Pten regulates endocytic trafficking of cell adhesion and Wnt signaling molecules to pattern the retina.

The retina is exquisitely patterned, with neuronal somata positioned at regular intervals to completely sample the visual field. Here, we show that phosphatase and tensin homolog (Pten) controls starburst amacrine cell spacing by modulating vesicular trafficking of cell adhesion molecules and Wnt proteins. Single-cell transcriptomics and double-mutant analyses revealed that Pten and Down syndrome cell adhesion molecule Dscam) are co-expressed and function additively to pattern starburst amacrine cell mosaics. Mechanistically, Pten loss accelerates the endocytic trafficking of DSCAM, FAT3, and MEGF10 off the cell membrane and into endocytic vesicles in amacrine cells. Accordingly, the vesicular proteome, a molecular signature of the cell of origin, is enriched in exocytosis, vesicle-mediated transport, and receptor internalization proteins in Pten conditional knockout (PtencKO) retinas. Wnt signaling molecules are also enriched in PtencKO retinal vesicles, and the genetic or pharmacological disruption of Wnt signaling phenocopies amacrine cell patterning defects. Pten thus controls vesicular trafficking of cell adhesion and signaling molecules to establish retinal amacrine cell mosaics.

Specific heterozygous variants in MGP lead to endoplasmic reticulum stress and cause spondyloepiphyseal dysplasia.

Matrix Gla protein (MGP) is a vitamin K-dependent post-translationally modified protein, highly expressed in vascular and cartilaginous tissues. It is a potent inhibitor of extracellular matrix mineralization. Biallelic loss-of-function variants in the MGP gene cause Keutel syndrome, an autosomal recessive disorder characterized by widespread calcification of various cartilaginous tissues and skeletal and vascular anomalies. In this study, we report four individuals from two unrelated families with two heterozygous variants in MGP, both altering the cysteine 19 residue to phenylalanine or tyrosine. These individuals present with a spondyloepiphyseal skeletal dysplasia characterized by short stature with a short trunk, diffuse platyspondyly, midface retrusion, progressive epiphyseal anomalies and brachytelephalangism. We investigated the cellular and molecular effects of one of the heterozygous deleterious variants (C19F) using both cell and genetically modified mouse models. Heterozygous 'knock-in' mice expressing C19F MGP recapitulate most of the skeletal anomalies observed in the affected individuals. Our results suggest that the main underlying mechanism leading to the observed skeletal dysplasia is endoplasmic reticulum stress-induced apoptosis of the growth plate chondrocytes. Overall, our findings support that heterozygous variants in MGP altering the Cys19 residue cause autosomal dominant spondyloepiphyseal dysplasia, a condition distinct from Keutel syndrome both clinically and molecularly.

Contributions of increased osteopontin and hypophosphatemia to dentoalveolar defects in osteomalacic Hyp mice.

X-linked hypophosphatemia (XLH) is an inherited disorder caused by inactivating mutations in the PHEX gene leading to renal phosphate wasting, rickets and osteomalacia. XLH is also associated with dentoalveolar mineralization defects in tooth enamel, dentin and cementum, and in alveolar bone, which lead to an increased prevalence of dental abscesses, periodontal disease and tooth loss. Genetic mouse experiments, and deficiencies in XLH patient therapies where treatments do not fully ameliorate mineralization defects, suggest that other pathogenic mechanisms may exist in XLH. The mineralization-inhibiting, secreted extracellular matrix phosphoprotein osteopontin (OPN, gene Spp1) is a substrate for the PHEX enzyme whereby extensive and inactivating degradation of inhibitory OPN by PHEX facilitates mineralization. Conversely, excess OPN accumulation in skeletal and dental tissues - for example in XLH where inactivating mutations in the PHEX gene limit degradation of inhibitory OPN, or as occurs in Fgf23-null mice - contributes to mineralization defects. We hypothesized that Spp1/OPN ablation in Hyp mice (a mouse model for XLH) would reduce dentoalveolar mineralization defects. Immunostaining revealed increased OPN in Hyp vs. wild-type (WT) alveolar bone, particularly in osteocyte lacunocanalicular networks where Hyp mice have characteristic hypomineralized peri-osteocytic lesions (POLs). Micro-computed tomography and histology showed that ablation of Spp1 in Hyp mice (Hyp;Spp1-/-) on a normal diet did not ameliorate bulk defects in enamel, dentin, or alveolar bone. On a high-phosphate diet, both Hyp and Hyp;Spp1-/- mice showed improved mineralization of enamel, dentin, and alveolar bone. Silver staining indicated Spp1 ablation did not improve alveolar or mandibular bone osteocyte POLs in Hyp mice; however, they were normalized by a high-phosphate diet in both Hyp and Hyp;Spp1-/- mice, although inducing increased OPN. Collectively, these data indicate that despite changes in OPN content in the dentoalveolar mineralized tissues, there exist other compensatory mineralization mechanisms that arise from knockout of Spp1/OPN in the Hyp background.

