The outstanding mechanical and chemical properties of dental enamel emerge from its complex hierarchical architecture. An accurate, detailed multiscale model of the structure and composition of enamel is important for understanding lesion formation in tooth decay (dental caries), enamel development (amelogenesis) and associated pathologies (e.g., amelogenesis imperfecta or molar hypomineralization), and minimally invasive dentistry. Although features at length scales smaller than 100 nm (individual crystallites) and greater than 50 µm (multiple rods) are well understood, competing field of view and sampling considerations have hindered exploration of mesoscale features, i.e., at the level of single enamel rods and the interrod enamel (1 to 10 µm). Here, we combine synchrotron X-ray diffraction at submicrometer resolution, analysis of crystallite orientation distribution, and unsupervised machine learning to show that crystallographic parameters differ between rod head and rod tail/interrod enamel. This variation strongly suggests that crystallites in different microarchitectural domains also differ in their composition. Thus, we use a dilute linear model to predict the concentrations of minority ions in hydroxylapatite (Mg ²⁺ and CO ₃ ²⁻ /Na ⁺ ) that plausibly explain the observed lattice parameter variations. While differences within samples are highly significant and of similar magnitude, absolute values and the sign of the effect for some crystallographic parameters show interindividual variation that warrants further investigation. By revealing additional complexity at the rod/interrod level of human enamel and leaving open the possibility of modulation across larger length scales, these results inform future investigations into mechanisms governing amelogenesis and introduce another feature to consider when modeling the mechanical and chemical performance of enamel.

We previously developed a recombinant growth factor-free, 3D-printed material comprised of hydroxyapatite (HA) and demineralized bone matrix (DBM) for bone regeneration. This material has demonstrated the capacity to promote re-mineralization of the DBM particles within the scaffold struts and shows potential to promote successful spine fusion. Here, we investigate the role of geometry and architecture in osteointegration, vascularization, and facilitation of spine fusion in a pre-clinical model. Inks containing HA and DBM particles in a poly(lactide-co-glycolide) elastomer were 3D-printed into scaffolds with varying relative strut angles (90° vs. 45° advancing angle), macropore size (0 vs. 500 vs. 1000 μm), and strut alignment (aligned vs. offset). The following configurations were compared with scaffolds containing no macropores: 90°/500μm/aligned, 45°/500μm/aligned, 90°/1,000μm/aligned, 45°/1,000μm/aligned, 90°/1,000μm/offset, 45°/1,000μm/offset. Eighty-four female Sprague-Dawley rats underwent spine fusion with bilateral placement of the various scaffold configurations (n=12/configuration). Osteointegration and vascularization were assessed using microComputed Tomography and histology, and spine fusion was assessed via blinded manual palpation. The 45°/1,000μm scaffolds with aligned struts achieved the highest average fusion score (1.61/2) as well as the highest osteointegration score. Both the 45°/1,000μm/aligned and 90°/1,000μm/aligned scaffolds elicited fusion rates of 100%, which was significantly greater than the 45°/500μm/aligned iteration (p<0.05). All porous scaffolds were fully vascularized, with blood vessels present in every macropore. Vessels were also observed extending from the native transverse process bone, through the protrusions of new bone, and into the macropores of the scaffolds. When viewed independently, scaffolds printed with relative strut angles of 45° and 90° each allowed for osteointegration sufficient to stabilize the spine at L4-L5. Within those parameters, a pore size of 500 μm or greater was generally sufficient to achieve unilateral fusion. However, our results suggest that scaffolds printed with the larger pore size and with aligned struts at an advancing angle of 45° may represent the optimal configuration to maximize osteointegration and fusion capacity. Overall, this work suggests that the HA/DBM composite scaffolds provide a conducive environment for bone regeneration as well as vascular infiltration. This technology therefore represents a novel, growth-factor-free biomaterial with significant potential as a bone graft substitute for use in spinal surgery.