Spine Dynamics: Are They All the Same?

Three-dimensional microfabrication using two-photon polymerization of biocompatible photopolymers is applied for producing scaffolds for tissue engineering of artificial cartilage. Different classes of photocrosslinkable polymeric materials including methacrylated oligo(lactide-co-caprolactone), urethanes, polyglycerines and biopolymers like hyaluronan, alginate and dextran were used to fabricate microstructured scaffolds based on CAD models. The biocompatibility of the materials was tested in cell culture experiments using bovine chondrocytes.

Two-Photon Polymerization for Microfabrication of Three-Dimensional Scaffolds for Tissue Engineering Application

Tissue-engineering therapies have shown early success in the clinic, however, the cell–biomaterial interactions that result in successful outcomes are not yet well understood and are difficult to observe. Here we describe a method for visualizing bone formation within a tissue-engineered construct in vivo, at a single-cell resolution, and in situ in three dimensions using two-photon microscopy. First, two-photon microscopy and histological perspectives were spatially linked using fluorescent reporters for cells in the skeletal lineage. In the process, the tissue microenvironment that precedes a repair-focused study was described. The distribution and organization of type I collagen in the calvarial microenvironment was also described using its second harmonic signal. Second, this platform was used to observe in vivo, for the first time, host cells, donor cells, scaffold, and new bone formation within cell-seeded constructs in a bone defect. We examined constructs during bone repair 4 and 6 weeks after implantation. New bone formed on scaffolds, primarily by donor cells. Host cells formed a new periosteal layer that covered the implant. Scaffold resorption appeared to be site specific, where areas near the top were removed and deeper areas were completely embedded in new mineral. Visualizing the in vivo progression of the cell and scaffold microenvironment will contribute to our understanding of tissue-engineered regeneration and should lead to the development of more streamlined and therapeutically powerful approaches.

/pdf/visualizing-osteogenesis-in-vivo-within-a-cell-scaffold-2rsmqjml85.pdf

Visualizing Osteogenesis In Vivo Within a Cell–Scaffold Construct for Bone Tissue Engineering Using Two-Photon Microscopy

PurposeNIR radiation in the range of about 800 nm is less absorbed by biological tissues and is suited for triggering photonic effects using femtosecond pulsed Ti: Sa lasers. Especially in the life...

Two-photon techniques in tissue engineering.

http://www1.udel.edu/chem/bahnson/chem645/Martini.pdf

Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time

In our experiments 2-Photon laser scanning microscopy (2PLSM) has been used to acquire 3-dimensional structural information on native unstained biological samples for tissue engineering purposes. Using near infrared (NIR) femtosecond laser pulses for 2-photon excitation and second harmonic generation (SHG) it was possible to achieve microscopic images at great depths in strongly (light) scattering collagen membranes (depth up to 300 μm) and cartilage samples (depth up to 460 μm). With the objective of optimizing the process of chondrocyte growth on collagen scaffolding materials for implantation into human knee joints, two types of samples have been investigated. (1) Both arthritic and non-arthritic bovine and human cartilage samples were examined in order to differentiate between these states and to estimate the density of chondrocytes. In particular, imaging depth, fluorescence intensity and surface topology appear promising as key information for discriminating between the non-arthritic and arthritic states. Human chondrocyte densities between 2-10 6 /cm 3 and 20-10 6 /cm 3 , depending on the relative position of the sample under investigation within the cartilage, were measured using an automated procedure. (2) Chondrocytes which had been sown out on different types of I/III-collagen membranes, were discriminated from the scaffolding membranes on the basis of their native fluorescence emission spectra. With respect to the different membranes, either SHG signals from the collagen fibers of the membranes or differences in the emission spectra of the chondrocytes and the scaffolding collagenes were used to identify chondrocytes and membranes.

/pdf/two-photon-laser-scanning-microscopy-on-native-cartilage-and-115ker422q.pdf

Two-photon laser scanning microscopy on native cartilage and collagen-membranes for tissue-engineering

Hyalin cartilage was investigated with 2-photon laser scanning microscope . NIR-2-photon-excitation and a specialized long distance objective lens allowed for autofluorescence and SHG measurements of the extracellular matrix up to 400 µm inside the sample.

/pdf/2-photon-laser-scanning-microscopy-on-native-human-cartilage-3vb43pnu41.pdf

2-photon laser scanning microscopy on native human cartilage

Native hyaline cartilage from a human knee joint was directly investigated with laser scanning microscopy via 2-photon autofluorescence excitation with no additional staining or labelling protocols in a nondestructive and sterile manner. Using a femtosecond, near-infrared (NIR) Ti:Sa laser for 2-photon excitation and a dedicated NIR long distance objective, autofluorescence imaging and measurements of the extracellular matrix (ECM) tissue with incorporated chondrocytes were possible with a penetration depth of up to 460 µm inside the sample. Via spectral autofluorescence separation these experiments allowed the discrimination of chondrocytes from the ECM and therefore an estimate of chondrocytic cell density within the cartilage tissue to approximately 0.2-2·10 7 /cm 3 . Furthermore, a comparison of the relative autofluorescence signals between nonarthritic and arthritic cartilage tissue exhibited distinct differences in tissue morphology. As these morphological findings are in keeping with the macroscopic diagnosis, our measurement has the potential of being used in future diagnostic applications.

/pdf/2-photon-laser-scanning-microscopy-on-native-human-cartilage-4s5ltbeqba.pdf

2-Photon Laser Scanning Microscopy on Native Human Cartilage

2-Photon Laser Scanning Microscopy on Native Human Cartilage and Collagen-Membranes for Tissue Engineering

The arrangement has an optical unit for controlling a laser pulse of a tissue (24) to be tested. A detection unit i.e. electron-multiplying charge-coupled device camera (26), detects fluorescent light from the tissue, and the optical unit comprises scanning mechanisms i.e. movable mirrors (12, 14), for turning the laser pulse into a bundle (20) of optical fibers. The optical fibers are discharged from a gradient lens-objective (22). An evaluation unit automatically evaluates the fluorescent light detected by the detection unit. The bundle comprises approximately 30000 fibers. An independent claim is also included for a method for producing a diagnostic relevant parameter of human cartilage-tissue in vivo.

Arrangement for producing diagnostic relevant parameter of human cartilage-tissue in vivo during e.g. screening tests, has evaluation unit automatically evaluating fluorescent light detected by detection unit

Michael Dickob

Papers

Two-photon laser scanning microscopy on native cartilage and collagen-membranes for tissue-engineering

2-photon laser scanning microscopy on native human cartilage

2-Photon Laser Scanning Microscopy on Native Human Cartilage

2-Photon Laser Scanning Microscopy on Native Human Cartilage and Collagen-Membranes for Tissue Engineering

Arrangement for producing diagnostic relevant parameter of human cartilage-tissue in vivo during e.g. screening tests, has evaluation unit automatically evaluating fluorescent light detected by detection unit