A novel use of a needle-free injection system for improved nucleic acid delivery and expression in vivo.

Transfection, a nonviral method of nucleic acid delivery, often exhibits poor efficiency in vivo. The needle-based in vivo delivery of transfection reagents can be invasive. Here, we report a noninvasive protocol for in vivo gene delivery via the needle-free MED-JET H4 MULTIJET (MJH4M) device using both "home-made" glucose-based and commercial transfection reagents. The objective of this study was to compare the relative transfection efficiencies of the needle-free system to that of the needle-based delivery method. We observed a 15-fold increase in transfection efficiency using the needle-free MJH4M device when compared to the needle-based delivery method. The highest transfection efficiency was achieved using a 5% glucose solution as the delivery vehicle.

A Bioglass-Poly(lactic-co-glycolic Acid) Scaffold@Fibrin Hydrogel Construct to Support Endochondral Bone Formation.

Bone tissue engineering using stem cells to build bone directly on a scaffold matrix often fails due to lack of oxygen at the injury site. This may be avoided by following the endochondral ossification route; herein, a cartilage template is promoted first, which can survive hypoxic environments, followed by its hypertrophy and ossification. However, hypertrophy is so far only achieved using biological factors. This work introduces a Bioglass-Poly(lactic-co-glycolic acid@fibrin (Bg-PLGA@fibrin) construct where a fibrin hydrogel infiltrates and encapsulates a porous Bg-PLGA. The hypothesis is that mesenchymal stem cells (MSCs) loaded in the fibrin gel and induced into chondrogenesis degrade the gel and become hypertrophic upon reaching the stiffer, bioactive Bg-PLGA core, without external induction factors. Results show that Bg-PLGA@fibrin induces hypertrophy, as well as matrix mineralization and osteogenesis; it also promotes a change in morphology of the MSCs at the gel/scaffold interface, possibly a sign of osteoblast-like differentiation of hypertrophic chondrocytes. Thus, the Bg-PLGA@fibrin construct can sequentially support the different phases of endochondral ossification purely based on material cues. This may facilitate clinical translation by decreasing in-vitro cell culture time pre-implantation and the complexity associated with the use of external induction factors.

A 6-bromoindirubin-3'-oxime incorporated chitosan-based hydrogel scaffold for potential osteogenic differentiation: Investigation of material properties in vitro.

Effective treatments for critical size bone defects remain challenging. 6-Bromoindirubin-3'-Oxime (BIO), a glycogen synthase kinase 3ß inhibitor, is a promising alternative for treatment of these defects since it aids in promoting osteogenic differentiation. In this study, BIO is incorporated into a new formulation of the guanosine diphosphate cross-linked chitosan scaffold to promote osteogenic differentiation. BIO incorporation was confirmed with 13C NMR through a novel concentration dependent peak around 41 ppm. The rapid gelation rate was maintained along with the internal structure's stability. The 10 µM BIO dose supported the control scaffold's microstructure demonstrating a suitable porosity and a low closed pore percentage. While pore sizes of BIO incorporated scaffolds were slightly smaller, pore heterogeneity was maintained. A proof-of-concept study with C2C12 cells suggested a dose-dependent response of BIO on early stages of osteogenic differentiation within the scaffold. These results support future work to examine BIO's role on osteogenic differentiation and biomineralization of encapsulated cells in the scaffold for bone regeneration.

