FP-00025

Section 1 - Basic information about you and your application:

Title of research project
Development of an advanced engineered in vitro model for the study of Intervertebral disc (IVD) degeneration pathways

Grant Type
The ORUK Early-career Research Fellowship

Research area
Diagnostic and treatment

Duration
24

Start date
November 1, 2023

Have you previously received funding from ORUK?
No.

Profession
Academic scientist

Your current job title/position
Postdoctoral Research fellow

Are you an early-career researcher (ECR)? (definition of ECR)
yes

Section 2 - Lay summary

Lay summary:

Low back pain (LBP) is estimated to affect the majority of the adult population (60-80%). In over 40% of cases, it progresses into a chronic disabling condition (Colombier P, 2014). In 2020, LBP affected 619 million individuals worldwide and it is projected that the prevalence of LBP will rise to 843 million cases by 2050 by population growth and ageing (Ferreira ML, 2023). LBP is strongly associated with the degeneration of the intervertebral disc (IVD), an anatomical structure made of a core of soft cartilage (nucleus pulposus, NP) surrounded by a ring of fibrous tissue (annulus fibrosus, AF). The disc function is to keep the vertebrae separated from each other to make the spine flexible and to act as a shock absorber upon compression. The IVD aging-driven degeneration or traumatic conditions like herniation, disrupt the IVD integrity and functions thus leading to LBP because of the compression of adjacent nerves as well as the invasion of new nerves within its non-innervated structure.

To date, the exact mechanisms triggering IVD degeneration have not been elucidated thus limiting the development of effective medical interventions to alleviate patients’ chronic pain. Therefore, understanding IVD changes, particularly those related to extracellular matrix constituents and cells, is key to identifying therapeutic targets that could potentially slow, halt or even reverse the age-related degenerative cascade.

My ambition is to provide the relevant research field with the first lab-bench IVD three-dimensional biomimetic model enabling the identification of biochemical pathways and cellular activities occurring in healthy and pathological IVD in relation to degeneration and consequently chronic pain. The model is expected to enable the accurate study, at the cellular level, of the effects that alterations of the anatomical structures, tissue macromolecular composition and biomechanics may have on IVD degeneration.

The research approach proposed here along with the data generated through the model will be a step-change to define my distinct research profile and establish my leadership in the field of IVD degeneration in vitro research. Indeed, the concepts underpinning this project, the methodology that will be developed and the results obtained at different levels (i.e. biochemical, cellular, biomechanical) will lead to my research in IVD being recognized internationally. This will also give me the opportunity to champion the cause of the ORUK, worldwide while contributing to the UK leadership in the life science sector.

This model has the potential to be applied to the training of young spine surgeons, providing them with a realistic simulation of IVD conditions as well as aiding in surgical planning, offering valuable insights into the specificities of individual cases. Indeed, its use for the identification of key biochemical pathways and cellular activities may help identify novel therapeutics.

Section 3 - Purpose of research

Purpose of research:

The aim of this project is to develop an in vitro model for the study of the cellular pathways leading to IVD degeneration. The model will be based on 3D scaffolds made of known macromolecules of the IVD and engineered as AF- and NP-mimicking structures in which tissue-specific cells will be encapsulated.

Objectives:

1. Design of biomimetic IVD structure

MRI scan data taken from healthy volunteers provided by Dr Nick Dowell (Clinical Imaging Science Centre, CISC, University of Sussex) will be collected and their data will inform the printing of 3D scaffolds through CAD software.

1. Bioprinting of cellularized healthy and degenerated IVD model:

The optimised anatomy-mimicking constructs will be printed in combination with the bioplotting of IVD- cells precisely located in the center of the NP-mimicking scaffold and throughout the concentric rings of the AF-mimicking structure.

1. Effect of mechanical stimulation on cell behavior:

The final construct will be tested in static and dynamic compression mode by a dedicated dynamic bioreactor to show the cells’ response to alterations of mechanical stimuli.

Deliverables

D1.1   A protocol outlining the parameters of the fabrication process, including optimal scaffold printing, bioplotting parameters and collagen/proteoglycan composition (Month 6).

D1.2 A protocol of IVD mimicking constructs (n=9) with varying concentrations of PGs mimicking healthy and degenerated conditions (Month 9).

D1.3 Report of characterisation data including imaging techniques, rheological data, and mechanical testing of the printed constructs (Month 12).

