Quantitative Monitoring of Extracellular Matrix Production in Bone Implants by 13 C and 31 P Solid-State Nuclear Magnetic Resonance Spectroscopy J. Schulz, 1 M. Pretzsch, 2 I. Khalaf, 1,3 A. Deiwick, 4 H. A. Scheidt, 3 G. Salis-Soglio, 2 A. Bader, 4 D. Huster 1,3 1 Institute of Medical Physics and Biophysics, University of Leipzig, D-04107 Leipzig, Germany 2 Department of Orthopedic Surgery, University of Leipzig, D-04103 Leipzig, Germany 3 Junior Research Group Structural Biology of Membrane Proteins, Institute of Biotechnology, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany 4 Cell Techniques and Applied Stem Cell Biology, Center for Biotechnology and Biomedicine, University of Leipzig, D-04103 Leipzig, Germany Received: 7 December 2006 / Accepted: 17 January 2007 / Online publication: 1 April 2007 Abstract. We used 31 P and 13 C solid-state nuclear magnetic resonance (NMR) spectroscopy to detect and analyze the major organic and inorganic components (collagen type I and bioapatite) in natural rabbit bone and b-tricalcium phosphate implants loaded with os- teogenically differentiated mesenchymal stem cells. High-resolution solid-state NMR spectra were obtained using the magic-angle spinning (MAS) technique. The 31 P NMR spectra of bone specimens showed a single line characteristic of bone calcium phosphate. 13 C cross- polarization (CP) MAS NMR spectra of bone exhibited the characteristic signatures of collagen type I with good resolution for all major amino acids in collagen. Quantitative measurements of 13 C- 1 H dipolar couplings indicated that the collagen segments are very rigid, undergoing only small amplitude fluctuations with cor- relation times in the nanosecond range. In contrast, di- rectly polarized 13 C MAS NMR spectra of rabbit bone were dominated by signals of highly mobile triglyce- rides. These quantitative investigations of natural bone may provide the basis for a quality control of various osteoinductive bone substitutes. We studied the forma- tion of extracellular bone matrix in artificial mesenchy- mal stem cell-loaded b-tricalcium phosphate matrices that were implanted into the femoral condyle of rabbits. The NMR spectra of these bone grafts were acquired 3 months after implantation. In the 31 P NMR spectra, b- tricalcium phosphate and bone calcium phosphate could be distinguished quantitatively, allowing recording of the formation of the natural bone matrix. Further, 13 C CPMAS allowed detection of collagen type I that had been produced in the implants. Comparison with the spectroscopic data from natural bone allowed assess- ment of the quality of the bone substitute material. Key words: Calcium phosphate — Collagen — Cross- polarization magic-angle spinning — Order parameter — Magic-angle spinning nuclear magnetic resonance Bone grafting surgery and the treatment of bone defects represent a routine procedure for orthopedic surgeons. Naturally, the best source for bone replacements is autologous tissue; however, complications with regard to donor site morbidity, quantity of tissue that can be obtained, and pain during recovery are not uncommon [1]. Therefore, in vivo bone tissue engineering using artificial biocompatible scaffolds instead of autologous bone grafts may represent an attractive alternative [2, 3]. Autologous cells can be seeded into these scaffolds and implanted into a bone defect of the patient either to heal it or to produce bone material for autologous trans- plantation [4]. Usually, the scaffolds used in bone tissue engineering are porous microcarriers that provide a large surface area for cell growth during propagation [5]. In particular, calcium phosphate ceramics have been shown to induce stable interfaces between the regener- ated bone and the surface of the scaffold material (bone bonding) [6]. Surface coating with cyclic RGD peptides has also been shown to stimulate osteoblast adhesion and proliferation [7]. In addition, some of these scaffold materials are biodegradable and can be resorbed with- out toxic degradation products. With the advent of new stem cell techniques, mesenchymal stem cells have been used instead of osteoblasts to colonize these scaffolds. While the cells participate in healing, they are also capable of forming new extracellular matrix (ECM) of the bone material. Mesenchymal stem cells are multi- potent cells, and in vitro osteogenic differentiation can be induced by the cell culture medium [8]. Once im- planted into the body, they will produce organic and inorganic ECM to finally form bone tissue. Although a few bone tissue engineering procedures are already in preclinical use, there is further need for in vitro and animal studies to improve the technology and fully exploit the potential of this very promising method. Yet, the monitoring of ECM production and the quan- J. S. and M. P. contributed equally to this study Correspondence to: D. Huster; E-mail: daniel.huster@bio- chemtech.uni-halle.de Calcif Tissue Int (2007) 80:275–285 DOI: 10.1007/s00223-007-9007-3