Identification of Transglutaminase Substrates from Porcine Nucleus Pulposus as Potential Amplifiers in Cross-Linking Cell Scaffolds Elke Gebauer, † Elke Goßla, † Carolin Kwas, † Denise Salzig, ‡ Alexandra Schmiermund, ‡ Peter Czermak, ‡ and Hans-Lothar Fuchsbauer* ,† † Department of Chemical Engineering and Biotechnology, University of Applied Sciences of Darmstadt, Schnittspahnstrasse 12, 64287 Darmstadt, Germany ‡ Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Wiesenstraße 14, 35390 Giessen, Germany ABSTRACT: Nucleus pulposus from the porcine intervertebral disc was separated chromatographically to discover substrates of microbial transglutaminase. Highly purified proteins were prepared, among them type II collagen, the major protein of the nucleus pulposus. Determination of substrates was performed by transglutaminase-mediated incorporation of biotinylated probes displaying several glutamine and lysine donor proteins. Type II collagen was only labeled if smaller nucleus pulposus proteins were present. One of the modulating proteins was serotransferrin, a lysine donor substrate of bacterial trans- glutaminase. An additional substrate was the carboxy-terminal propeptide of type II collagen, chondrocalcin. Chondrocalcin, a regulator of type II collagen fibrillogenesis, occurs abundantly in juvenile cartilage and nucleus pulposus. Accordingly, the protein may be regarded as an excellent additive for the preparation of injectable stem cells in nucleus pulposus-like matrices cross-linked by microbial transglutaminase. ■ INTRODUCTION A promising approach of minimal invasive surgery may be the application of stem cells or mature tissue cells in appropriate extracellular matrices (ECMs). Stem cells need the correspond- ing signals to differentiate into specific tissue cells and to produce compounds of their own ECM. Differentiation may be induced by the ECM, nourishment with essential nutrients, oxygen, or mechanical stress. 1-3 In the past, materials such as alginate, agarose, fibrin, collagen, gelatin, or hyaluronic acid have been used to produce hydrogels without substantial success. 4 Drawbacks of alginate and agarose are minor biodegradability and low cell adhesion. The other biopolymers are better suited to be decomposed and to attach to cells. However, most of them are not enabled to supply stimulating signals for cell differentiation, in particular, if they are not typical components of the ECM. Collagens, besides proteogly- cans, the most important building blocks of the ECM, are nearly insoluble in water so that gelatin has been frequently preferred for preparing cell sca ffolds. 5 However, any modification alters the properties of the proteins and may cause erroneous signals. This may be equally true for hyaluronic acid that is as part of proteoglycans often degraded. Injectable materials may be particularly suited in the treatment of moderately injured intervertebral discs (IVDs). In the human body, there are at least 23 IVDs joining the vertebral bodies. Their main task is the constant transfer of loads arising from body weight and muscle activity through the spinal column. Moreover, they provide flexibility, flexion, and torsion. The complex IVD structure consists of a thick outer ring of fibrous cartilage, the so-called annulus fibrosus (AF). The inner part, termed nucleus pulposus (NP), is more jelly like. NP is covered above and below by cartilage end plates. 6 With increasing age and degeneration, the IVD alters collagen and proteoglycan composition and morphology. A more disorganized ECM results in frequently forming clefts with fissures. The major changes are loss of proteoglycans and thus hydration. Moreover, the composition of collagens varies with degeneration of the ECM. There are indications that the modified IVD is the result of increased hydrolytic activity displayed by fragmentation of collagens, proteoglycans, and fibronectin. 6 Poor nutrient supply and inappropriate mechan- ical load are thought as well to be responsible for the onset of degeneration. 7,8 The degenerative stages are often correlated with back pain, and in severe cases disc protrusion or discus prolaps occurs. Symptoms can worsen from heavy pain to numbness and palsy. Conventional therapies are focused on reducing symptoms by administering anti-inflammatory drugs, muscle relaxants, or opioid analgesics. 9 However, all these methods are not suited to regenerate damaged tissues and must be replaced by a more causal therapy. Recently, we analyzed the in vitro differentiation and proliferation of human mesenchymal stem cells (hMSCs) in porcine gelatin type A and a mixture of Received: February 6, 2013 Revised: March 4, 2013 Published: March 18, 2013 Article pubs.acs.org/Biomac © 2013 American Chemical Society 1564 dx.doi.org/10.1021/bm400188r | Biomacromolecules 2013, 14, 1564-1571