ECM Monitoring in Scaffolds


 

Optically-based analytics of extracellular matrix production in cell-seeded artificial scaffolds

The central dogma in Tissue Engineering is about seeding artificial, biocompatible scaffolds (made from biomaterials) with cells. Those cells are expected to populate into the scaffold, degrade the scaffold matrix and replace the latter by a natural tissue matrix (extracellular matrix, ECM) (see figure, A). This tissue ECM consists primarily of collagens, elastins and other extracellular scaffolding proteins that anchor the cells into the de novo built tissue. The optimization of this matrix production is a primary challenge in tissue process engineering since only correctly embedded cells express their optimal function and biomechanical stability. Thus, readouts are required that are capable to visualize and quantify the ECM production and the cell-scaffold junction deep within the seeded scaffolds. ECM proteins can be assessed immune-optically or biochemically however, during these procedure, the construct is usually fixed and no longer viable. We use multiphoton microscopy and take advantage of an intrinsic physical effect, frequency doubling, inherent to collagen-I protein fibrils to visualize collagen matrix. High energetic pulsed infrared laser light is deflected into the probe and emitted light of double frequency (half wavelength) is optically detected in backscattered direction to visualize built collagen-I (see figure, B). By optically slicing through the probe we can a minimally-invasive reconstruction of the 3D aspect of the ECM production and optimize this process. In particular, optimization of the environmental and chemical milieu of the medium are crucial. We investigate which supplementation factors to cell culture medium enhance ECM production in scaffolds. In current studies, we found a much stronger production of collagen fibrils under conditions of reduced serum and increased ascorbic acid supplementation (see figure, C).

Muscle Biomechanics

Figure: Extracellular matrix (ECM) production in cell-seeded artificial scaffolds using minimally invasive optical metrology for optimizing process engineering. A, central dogma in Tissue Engineering: artificially produced 3D scaffolds from biodegradable biomaterials and given porosity are seeded with cells (usually stem cells). Those migrate into the scaffold, proliferate and degrade the scaffold components. The scaffold is then successively replaced by cell-produced ECM. B, ECM components, in particular, collagen-I, can non-invasively be visualized deep within the scaffold without any external labelling, using its frequency doubling effect (Second Harmonic Generation) in multiphoton excitation. C, recent results of our process optimization study to enhance ECM production in 3D cell pellets. Under conditions of serum deprivation (1%, 0% FBS) and ascorbic acid supplementation (aa), collagen fibril production and orientation are optimal (from Vielreicher et al. 2015).

Literatur:

Vielreicher M, Schürmann S, Detsch R; Schmidt MA, Buttgereit A, Boccaccini A, Friedrich O (2013) Taking a deep look: modern microscopy technologies to optimize the design and functionality of biocompatible scaffolds for tissue engineering in regenerative medicine. J R Soc Interface 10(86):20130263.

Vielreicher M, Gellner M, Rottensteiner U, Horch RE, Arkudas A, Friedrich O (2015) Multiphoton microscopy analysis of extracellular collagen I network formation by mesenchymal stem cells. J Tissue Eng Regen Med, in press.