Prevention of Arterial Elastocalcinosis: Differential Roles of the Conserved Glutamic Acid and Serine Residues of Matrix Gla Protein.

BACKGROUND: Inactivating mutations in matrix Gla protein (MGP) lead to Keutel syndrome, a rare disease hallmarked by ectopic calcification of cartilage and vascular tissues. Although MGP acts as a strong inhibitor of arterial elastic lamina calcification (elastocalcinosis), its mode of action is unknown. Two sets of conserved residues undergoing posttranslational modifications-4 glutamic acid residues, which are γ-carboxylated by gamma-glutamyl carboxylase; and 3 serine residues, which are phosphorylated by yet unknown kinase(s)-are thought to be essential for MGP's function. METHODS: We pursued a genetic approach to study the roles of MGP's conserved residues. First, a transgenic line (SM22a-GlamutMgp) expressing a mutant form of MGP, in which the conserved glutamic acid residues were mutated to alanine, was generated. The transgene was introduced to Mgp-/- mice to generate a compound mutant, which produced the mutated MGP only in the vascular tissues. We generated a second mouse model (MgpS3mut/S3mut) to mutate MGP's conserved serine residues to alanine. The initiation and progression of vascular calcification in these models were analyzed by alizarin red staining, histology, and micro-computed tomography imaging. RESULTS: On a regular diet, the arterial walls in the Mgp-/-; SM22α-GlamutMgp mice were not calcified. However, on a high phosphorus diet, these mice showed wide-spread arterial calcification. In contrast, MgpS3mut/S3mut mice on a regular diet recapitulated arterial calcification traits of Mgp-/- mice, although with lesser severity. CONCLUSIONS: For the first time, we show here that MGP's conserved serine residues are indispensable for its antimineralization function in the arterial tissues. Although the conserved glutamic acid residues are not essential for this function on a regular diet, they are needed to prevent phosphate-induced arterial elastocalcinosis.

Keutel Syndrome, a Review of 50 Years of Literature.

Keutel syndrome (KS) is a rare autosomal recessive genetic disorder that was first identified in the beginning of the 1970s and nearly 30 years later attributed to loss-of-function mutations in the gene coding for the matrix Gla protein (MGP). Patients with KS are usually diagnosed during childhood (early onset of the disease), and the major traits include abnormal calcification of cartilaginous tissues resulting in or associated with malformations of skeletal tissues (e.g., midface hypoplasia and brachytelephalangism) and cardiovascular defects (e.g., congenital heart defect, peripheral pulmonary artery stenosis, and, in some cases, arterial calcification), and also hearing loss and mild developmental delay. While studies on Mgp -/- mouse, a faithful model of KS, show that pathologic mineral deposition (ectopic calcification) in cartilaginous and vascular tissues is the primary cause underlying many of these abnormalities, the mechanisms explaining how MGP prevents abnormal calcification remain poorly understood. This has negative implication for the development of a cure for KS. Indeed, at present, only symptomatic treatments are available to treat hypertension and respiratory complications occurring in the KS patients. In this review, we summarize the results published in the last 50 years on Keutel syndrome and present the current status of the knowledge on this rare pathology.

Elastin calcification in in vitro models and its prevention by MGP's N-terminal peptide.

Medial calcification has been associated with diabetes, chronic kidney disease, and genetic disorders like pseudoxanthoma elasticum. Recently, we showed that genetic reduction of arterial elastin content reduces the severity of medial calcification in matrix Gla protein (MGP)-deficient and Eln haploinsufficient Mgp-/-;Eln+/- mice. This study suggests that there might be a direct effect of elastin amount on medial calcification. We studied this using novel in vitro systems, which are based on elastin or elastin-like polypeptides. We first examined the mineral deposition properties of a transfected pigmented epithelial cell line that expresses elastin and other elastic lamina proteins. When grown in inorganic phosphate-supplemented medium, these cells deposited calcium phosphate minerals, which could be prevented by an N'-terminal peptide of MGP (m3pS) carrying phosphorylated serine residues. We next confirmed these findings using a cell-free elastin-like polypeptide (ELP3) scaffold, where the peptide prevented mineral maturation. Overall, this work describes a novel cell culture model for elastocalcinosis and examines the inhibition of mineral deposition by the m3pS peptide in this and a cell-free elastin-based scaffold. Our study provides strong evidence suggesting the critical functional roles of MGP's phosphorylated serine residues in the prevention of elastin calcification and proposes a possible mechanism of their action.

