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Rational Bioactive Materials Design for Tissue Regeneration

Periodic Report Summary 5 - BIODESIGN (Rational Bioactive Materials Design for Tissue Regeneration)

Project Context and Objectives:
The RATIONAL DESIGN of bio-interactive materials is critically dependent on the understanding of how relevant cells interact with synthetic or natural materials as tissues form and remodel in-vivo. Through this understanding, it will then be feasible to adapt and apply, these natural mechanisms to our synthetic biomaterials such that they operate within the cell-tissue constructs. The challenge has been that the current biomaterials research-design paradigm has been poorly suited to the needs of tissue engineering and regenerative medicine. Within “the tissue-cell constructs” means that the fabric of the biomaterial must not just adhere to cells, hold spaces for cells due to porosity or gradually dissolve away, delivering surprise unexpected mechanical drop in materials strength and the corresponding increase in loading of tissues as they disintegrate. In effect, the use of unnaturally rigid structures, random pores, and materials designed for uncontrolled cell-independent dissolution have not been sufficient to trigger natural regeneration processes. In BIODESIGN, the next generation materials have been developed to be sensitive to cell-enzyme degradation, suitable for cells to move rapidly through as well as over, provide compliant directional attachment for more than one cell surface at a time.
The fault-line has been that current low-risk solutions assume a start-point based on materials/structures introduced >30yrs ago for the prosthetics era, whether they are suitable or not. Worse yet, current experimental options to break this paradigm are limited, as they have been relying on similarly inappropriate in vivo/in vitro test systems.
The solution has been to enable innovative alternatives by first developing brand new, purpose-built and rational test-experimental systems for developing next generation biomaterials for regenerative medicine. Molecular and physical information coded within the extracellular milieu has in BIODESIGN been used and designed into new 3D supports, head-to-head with test systems which during last year (a) allow meaningful and relevant high throughput in vitro screening of variant biomaterials, before animal testing and (b) measure bio-functional outcomes directly by tailored imaging and (c) have been successfully evaluated in animal models.
For efficient industrial uptake and clinical use, tissue-engineering constructs must not only be functional within current expectation but also be cost-effective. This introduces a tension between the need for open-ended sophistication with ease of production. This tension has been (at least for bone) balanced where we have a rational basis for selecting out those factors, from the broad array of extracellular influences, which we must control.
To accomplish this, the following work components have been put in place:

✓ A methodology to correlate biomaterial design with functional in vivo outcomes.
✓ Defined and non-toxic chemistries that allow assembly of a broad range of scaffold matrix components.
✓ Functional matrix components allowing assembly in vivo after injection.
✓ Functional matrix components that control the delivery of biological cues such as growth factors and oligonucleotides.
✓ Scaffold components that allow in situ and in vivo imaging.
✓ In vitro evaluation methods ranging from 2D to 3D, over screening techniques to more in vivo mimetic.
✓ In vivo assessment methods allowing studies of correlation with in vitro outcomes, limiting the number of animals required.
✓ Methods, based on correlation analysis, to select tests that can be predictive of in vivo animal outcome, at least for bone.

As a result of the objectives, the scientific work has been divided into distinct but interdependent areas to which individual partners have brought in specific scientific expertise and resources that are collectively leveraged to achieve our aims. These include:
1. Correlation analysis for rational design
2. Scaffold design
3. In-vitro scaffold model development and evaluation
4. Tissue-specific in vitro and in vivo scaffold assessment and imaging
5. Selecting in vitro screens for 3R s (Replacement, Refinement, and Reduction)

Area 1 has assembled, analysed and drawn conclusions from the present limitations seen for biomaterials in clinical trials or large animal testing for engineering of bone. The aim has been to understand the link between scaffold design and the final clinical outcome and to translate knowledge between biomaterials scientists and the clinical users. So far this has not been appreciated and has been difficult to promote, but with regenerative medicine moving to the forefront of therapeutic strategies, integrating those project specialists who have performed these studies, including human clinical studies, has now been achieved within the consortium.

