Prof. Daniel Huster1 and Dr. Mathias Pretzsch2
1Institute of Medical Physics and Biophysics and 2Department of Orthopedics, University of Leipzig, Leipzig, Germany email@example.com
Bone is a composite material with a remarkable combination of elasticity and stability. The tissue is composed of extracellular inorganic (≈50–60 wt. %) and organic (≈30–40 wt. %) matter, water (≈10 wt. %), and several cell types. This extracellular matrix (ECM) is produced by cells called osteoblasts, and mostly consists of bioapatite and collagen. The bone mineral can be characterized as a carbonated apatite consisting of hydroxyapatite (HA, Ca10(PO4)6(OH)2) with about 4–8% CO3 2– and other trace elements. Collagen Type I is the largest component of the bone tissue. The platelet-like bioapatite crystals are inserted in a parallel fashion into the collagen fibrils, replacing the water found in other tissue collagens (Figure 1).
Figure 1. Schematic drawing of a mineralized collagen fiber. Crystalline bone mineral (green) is incorporated where collagen triple helices (white cylinders) meet.
In principle, bone has a good self-healing capacity. However, for defects larger than a certain size (“critical size defect”) spontaneous healing of bone injury is not possible. Such defects can occur in many diseases such as osteoarthritis, bone cysts and tumors, or as a result of surgical procedures, for instance osteolyses associated with loosened endoprostheses or osteotomies. The gold standard for the treatment of bone defects is an autologous bone transplantation; however, disadvantages of the method include follow-up operations that are necessary in addition to the bone harvesting from the iliac crest, which are connected with significant comorbidity.1 Availability of natural bone is restricted and often not sufficient to heal large bone defects, while maintenance of an extensive bone bank is expensive and complicated by long-term tissue preservation issues. To overcome these difficulties, a number of synthetic and partial synthetic bone substitute materials have been developed. In clinical practice, the most important class of materials are HA ceramics, because the inorganic component of the bone matrix consists largely of HA. Materials such as HA obtained typically from marine coral,2 glass-reinforced HA,3 brushite,4 tricalcium phosphate,5 and mixtures of these materials (composites)6 are in use. Of central importance is the structure of the ceramics because an interconnecting pore system is required for reasonable osseous integration.7 A resorption of pure HA ceramics and substitution with bone (restitutio ad integrum) does not take place. However, bioceramics made of HA and tricalcium phosphate (TCP) provide better scaffold resorption.8
While there are a number of available bone substitution materials with sufficient bioactivity to treat small defects, it remains difficult to stimulate formation of bone ECM, necessary to treat larger injuries. This ECM re-growth, or osteoinductive effect, can be achieved by seeding implants with mesenchymal stem cells (MSCs).10 MSCs are easily harvested from the iliac crest and are suitable for regenerative therapies due to their high duplication capacity (up to forty times), ability to withstand preservation by freezing and capacity to build new tissue in a defect.11 In numerous animal12,13 and even human14 in-vivo studies, improved healing of critical size defect was observed using HA implants seeded with MSCs. However, reliable regenerative bone therapy remains a challenge with additional experiments needed to address the slow HA implant degredation15, choice of optimal MSCs and implant materials, as well as surgical procedures an patient follow-up.
Quantitative analytical tools need to be developed to monitor the formation of bone ECM in the various implant materials under appropriate conditions. We have used solidstate NMR spectroscopy (at a magnetic field of 17.6 T) to study the formation of ECM in bone implants. To this end, b-TCP (Ca3(PO4)2) ceramics were loaded with osteogenically differentiated MSCs to heal a critical size defect in the femoral condyle (knee joint) of a rabbit model.16 The MSC’s were isolated from aspirated bone marrow, cultured, and osteogenically differentiated. Porous b-TCP cylinders (6 mm 3 10 mm) were seeded with the MSCs for up to 7 days. The cylinders were transplanted into a 6 mm hole that was drilled into the rabbit bone (metaphyse of the distal femoral condyle). The animals were sacrificed after three months and the bone implants were removed for analysis.
Typically, the NMR spectra of solid materials are characterized by broad, anisotropic line shapes. However, the application of magic angle spinning (MAS) averages the anisotropic contribution to the chemical shift and collects the spectral intensity into a narrow centerband and a number of spinning sidebands. In the centerband of the NMR spectrum, the characteristic signals are separated according to their isotropic chemical shifts. Depending on the nucleus that is observed in these spectra, the organic and inorganic components of the newly formed bone ECM could be detected. Figure 2 shows 31P NMR spectra of (a) rabbit bone, (b) pure b-TCP, and (c) a rabbit implant removed after three months. The NMR spectra of rabbit bone and TCP consist of a single or two lines, respectively. The 31P NMR signal of the rabbit implant can be described by a superposition of the line shapes of rabbit bone bioapatite and the b-TCP matrix material. This spectrum indicates that (i) the characteristic carbonated bioapatite mineral was synthesized in the implant and (ii) the b-TCP matrix material was not fully resorbed after three months of implantation.
