degeneration process involves the alteration of the nucleus pulposus (NP) via cell-mediated enzymatic breakdown of key extracellular matrix (ECM) components. Thus, the development of injectable and biomimetic biomaterials that can instruct the regenerative cell component to produce tissue-specific ECM is pivotal for IVD repair. Chondroitin sulfate (CS) and type II collagen are the primary components of NP tissue and together create the ideal environment for cells to deposit de-novo matrix. Given their high matrix synthesis capacity potential post-expansion, nasal chondrocytes (NC) have been proposed as a potential cell source to promote NP repair. The overall goal of this study was to assess the effects of CS incorporation into disc derived self-assembled ECM hydrogels on the matrix deposition of NCs. Results showed an increased sGAG production with higher amounts of CS in the gel composition and that its presence was found to be critical for the synthesis of collagen type II. Taken together, our results demonstrate how the inclusion of CS into the composition of the material aids the preservation of a rounded cell morphology for NCs in 3D culture and enhances their ability to synthesise NP-like matrix.
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Dégénérescence de disque intervertébral , Disque intervertébral , Nucleus pulposus , Chondroïtines sulfate , Humains , Hydrogels/pharmacologie , RégénérationRÉSUMÉ
Extracellular matrix (ECM)-derived implants hold great promise for tissue repair, but new strategies are required to produce efficiently decellularized scaffolds with the necessary porosity and mechanical properties to facilitate regeneration. In this study, we demonstrate that it is possible to produce highly porous, elastic, articular cartilage (AC) ECM-derived scaffolds that are efficiently decellularized, nonimmunogenic, and chondro-permissive. Pepsin solubilized porcine AC was cross-linked with glyoxal, lyophilized and then subjected to dehydrothermal treatment. The resulting scaffolds were predominantly collagenous in nature, with the majority of sulphated glycosaminoglycan (sGAG) and DNA removed during scaffold fabrication. Four scaffold variants were produced to examine the effect of both ECM (10 or 20 mg/mL) and glyoxal (5 or 10 mM) concentration on the mechanical and biological properties of the resulting construct. When seeded with human infrapatellar fat pad-derived stromal cells, the scaffolds with the lowest concentration of both ECM and glyoxal were found to promote the development of a more hyaline-like cartilage tissue, as evident by increased sGAG and type II collagen deposition. Furthermore, when cultured in the presence of human macrophages, it was found that these ECM-derived scaffolds did not induce the production of key proinflammatory cytokines, which is critical to success of an implantable biomaterial. Together these findings demonstrate that the novel combination of solubilized AC ECM and glyoxal crosslinking can be used to produce highly porous scaffolds that are sufficiently decellularized, highly elastic, chondro-permissive and do not illicit a detrimental immune response when cultured in the presence of human macrophages.
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Chondrocytes/cytologie , Réactifs réticulants/composition chimique , Élasticité , Matrice extracellulaire/métabolisme , Glyoxal/pharmacologie , Orthopédie , Ingénierie tissulaire/méthodes , Structures d'échafaudage tissulaires/composition chimique , Animaux , Cartilage articulaire/cytologie , Chondrocytes/effets des médicaments et des substances chimiques , Chondrogenèse , Cytokines/biosynthèse , Matrice extracellulaire/effets des médicaments et des substances chimiques , Femelle , Humains , Macrophages/effets des médicaments et des substances chimiques , Macrophages/métabolisme , Porosité , Solubilité , SuidaeRÉSUMÉ
Hydrogels, such as fibrin, offer a promising delivery vehicle to introduce cells into the intervertebral disc (IVD) to regenerate damaged disc tissue as a potential treatment for low back pain. However, fibrin lacks key extracellular matrix (ECM) components, such as collagen (Col) and hyaluronan (HA), normally found in native nucleus pulposus (NP) tissue. The overall aim of this work was to create a fibrin-based hydrogel, by incorporating Col and HA into the matrix to enhance NP-like matrix accumulation using articular chondrocytes (CC). Firstly, we assessed the effect of fibrin concentrations on hydrogel stability, and the viability and proliferation kinetics of articular chondrocytes. Secondly, we investigated the effect of incorporating Col and HA to enhance NP-like matrix accumulation, and finally, examined the influence of various HA concentrations. Results showed that increasing fibrin concentration enhanced cell viability and proliferation. Interestingly, incorporation of HA promoted sGAG accumulation and tended to suppress collagen formation at higher concentrations. Taken together, these results suggest that incorporation of ECM components can enhance the bioactivity of fibrin-based hydrogels, which may help advance the clinical potential of commercial cell and biomaterial ventures in the treatment of IVD regeneration.
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Cell delivery and leakage during injection remains a challenge for cell-based intervertebral disc regeneration strategies. Cellular microencapsulation may offer a promising approach to overcome these limitations by providing a protective niche during intradiscal injection. Electrohydrodynamic spraying (EHDS) is a versatile one-step approach for microencapsulation of cells using a high voltage electric field. The primary objective of this work was to characterise key processing parameters such as applied voltage (0, 5, 10 or 15 kV), emitter needle gauge (21, 26 or 30 G), alginate concentration (1%, 2% or 3%) and flow rate (50, 100, 250 or 500 µl min-1) to regulate the size and morphology of alginate microcapsules as well as subsequent cell viability when altering these parameters. The effect of initial cell seeding density (5, 10 and 20 × 106 cells ml-1) on subsequent matrix accumulation of microencapsulated articular chondrocytes was also evaluated. Results showed that increasing alginate concentration and thus viscosity increased overall microcapsule size but also affected the geometry towards ellipsoidal-shaped gels. Altering the electric field strength and needle diameter regulated microcapsule size towards a smaller diameter with increasing voltage and smaller needle diameter. Needle size did not appear to affect cell viability when operating with lower alginate concentrations (1% and 2%), although higher concentrations (3%) and thus higher viscosity hydrogels resulted in diminished viability with decreasing needle diameter. Increasing cell density resulted in decreased cell viability and a concomitant decrease in DNA content, perhaps due to competing nutrient demands as a result of more closely packed cells. However, higher cell densities resulted in increased levels of extracellular matrix accumulated. Overall, this work highlights the potential of EHDS as a controllable and versatile approach to fabricate microcapsules for injectable delivery which can be used in a variety of applications such as drug development or cell therapies.