Area 2 has developed methods to prepare scaffolds, in a modular context, in the form of (i) injectable soft gels, (ii) compliant ECM composites and (iii) load bearing ceramics. Materials prepared using these novel methods have now been used for specific evaluation purposes e.g. monitoring degradation, studying the effect of material physical properties of cell and tissue behaviors, and for implantation to correlate in vitro assessment methods with in vivo outcome.

Area 3 has developed test methods to evaluate the potency and function of cell/scaffold products before clinical use. The structure and function of specific products can now drive product-testing matrices, which are individualized for each cell/scaffold construct. We have assembled, developed and for bone applied in vitro screening tools for scaffold materials development in vitro so that reliable and convenient protocols for monitoring engineered tissues can be used on-line and non-destructively.

Area 4 has developed methods for imaging in vitro and ex vivo tissue engineered structures, with capabilities for determining the scaffold parameters that can be measured before animal experiments have to be conducted and that are predictive of the scaffold’s in vivo performance. These parameters are now being used beyond the scope of BIODESIGN to correlate the scaffold’s in vivo performance without further animal testing. Before a novel matrix is used for ex vivo/in vivo tests, a set of biocompatibility tests has been conducted.

Area 5 has established correlations between in vitro evaluation tools that mimic the in vivo milieu (i.e. bioreactors) and the in vivo outcomes in the animal models, which helps to reduce the ethical challenge and costly use of animal models. Advanced in vitro tissue models has been developed, to use the native morphological, mechanical and ECM carried cues. These have been employed in bioreactor systems using scaffolds from area 2 and external stimuli and advanced monitoring techniques from area 3 for tissue mimetic models.

Project Results:
Area 1. Correlation analysis for rational design
The consortium has assembled, analysed and drawn conclusions from the present limitations seen for biomaterials in clinical trials and large animal models for bone, skeletal muscle, and cardiac muscle regeneration. This has resulted in a better understanding of the links between scaffold design and outcome, as well as where a lack of knowledge is prevalent. For bone regeneration, correlations are found between transport properties (porosity and permeability) of the matrix and functional outcome and have been summarised in an extensive review paper. For skeletal muscle and cardiac muscle current data for clinical translation are still too few to draw conclusions regarding any possible correlations.

Area 2. Scaffold design
The procedure for making the gels is premised on a modular synthesis of rationally conceived in vivo injectable extracellular matrix (ECM) mimics. The consortium has developed, made commercial and clinically available such gels. Similarly, bone conductive and inductive ceramics have been developed where the critical parameter of physical strength has been improved dramatically (>10X). These transformations permit the simultaneous assembly of multicomponent systems with a defined connectivity between the components. A set of standard materials has been prepared and evaluated in in-vitro and in-vivo models. Biomaterials allowing for delivery of DNA and RNA, following the principles of potentially translatable to humans, has been developed, laid the basis for a start-up company and demonstrated initial feasibility in vivo.

Area 3. In-vitro scaffold model development & evaluation
In-vitro scaffold model development & evaluation
In vitro and in vivo correlation can only be possible by standardising the variables. Each group in the Biodesign consortium had their own methodologies and resources available. BIODESIGN has therefore assembled information regarding the cell types and techniques used by each group. All institutions involved in providing cells for the project has standardised the cell types and culture techniques appropriate for those cell types. It became possible to exchange cell types and test that the methodologies described within each institution are repeatable within separate institutions. A selected set of screening tools has been selected and used for a screeing of a selected set of biomaterials to evaluate predictability. Standard Operating Procedures (SOPs) have been tested in several laboratories to ensure validity and been implemented so the same procedures were used by all labs performing correlating evaluations. Thereby, we set the standards for future work on the correlation between in-vitro and in-vivo.