Figure 2. 161.9 MHz solid-state 31P MAS NMR spectra of (a) rabbit bone, (b) pure b-TCP, and (c) a rabbit implant removed after 3 months. Spectrum (c) can be simulated from a superposition of spectra (a) and (b) at a 49:51 ratio. The NMR experiments were carried out in a 4-mm MAS rotor using a Hahn echo pulse sequence at 37 °C and a MAS frequency of 8 kHz. Spectra were acquired with a 1.9 μs 90° pulse and a relaxation delay of 400 s.
The solid-state NMR results can be quantified to measure the composition of the implant material, provided a few prerequisites are fulfilled. First, all 31P nuclei need to be polarized identically, as in a single pulse excitation experiment. Second, the chemical shift anisotropies of the signals must be similar; otherwise all sideband intensities have to be included into the analysis. Third, the spin-lattice relaxation times of the molecular species must be at least five times shorter than the repetition time of the experiment. Under these conditions, the line shape of the implant spectrum can be decomposed into a superposition of rabbit bone and the b-TCP spectrum at a 49:51 ratio.
From the 31P NMR spectrum of the bone implant, the contribution from the apatite can be filtered out. Since bone apatite contains hydroxyl groups, the 31P NMR spectrum can be obtained by the cross-polarization (CP) technique, in which, first, the 1H nuclei are polarized and subsequently, the polarization is transferred to 31P. While no 31P CP MAS spectrum of b-TCP can be acquired due to the absence of 1H nuclei, the 31P CP MAS of a rabbit implant consists only of a single line, while the directly polarized 31P NMR spectrum of the same implant yields the superposition of the b-TCP and the rabbit bone NMR spectrum (Figure 3). Thus, it could be demonstrated that the newly formed inorganic bone ECM in the implant is indeed bioapatite containing hydroxyl groups.
Figure 3. 161.9 MHz 31P MAS NMR spectra of a b-TCP implant removed from a rabbit’s femoral condyle after three months. Spectrum (a) was directly polarized using a single 90° pulse, while spectrum (b) was cross-polarized using a contact time of 1408 μs. All spectra were measured at 37 °C.
In addition to the inorganic contribution of bone ECM, solidstate NMR can also detect the organic component in the implants using 13C NMR. Because of the lack of 13C in b-TCP, no 13C NMR spectrum can be acquired (not shown). However, the rabbit bone implants recovered after three months showed a 13C CP MAS NMR spectrum with characteristic signals from protein amino acids (Figure 4). For comparison, the NMR spectra of rabbit bone and collagen type I are given. Clearly, a good correspondence is visible between the three NMR spectra indicating that the 13C NMR spectrum of the implant can be explained solely by the organic collagen component. According to the isotropic chemical shifts of the signals in the 13C NMR spectra, the most abundant amino acids of collagen type I (glycine, alanine, proline, hydroxyproline, and glutamate) can be identified. The crucial peak confirming that the NMR spectrum of the bone implant indeed refers to collagen is the HyPro Cg peak at 71.1 ppm. There is no other aliphatic 13C NMR signal of a regular amino acid with this chemical shift indicating that collagen type I was synthesized by the osteoblasts in the implant. Furthermore, the isotropic chemical shifts of the peaks in the spectra of the implant, native rabbit bone, and isolated collagen type I are identical indicating that no alterations of the collagen structure in the b-TCP implants occurred as isotropic chemical shifts are a very sensitive marker of protein secondary structure.
Figure 4. 1H decoupled 188.5 MHz 13C CP MAS NMR spectra of (a) native rabbit bone, (b) a b-TCP implant removed from a rabbit’s femoral condyle after three months, and (c) isolated collagen type I. Spectra were recorded at 37 °C and a MAS frequency of 7 kHz. The peak assignment was taken from the literature.17,18
In summary, it was shown that hydroxylated apatite and collagen was produced in implants of inorganic ceramics that were loaded with MSCs. The 31P NMR analysis of the inorganic ECM formation in the implants allows quantitative detection of bone mineral formation. The 13C NMR analysis of collagen represents a unique fingerprint of the molecule and its dynamic properties. These results suggest that solid-state NMR is a useful analytical tool to monitor the formation of bone ECM quantitatively. The method can be used to overcome challenges faced in bone engineering including the choice of the appropriate scaffold material, possible surface modifications of these scaffolds, and the right growth conditions. Since tissue engineering of bone faces increasing demand, current procedures may benefit from an atomistic and quantitative understanding of bone synthesis in implant materials by solid-state NMR spectroscopy.
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The study was supported by the DFG (HU 720/7-1).