Quantitative Monitoring of Extracellular Matrix Production in Bone Implants by 13C and 31P Solid-State Nuclear Magnetic Resonance Spectroscopy

We used ³¹P and ¹³C 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 β-tricalcium phosphate implants loaded with osteogenically differentiated mesenchymal stem cells. High-resolution solid-state NMR spectra were obtained using the magic-angle spinning (MAS) technique. The ³¹P NMR spectra of bone specimens showed a single line characteristic of bone calcium phosphate. ¹³C 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 ¹³C-¹H dipolar couplings indicated that the collagen segments are very rigid, undergoing only small amplitude fluctuations with correlation times in the nanosecond range. In contrast, directly polarized ¹³C MAS NMR spectra of rabbit bone were dominated by signals of highly mobile triglycerides. These quantitative investigations of natural bone may provide the basis for a quality control of various osteoinductive bone substitutes. We studied the formation of extracellular bone matrix in artificial mesenchymal stem cell-loaded β-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 ³¹P NMR spectra, β-tricalcium phosphate and bone calcium phosphate could be distinguished quantitatively, allowing recording of the formation of the natural bone matrix. Further, ¹³C CPMAS allowed detection of collagen type I that had been produced in the implants. Comparison with the spectroscopic data from natural bone allowed assessment of the quality of the bone substitute material. J. S. and M. P. contributed equally to this study