Community Research and Development Information Service - CORDIS

Periodic Report Summary 4 - BIODESIGN (Rational Bioactive Materials Design for Tissue Regeneration)

Project Context and Objectives:
1. Project Objectives

The development of biofunctional scaffolds for regenerative medicine today is mostly based on perceived and limited design criteria often using a single point approach after lengthy animal trials. The outcome after in-vitro and in-vivo evaluation is often disappointing resulting in tedious iteration processes. The overall objective of the Biodesign project is to develop new, and rational design criteria for advanced biomaterials used as scaffolds for bone, skeletal muscle and cardiac muscle. This involves defined chemistries with physical characterization with the aim to be generally applicable to many scaffold components. These are being evaluated in a multiparametric in-vitro environment that mimics the complexity of the natural tissue prior entering into animal models to reduce the number of animals required and hence the cost for bringing new biomaterials to the clinic.
To address this the following work components are now 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 evaluation 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.

As a result of the objectives, the scientific work has been divided into distinct but interdependent areas to which individual partners bring 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 & evaluation
4. Tissue specific in vitro and in vivo scaffold evaluation and imaging
5. Selecting in vitro screens for 3R s (Replacement, Refinement and Reduction)

Area 1 assembles, analyses and draws conclusions from the present limitations seen for biomaterials in clinical trials or large animal testing. The aim is to understand the link between scaffold design and the final clinical outcome and to translate knowledge between biomaterials scientists and the clinical users. Presently this is not appreciated and is 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 in the consortium.

Area 2 designs 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 developed and used for specific evaluation purposes e.g. monitoring degradation, studying effect of material properties on cell and tissue behaviours, and for implantation to correlate in vitro evaluation methods with in vivo outcome.

Area 3 develops test methods to evaluate the potency and function of cell/scaffold products before clinical use. The structure and function of specific products now drives product-testing matrices, which are individualized for each cell/scaffold construct. We have assembled, developed and for bone started to apply in vitro screening tools for screening 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 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 establishes correlations between in vitro evaluation tools that mimic the in vivo milieu (i.e. bioreactors) and the in vivo outcomes in the animal models, that helps reduce the ethical challenge and costly use of animal models. Advanced in vitro tissue models has been developed partially based on decellularized tissue, to use the native morphological, mechanical and ECM carried cues. These are beeing 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:
2. Work performed since beginning of project

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 final 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 has been summarised in an extensive review paper (under revision). For skeletal muscle and cardiac muscle current data 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 designed 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 delivered for evaluation 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 and demonstrated initial fesibility.

Area 3. 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. We have therefore assembled information regarding the cell types and techniques used by each group. All institutions involved in providing cells for the project have described the cell types and culture techniques appropriate for those cell types. It is now possible to exchange cell types and test that the methodologies described within each institution are repeatable within separate institutions. A selected set of screeining tools have been selected and used for a ”test” 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 are now being used by all labs performing correlating evaluations.

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 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 stimulations and performed tests with selected materials. Drug release kinetics and potential 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 relevant 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 is 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:
3. Expected final results and potential impact

Bioscaffolds research has been 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 that are bioactive alone, or are functionalized through the addition of factors or cells that can be applied by 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 own bottlenecks. Bioactive scaffolds are assessed at the pre clinical 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 designed to address this specific 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) Achieve radical innovations in state-of-the-art biomaterials and to design highly performing bioinspired materials learning from natural processes.
(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

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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