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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: 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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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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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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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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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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Modular biofabrication strategies using microtissues or organoids as biological building blocks have great potential for engineering replacement tissues and organs at scale. Here we describe the development of a biofabrication strategy to engineer osteochondral tissues by spatially localising phenotypically distinct cartilage microtissues within an instructive 3D printed polymer framework. We first demonstrate that immature cartilage microtissues can spontaneously fuse to form homogeneous macrotissues, and that combining less cellular microtissues results in superior fusion and the generation of a more hyaline-like cartilage containing higher levels of sulphated glycosaminoglycans and type II collagen. Furthermore, temporally exposing developing microtissues to transforming growth factor-ß accelerates their volumetric growth and subsequent capacity to fuse into larger hyaline cartilage grafts. Next, 3D printed polymeric frameworks are used to further guide microtissue fusion and the subsequent self-organisation process, resulting in the development of a macroscale tissue with zonal collagen organisation analogous to the structure seen in native articular cartilage. To engineer osteochondral grafts, hypertrophic cartilage microtissues are engineered as bone precursor tissues and spatially localised below phenotypically stable cartilage microtissues. Implantation of these engineered grafts into critically-sized caprine osteochondral defects results in effective defect stabilisation and histologically supports the restoration of a more normal articular surface after 6 months in vivo. These findings support the use of such modular biofabrication strategies for biological joint resurfacing.
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Cartílago Articular , Cabras , Animales , Colágeno , Colágeno Tipo II , Glicosaminoglicanos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Factores de Crecimiento TransformadoresRESUMEN
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.