D2.1 SOPs summarizing the successful incorporation of cells within the constructs (Month 16).

D2.2 Report of cell behavior within the printed constructs (Month 18).

D3.1 A protocol for mechanical stimulation procedure on 3D cellularized constructs including the setup of dynamic bioreactor (Month 20).

D3.2 Report including data on the effect of mechanotransduction on cell behavior (Month 23).

D3.3  Final report including scientific data and demonstration of dissemination activities (Month 24).

Section 4 - Background to investigation

Background to investigation:

IVD is a complex structure that effectively withstands spinal compression while facilitating the motion of intervertebral segments. IVD is a highly hydrated tissue and is mainly composed of extracellular matrix molecules including collagens and proteoglycans (PGs). In a young and healthy disc, the nucleus pulposus (NP) constitutes a highly plastic and hydrated region, while the annulus fibrosus (AF) consists of a network of collagen fibers arranged in sheets around the nucleus. This arrangement provides tensile strength as well as confinement to the NP, effectively limiting bulging.

During disc degeneration and ageing, significant changes are observed in the IVD at both cellular and tissue levels. Loss of cell density and transition toward chondrocyte-like cells results in less effective NP-specific matrix synthesis. Changes in the composition and mechanical properties of the surrounding environment has an effect on NP cell function and behavior.

The alteration in matrix turnover during degeneration leads to dehydration and progressive ECM disorganization which promotes mechanical failure and annular tears. Over time, type II collagen is replaced by type I collagen in the NP and aggrecan content decreases. Cytokines may also be released, further affecting cell activity and tissue homeostasis. This imbalance results in reduced disc height, hernia formation and spinal pain. The release of growth factors, cytokines and other signaling molecules is believed to cause neovascularization and neoinnervation within degenerative IVD.

Understanding IVD physiology and pathology (particularly in terms of IVD matrix constituents and their alterations in development and disease) is key to unveiling molecular cues that might be used to slow, halt or reverse the age-associated degenerative cascade. Its strong association with lower back pain imposes a significant socioeconomic burden. In vitro models providing insights into the biochemical and cellular pathological pathways of IVD degeneration are advocated tools to foster the development of novel pharmaceutical treatments.

Since my appointment as the project Research Fellow, I have made significant progress toward the development of the model. The expertise I gained during my PhD and previous postdoc in the fabrication and characterization of 3D constructs as well as conducting in vitro cell experiments provided me with the opportunity to secure the prestigious John Shepperd fellowship at UoB focusing on in vitro studies of IVD degeneration. Previously, I had successfully obtained my PhD in Biomaterials Engineering from the University of Oxford, where I investigated novel tissue engineering approaches to address the limitations in the current treatments of osteochondral defects. Following my doctoral studies, I worked as a postdoctoral research associate at the Tehran University of Medical Sciences on collagen-based hydrogels for cartilage tissue engineering. This project was conducted in collaboration with the UCL Biomedical Institute at the Royal National Orthopaedic Hospital (London). Subsequently, I worked as a research associate scientist at UCL Biomedical Institute where I investigated the potential of exosome-like nanovesicles for the treatment of osteoarthritis.

During my fellowship at UoB, with the support of my second mentor Dr Derek Covill, I have been able to print 3D molds able to guide the gelification of collagen type I and type II as well as of proteoglycans in 3D structures mimicking those of the nucleus pulposus and annulus fibrosus. More importantly, we have been able to reproduce 3D structures where the percentages of collagens and proteoglycans can be changed to mimic IVD healthy and degenerated conditions, whereby a decreased presence of proteoglycans (PGs) aims to mimic an ageing/degenerated IVD (Fig 1).

Besides, I have focused on the impact of PGs reduction, on the physicochemical properties and cellular behavior of the NP- and AF-mimicking constructs. To this end, 3D IVD-mimicking constructs were developed using collagen type I, II and varying concentrations of PGs. A comprehensive physicochemical characterization was conducted by scanning electron microscopy (Fig 2), FTIR, rheological tests and water retention properties to define the microenvironment of NP and AF cells in the healthy and diseased disc showing changes in porosity, crosslinking and mechanical properties (Fig 3). Human NP and AF cells were encapsulated within the collagen-PGs hydrogels, and morphological analysis was performed along with assessing the synthesis of key components of the IVD extracellular matrix (Fig 4). Immunohistochemistry staining was assessed by confocal microscopy to study the cell-matrix adhesion pathway and cellular regulators such as hypoxia-inducible factor (HIF1-⍺) (Fig 5).

