Probes for SPP1

The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank−/−) mice, featuring reduced PPi, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank−/− mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240 days postnatal (dpn) indicated normal histological structures in Spp1−/− comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90 dpn revealed significantly increased volumes and tissue mineral densities of Spp1−/− mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1−/− mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1−/− vs. WT mice at 26 dpn. We genetically deleted Spp1 on the Ank−/− mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank−/− mice. Ank−/−; Spp1−/−double deficient mice did not exhibit greater hypercementosis than that in Ank−/− mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.

Previous studies revealed that cementum formation is tightly regulated by inorganic pyrophosphate (PPi), a mineralization inhibitor. Local PPiconcentrations are determined by regulators, including ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which increases PPiconcentrations by adenosine triphosphate hydrolysis. Orthodontic forces stimulate alveolar bone remodelling, leading to orthodontic tooth movement (OTM). To better understand how disturbed mineral metabolism and the resulting altered periodontal structures affect OTM, we employed Enpp1 mutant mice that feature reduced PPi and increased cervical cementum in a model of OTM induced by a stretched closed-coil spring ligated between the maxillary left first molar and maxillary incisors. We analyzed tooth movement, osteoclast/odontoclast response, and tooth root resorption by micro-computed tomography, histology, histomorphometry, and immunohistochemistry. Preoperatively, we noted an altered periodontium in Enpp1 mutant mice, with significantly increased periodontal ligament (PDL) volume and thickness, as well as increased PDL-bone/tooth root surface area, compared to wild-type (WT) controls. After 11 d of orthodontic treatment, Enpp1 mutant mice displayed 38% reduced tooth movement versus WT mice. Molar roots in Enpp1 mutant mice exhibited less change in PDL width in compression and tension zones compared to WT mice. Root resorption was noted in both groups with no difference in average depths, but resorption lacunae in Enpp1 mutant mice were almost entirely limited to cementum, with 150% increased cementum resorption and 92% decreased dentin resorption. Osteoclast/odontoclast cells were reduced by 64% in Enpp1 mutant mice, with a predominance of tartrate-resistant acid phosphatase (TRAP)-positive cells on root surfaces, compared to WT mice. Increased numbers of TRAP-positive cells on root surfaces were associated with robust immunolocalization of osteopontin (OPN) and receptor-activator of NF-κB ligand (RANKL). Collectively, reduced response to orthodontic forces, decreased tooth movement, and altered osteoclast/odontoclast distribution suggests Enpp1 loss of function has direct effects on clastic function/recruitment and/or indirect effects on periodontal remodeling via altered periodontal structure or tissue mineralization.