Tissue nonspecific alkaline phosphatase (TNAP/Alpl) is associated with cell stemness; however, the function of TNAP in mesenchymal progenitor cells remains largely unknown. In this study, we aimed to establish an essential role for TNAP in bone and muscle progenitor cells. We investigated the impact of TNAP deficiency on bone formation, mineralization, and differentiation of bone marrow stromal cells. We also pursued studies of proliferation, mitochondrial function and ATP levels in TNAP deficient bone and muscle progenitor cells. We find that TNAP deficiency decreases trabecular bone volume fraction and trabeculation in addition to decreased mineralization. We also find that Alpl^−/− mice (global TNAP knockout mice) exhibit muscle and motor coordination deficiencies similar to those found in individuals with hypophosphatasia (TNAP deficiency). Subsequent studies demonstrate diminished proliferation, with mitochondrial hyperfunction and increased ATP levels in TNAP deficient bone and muscle progenitor cells, plus intracellular expression of TNAP in TNAP+ cranial osteoprogenitors, bone marrow stromal cells, and skeletal muscle progenitor cells. Together, our results indicate that TNAP functions inside bone and muscle progenitor cells to influence mitochondrial respiration and ATP production. Future studies are required to establish mechanisms by which TNAP influences mitochondrial function and determine if modulation of TNAP can alter mitochondrial respiration in vivo.

Bone growth plate abnormalities and skull shape defects are seen in hypophosphatasia, a heritable disorder in humans that occurs due to the deficiency of tissue nonspecific alkaline phosphatase (TNAP, Alpl) enzyme activity. The abnormal development of the cranial base growth plates (synchondroses) and abnormal skull shapes have also been demonstrated in global Alpl^−/− mice. To distinguish local vs. systemic effects of TNAP on skull development, we utilized P0-Cre to knockout Alpl only in cranial neural crest-derived tissues using Alpl flox mice. Here, we show that Alpl deficiency using P0-Cre in cranial neural crest leads to skull shape defects and the deficient growth of the intersphenoid synchondrosis (ISS). ISS chondrocyte abnormalities included increased proliferation in resting and proliferative zones with decreased apoptosis in hypertrophic zones. ColX expression was increased, which is indicative of premature differentiation in the absence of Alpl. Sox9 expression was increased in both the resting and prehypertrophic zones of mutant mice. The expression of Parathyroid hormone related protein (PTHrP) and Indian hedgehog homolog (IHH) were also increased. Finally, cranial base organ culture revealed that inorganic phosphate (P_i) and pyrophosphate (PP_i) have specific effects on cell signaling and phenotype changes in the ISS. Together, these results demonstrate that the TNAP expression downstream of Alpl in growth plate chondrocytes is essential for normal development, and that the mechanism likely involves Sox9, PTHrP, IHH and PP_i.

Severity of craniosynostosis in humans varies widely even in patients with identical genetic mutations. In this study we compared RNA sequencing data from cranial tissues of a severe form of Crouzon craniosynostosis syndrome (C57BL/6 FGFR2 ^C342Y/+ mice) with those of a less severe form of Crouzon craniosynostosis (BALB/c FGFR2 ^C342Y/+ mice) to identify genetic modifiers that influence craniosynostosis phenotype severity. Comparison of the mice revealed neonatal onset of coronal suture fusion in the form of suture obliteration in C57BL/6 mice (88% incidence, p<.001 between genotypes). Coronal suture fusion in the form of point fusions across the suture occurred at approximately 4 weeks after birth, with less severe skull shape abnormalities, in BALB/c mice. Substantially fewer genes were differentially expressed in BALB/c FGFR2 ^+/+ vs. FGFR2C ^342Y/+ mice (87 out of 15,893 expressed genes) than C57BL/6 FGFR2C ^+/+ vs. FGFR2C ^342Y/+ mice (2,043 out of 19,097 expressed genes). Further investigation revealed differential expression of coronal suture fusion associated genes, eph/ephrin boundary genes, cell proliferation genes, osteoblast differentiation genes and epigenetic regulators, among others. The most striking pattern in the data was the minimal change in gene expression seen for most genes in BALB/c FGFR2 ^+/+ vs. FGFR2C ^342Y/+ mice. Analysis of protein processing and lysosomal components support the hypothesis that the craniosynostosis phenotype is less severe in BALB/c mice because the mutant FGFR2C ^342Y protein is not expressed to the same extent as that seen in C57BL/6 mice. Together, these results suggest that a strategy aimed at increasing degradation of the mutant receptor or downstream signaling inhibition could lead to diminished phenotype severity.

Craniosynostosis is the premature fusion of skull bones due to loss of stem/progenitor cells located in non-mineralized tissue between growing cranial bones of infants. We generated scaffolds from a biodegradable biomaterial with small interconnected pores (125–250 μm diameter), previously shown to maintain stemness of a mesenchymal cell population, to further develop a method for the creation of an artificial cranial bone stem cell niche. Polymer scaffolds of consistent pore size were fabricated using a molecular-sieved sugar sphere casting technique with poly-l-lactic acid. A rectangular surgical defect within the parietal bone of juvenile mice was created. The three groups included sham animals with surgery but no scaffold, experimental animals with surgery plus an implanted cell-free scaffold, and experimental animals with surgery plus an implanted bone mesenchymal cell-seeded scaffold. Healing at the surgical site was evaluated at 4 and 12 weeks after surgery by micro-CT and histology. Surgical site bone volume fraction and bone mineral density were significantly greater at twelve than four weeks in the sham group but not in either of the scaffold groups. At twelve weeks, the surgical site bone volume fraction and bone mineral density were significantly lower in the cell-seeded scaffold as compared to the sham animal group. At twelve weeks, the anterior and middle cranial vault widths were significantly greater in the cell-seeded scaffold as compared to the sham animal group on the surgery side of the skulls. Less mineralization was evident within the cell-seeded than the cell-free scaffolds by histology. Based on these findings, scaffolds of sufficiently small pore size seeded with autologous bone mesenchymal stem cells could function as an artificial cranial stem cell niche to inhibit surgical-site mineralization and promote cranial growth.