Cells showed an elongated morphology with evidence of mitosis in the absence of PGs, while they adopted a more rounded morphology when surrounded by PGs matrix. These observations are aligned with the degeneration process where ageing human NP cells undergo phenotypical changes shifting from a chondrocyte-like round morphology to an elongated fibroblast-like morphology.

A higher amount of collagen and PGs appeared to be synthesized by NP cells at increasing PG content. This indicates that the increased PGs content of hydrogels creates a more favorable environment for the NP cells to synthesize and secrete these extracellular matrix components thus preserving the IVD healthy conditions.

Likewise, immunohistochemistry analysis confirmed that the expression of the integrin-b1 receptor known to promote the adhesion to the collagen type II fibers was higher in the absence or at lower PG amounts, whereas the expression of the CD44 hyaluronic acid receptors was increased. These receptor-mediated interactions can have significant effects on gene expression. The findings also indicate that the increased PGs content leads to the higher expression of hypoxia-inducible factor (HIF-1a) by NP cells suggesting a potential regulatory role of PGs in HIF signaling. The effect of PGs on the expression of HIF-1a by NP calls is not established in the existing research.

My findings reveal that the reduction in PGs content of NP tissue, known to occur during the IVD degeneration process, affects the physicochemical properties of the tissue and the subsequent cell behaviors in terms of NP cell phenotype and expression of transcriptional markers such as HIF1-α. I presented these findings at the Termis EU Conference 2023, and I am also presenting at the ESB conference held in September 2023. Currently, we are in the process of submitting these promising results to the Bioactive Materials journal and generating data from the testing of AF-mimicking constructs.

Section 5 - Plan of investigation

Plan of investigation:

We are planning to achieve the proposed 3D in vitro model by dividing the project into 4 main work packages outlined as follows:

WP1. Development of biomimetic multi-concentric IVD structure via bioprinting to closely mimic the NP and AF tissues (Months 1-9):

MRI scan data taken from n=9 healthy volunteers divided in 3 age groups (18-25, 35-50, 60-70 years old) provided by Dr Nick Dowell (CISC, University of Sussex) will be collected and their data will be linked to the bioplotter through computer-aided design (CAD) software (BioScaffolder, Gesim, Germany). Collagen type I and type II will be co-printed with PG at different component ratios in the form of an intervertebral disc including both annulus fibrosus and nucleus pulposus compartments. The concentration of PGs to be used will be based on previous in vitro studies (see preliminary data above) as well as on MRI data processed by an optimized method that enables the quantification of PGs in tissues. This phase of the work will show the potential of 3D printing to be developed into a tissue engineering construct sustaining cell functions and new tissue deposition within the printed scaffold. A process of reiterated characterization of the printed scaffolds will enable the optimization of the printing parameters to inform the protocol of cell integration into the scaffolds as described in WP2.

WP2. Bioprinting of cellularized healthy and degenerated IVD model (Months 8-18):
The optimised anatomy-mimicking constructs will be printed in combination with IVD-specific cells precisely located either in the center of the NP (human nucleus pulposus primary cells) or throughout the concentric rings mimicking the AF (i.e. human annulus fibrosus primary cells). Using a method previously developed by the scientists at the CRMD, there will be also the potential of mimicking physiological and pathological angiogenesis at the periphery (i.e. outer section of the annulus) and in the core (i.e. avascular nucleus pulposus). To mimic the degenerating conditions, scaffolds will be seeded at their periphery by both vascular endothelial cells (i.e. human umbilical vascular endothelial cells, HUVEC) and nerve cells to assess their ability to penetrate within the gels on the basis of collagens/PG ratio variations. Viability, morphology and secretion of either pro-inflammatory cytokines or growth factors, particularly those known to promote innervation, by the encapsulated IVD cells and determined by enzyme-linked immunoassays (ELISA) will determine the effect of the ECM composition to induce either physiological remodeling of the tissue (i.e. growth factor secretion) or its degeneration (i.e. pro-inflammatory cytokine secretion). Immunohistochemistry staining assessed by confocal microscopy will enable the study of cell morphology, whereas other ELISA tests will establish their contribution toward the physiological or degenerative status of the engineered IVD. HUVEC and peripheral nerve cells, seeded at the surface of the engineered IVD will be studied for their ability to invade the IVD compartments at the different ECM compositions described above. Data on HUVEC angiogenic sprouting and axonal growth of nerve cells will be linked to the biochemical signaling released by the IVD cells.