Area 4. Tissue-specific in vitro and in vivo scaffold evaluation and imaging
Imaging systems to monitor scaffold degradation along with tissue formation have been established and successfully evaluated. Data for ex vivo models of bone repair has been generated e.g. using chick bones as a culture template. A tissue engineering porous scaffold implanted into a living load-bearing tissue like the bone is subjected to two main types of mechanical stimulations: shear stresses coming from the fluid flow inside the scaffold and mechanical loading directly transmitted from the surrounding tissue. In this work package, we established procedures for characterizing mechanical stimulation and performed tests with selected materials. Drug release kinetics and possible control parameter were also investigated and successfully demonstrated feasibility to maximize drug loading potential and duration of release resulting in verification in animal trials using selected materials and correlating in vitro screens. Interestingly potentially new candidates to replace BMP-2 has been identified.

Area 5. Selecting in vitro screens for 3R
Current experimental models of tissue formation limit the dissection of complex cellular responses. In vitro assays are highly controllable, but do not capture tissue/host relations or relevant tissue architecture and physiology. In vivo model systems provide the appropriate organism contexts but cannot readily be manipulated. The central premise is that an ex vivo organotypic tissue system can provide a hybrid environment between the in vivo, and in vitro monoculture conditions to study cell invasion, proliferation, and fate. The objective in this work package has been to utilize ex vivo organ culture models (chick) as well as physiologically relevant bioreactor approaches to generate 3D model tissues. The models have now successfully been used to investigate selected biomaterials including decellularised tissue matrices as well as cells (skeletal muscle/myofibres, cardiac muscle and stromal lineages including bone and cartilage) of interest. The scale of this tissue allows for real-time imaging over weeks in culture.

Potential Impact:
Bioscaffolds research has significantly changed by the simultaneous development of biopharmaceuticals and the more profound knowledge of developmental processes and mechanisms in biological systems. The previous paradigm of developing scaffolds for tissues with easy access (such as skin) or with very similar chemical composite structures to the tissue being repaired (CaPO4 scaffolds and bone) has been increasingly replaced by advanced scaffold types. These are bioactive alone or are functionalized through the addition of factors or cells that can be applied by open surgery or injection to aid cell function and host repair. This has defined and refined tissue engineering or regenerative medicine concepts, which now encourage tissue repair using scaffolds that serve a temporary purpose, similar to a development process or target a critical component of the repairing tissue itself such as its blood supply through endogenous cell stimulation.
As a result of these exciting advances, there has been a clouding of what were once distinct regulatory fields (medical device versus medicine), which has created its bottlenecks. Bioactive scaffolds are assessed at the preclinical to clinical transition using the same criteria once reserved exclusively for New Chemical Entities and New Biopharmaceuticals which necessitate extensive animal studies and associated high costs.
This is not an optimal situation in consideration of the pre-investments made and matched with the sequential time and costs of the steps needed, therefore this call, NMP.2010.2.3-1 Development of standard scaffolds for the rational design of bioactive materials for tissue regeneration has been developed to address this particular issue with the following required impacts:

Principal and major impacts:

(i) Development of new, rational design criteria for advanced biomaterials/implants, whereby the specific nano/micro-scale properties, as well as the presentation of signaling molecules, are specifically targeted for a defined clinical use;
(ii) Radical innovations in state-of-the-art biomaterials and to design highly performing bioinspired materials learning from natural processes. The partners of BIODESIGN have created new business that already by the project end date has multiple times higher market value than the total project investments.
(iii) Reduction of our reliance on complex and costly in vivo experiments to predict the performance of bioactive materials;
(iv) Enhanced competitiveness of the biomaterials and biomedical industries in the EU as seen by commercial and clinical translation.

When newly designed materials are translated into clinical applications as therapeutic tissues, we run up against the roadblocks of reality. These are extensive animal trials and reduced predictability, too much inflammatory or immune response too little strength or survival time and unexpected patient responses. To locate new and effective development routes, BIODESIGN has integrated systematic materials development with robust in-vitro screening models that corroborate with the in-vivo outcome, as predictive tools. By applying standard fabrication concepts to the tissue outcome, it is at last possible to reduce the path-length from materials to functional tissue, at least for bone but likely very soon for skeletal and cardiac muscle.
Through successful preclinical evaluation during the first years, a series of animal trials has been performed towards the development of products for participating companies. Participating companies have grown/expanded, some done an exit to bring in additional funding and also new companies have been created as a result of research work within Biodesign.


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