WP3. Effect of mechanical stimulation on cell behavior (Months 19-24):
The final construct will be tested in dynamic compression mode by the mechanical test instrument (Bose ElectroForce) to show the cells response to mechanical stimuli similar to those of the spine. The mechanotransduction effects on the encapsulated cells will also be analysed upon spiking of the tissue culture medium with pro-inflammatory cytokines to simulate inflammation. After stimulation at different frequencies and cycles of compression, the cells will be analysed as reported in WP2. Data on mechanotransduction will be compared with those obtained in WP2 in the absence of mechanical stimuli. A qPCR test will be carried out to study the expression of both cytokines and extracellular matrix proteins in all the above-described conditions.

WP4. Project management (Month 1 – 24):

1. Weekly meeting with Prof Santin to discuss the project’s overall progress and assess the risk management plan (see risk registry). 2. Monthly meetings with collaborator teams reviewing scientific work through presentations. 3. Review of the reports to be submitted to the ORUK. 4. Revision of the risk registry and mitigation plan. 5. Planning and implementation of disseminating activities including the publication of scientific papers as well as attendance at conferences. Opportunities for commercial exploitation of the model as a tool for IVD surgery planning will be considered as a service to be provided to hospitals through the establishment of a start-up company or IP licensing agreement.

Risks and mitigations: If the bioprinting of the construct fails because of unforeseen reasons, we will still be able to produce multi-concentric structures by using molds with various radii and implementing multiple steps of casting and gelation. If mechanical stimulus testing faces any challenges, the alternative experiments would be the study of cell behavior in hypoxia conditions/or in transwell inserts with diffusion/penetration of medium to cells to simulate in vivo conditions as closely as possible.

The accurate analysis of cells, when encapsulated or invading an IVD-mimicking construct, could be impaired by the difficulty of the reagents to penetrate the scaffolds. In such a case the scaffold will be sectioned into relatively thin slices to remediate this problem.The breakthrough character of this model may not find the end users ready to immediately adopt it for surgery planning. Therefore, the model will be commercially exploited as a research tool worldwide.

Section 6 - Research environment and resources

Research environment and resources:

This research will be carried out at the Centre for Regenerative Medicine and Devices (CRMD), University of Brighton. The CRMD possesses state-of-the-art facilities in chemistry, material science, engineering, and molecular and cell biology. The Centre is a multidisciplinary research environment, with a blend of more than 30 permanent internationally recognized scientists, early career researchers, and industrial collaborators, and it has a regular uptake of Ph.D. and MRes students recruited in the UK and worldwide. The CRMD has an exemplary track record and interest in the development of biomimetic biomaterials, proved by successful EC and EPSRC-funded projects (>£5M). The UoB has a strong commitment to supporting early career researchers (“Concordat to Support the Career Development of Researchers”), proved by the resources and perspectives in support of my Fellowship. I will integrate with the current research network and facilities by bringing my expertise and skills for hydrogel design and characterisation to synergistically contribute to the success of the Centre and enable the establishment of new collaborations internally, nationally, and internationally. The CRMD Director, Professor Matteo Santin and his team have collected over 100 scientific outputs, over £ 4 million in competitive funding and a number of academic accolades. Prof Santin’s lab is equipped with the essential machinery for this project, including a bioprinter, mechanotransduction machine, microfluidic machine and clean-room-grade laminar flow cabinets.  To further enhance our research capabilities, we have established valuable collaborations with Dr Derek Covill from the Department of Engineering who brings valuable engineering insights and guidance to our work. Additionally, we have partnered with Dr Nick Dowel from the Clinical Imaging Science Centre, University of Sussex who provides us with MRI scans of healthy volunteers enabling us to obtain essential imaging data for our study.  Furthermore, this research will be supported by a philanthropic donation of Mr John Shepperd which will contribute significantly to the funding of this research endeavor.

Section 7: Research impact

Who will benefit from this research?

The full development of the model will benefit researchers studying the causes of intervertebral disc (IVD) degeneration and spine surgeons who can use this model for training and operation-planning purposes. The utilization of this model by pharmaceutical companies for drug testing purposes can offer significant benefits, including a reduction in animal study size, costs and ethical concerns.

How can your research be translated in real-life?

1.     Therapeutic targets: the model can aid in the development of therapeutic targets, such as novel drugs, regenerative and nanomedicine as tissue-engineered scaffolds, gene and cell therapies. Researchers can test different strategies to promote disc regeneration including the use of stem cells, growth factors or scaffold-based approaches within the model. Pharmaceutical companies can use the model to screen and test compounds aimed at treating disc degeneration to assess their therapeutic potential and side effects before proceeding to animal studies and clinical trials.

2.     Personalised medicine: The model can be used to study patient-specific responses to different treatment options by integrating cells derived from patients (i.e. induced pluripotent stem cells). researchers can simulate the degenerative process and test various therapies to determine the most effective treatment approach tailored to the patient’s condition. The patient-tailored printed model can support surgeons in the planning of IVD operations and the training of young surgeons.

How will your research be beneficial for ORUK and its purpose?

1. Improved understanding of IVD degeneration (particularly in terms of IVD matrix components and their changes in development and disease): The focus of this research is to unveil the underlying mechanisms and factors contributing to disc degeneration and lower back pain. This can advance musculoskeletal knowledge and inform the development of preventative measures and treatment strategies for degenerative disc disease.
2. Enhanced treatment options and improved patients’ quality of life: This study can help identify potential therapeutic intervention targets. This includes investigating the efficacy of drugs, biological agents, and tissue engineering approaches to promote disc repair.
3. Optimisation of surgical techniques: This model can contribute to the optimization of surgical techniques used in spine procedures. By providing a better understanding of biomechanical properties, the model can assist in developing, more effective and potentially less invasive surgical approaches.

Section 8: Outreach and engagement

1. Identify our target audience: identify the specific non-academic audience we want to reach, such as patients with disc-related conditions, their families, or the general public interested in spinal health. Understand their level of knowledge and specific concerns to tailor our communication accordingly.
2. Co-creation of dissemination plans with patient advocacy groups focused on spinal health.
3. Simplify complex scientific concepts regarding disc degeneration and using visuals and infographics diagrams and animation to represent the IVD degeneration process and its effects.
4. Relate scientific findings obtained by the model to real-life implications for patients and discuss the potential symptoms, consequences, and available treatment options by taking advice from our project adviser, specialist, and spine surgeon Mr John Shepperd.
5. Practical tips and recommendations can also be provided by Mr John Shepperd: these include advice that can be offered to help individuals prevent or manage disc degeneration. These include lifestyle modifications, exercises, ergonomic practices, and self-care techniques. This empowers the practical impact of the research. Engaging posts, videos or blog articles will be used to share key information.
6. Use of social media and online platforms to disseminate the findings to a broader non-academic audience.

Section 9: Research budget

Requested funding from ORUK

University fees (if any)
£0

Salary
£106800

Consumables
£1200

Publications
£1000

Conference attendance
£1000

Other items
£

Total 'requested fund'
£110000



Other items




Other secured funds

Internal funding
£0

Partner (University)
£0

Partner (Commercial)
£0

Partner (Charity)
£8000

Other sources
£0

Total 'other funds)
£8000

Section 10: Intellectual property and testing on animal

Is there an IP linked to this research?
No

Who owns and maintains this patent?


Does your research include procedures to be carried out on animals in the UK under the Animals (Scientific Procedures) Act?
No

If yes, have the following necessary approvals been given by:

The Home office(in relation to personal, project and establishment licences)?


Animal Welfare and Ethical Review Body?


Does your research involve the use of animals or animal tissue outside the UK?
No

Does the proposed research involve a protected species? (If yes, state which)



Does the proposed research involve genetically modified animals?


Include details of sample size calculations and statistical advice sought. Please use the ARRIVE guidelines when designing and describing your experiments.


There should be sufficient information to allow for a robust review of any applications involving animals. Further guidance is available from the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs), including an online experimental design assistant to guide researchers through the design of animal experiments.

Please provide details of any moderate or severe procedures


Why is animal use necessary, are there any other possible approaches?


Why is the species/model to be used the most appropriate?