PHOSPHO1 is a skeletal regulator of insulin resistance and obesity.

BACKGROUND: The classical functions of the skeleton encompass locomotion, protection and mineral homeostasis. However, cell-specific gene deletions in the mouse and human genetic studies have identified the skeleton as a key endocrine regulator of metabolism. The bone-specific phosphatase, Phosphatase, Orphan 1 (PHOSPHO1), which is indispensable for bone mineralisation, has been recently implicated in the regulation of energy metabolism in humans, but its role in systemic metabolism remains unclear. Here, we probe the mechanism underlying metabolic regulation by analysing Phospho1 mutant mice. RESULTS: Phospho1-/- mice exhibited improved basal glucose homeostasis and resisted high-fat-diet-induced weight gain and diabetes. The metabolic protection in Phospho1-/- mice was manifested in the absence of altered levels of osteocalcin. Osteoblasts isolated from Phospho1-/- mice were enriched for genes associated with energy metabolism and diabetes; Phospho1 both directly and indirectly interacted with genes associated with glucose transport and insulin receptor signalling. Canonical thermogenesis via brown adipose tissue did not underlie the metabolic protection observed in adult Phospho1-/- mice. However, the decreased serum choline levels in Phospho1-/- mice were normalised by feeding a 2% choline rich diet resulting in a normalisation in insulin sensitivity and fat mass. CONCLUSION: We show that mice lacking the bone mineralisation enzyme PHOSPHO1 exhibit improved basal glucose homeostasis and resist high-fat-diet-induced weight gain and diabetes. This study identifies PHOSPHO1 as a potential bone-derived therapeutic target for the treatment of obesity and diabetes.

In vitro and in vivo investigation of osteogenic properties of self-contained phosphate-releasing injectable purine-crosslinked chitosan-hydroxyapatite constructs.

Bone fracture repair is a multifaceted, coordinated physiological process that requires new bone formation and resorption, eventually returning the fractured bone to its original state. Currently, a variety of different approaches are pursued to accelerate the repair of defective bones, which include the use of 'gold standard' autologous bone grafts. However, such grafts may not be readily available, and procedural complications may result in undesired outcomes. Considering the ease of use and tremendous customization potentials, synthetic materials may become a more suitable alternative of bone grafts. In this study, we examined the osteogenic potential of guanosine 5'-diphosphate-crosslinked chitosan scaffolds with the incorporation of hydroxyapatite, with or without pyrophosphatase activity, both in vitro and in vivo. First, scaffolds embedded with cells were characterized for cell morphology, viability, and attachment. The cell-laden scaffolds were found to significantly enhance proliferation for up to threefold, double alkaline phosphatase activity and osterix expression, and increase calcium phosphate deposits in vitro. Next, chitosan scaffolds were implanted at the fracture site in a mouse model of intramedullary rod-fixed tibial fracture. Our results showed increased callus formation at the fracture site with the scaffold carrying both hydroxyapatite and pyrophosphatase in comparison to the control scaffolds lacking both pyrophosphatase and hydroxyapatite, or pyrophosphatase alone. These results indicate that the pyrophosphatase-hydroxyapatite composite scaffold has a promising capacity to facilitate bone fracture healing.

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia.

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (Pi ) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn-/- ) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn-/- mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn-/- mice were fed a high-Pi diet, serum Pi concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-Pi diet, potentially indicating a role for elevated inhibitory pyrophosphate (PPi ) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum Pi , immunohistochemistry for OPN on Hyp mice fed a high-Pi diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum Pi may be attributable to the well-known upregulation of mineralization-inhibiting OPN by Pi , and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic Pi -regulating factors. © 2020 American Society for Bone and Mineral Research.

Mineralized tissues in hypophosphatemic rickets.

Hypophosphatemic rickets is caused by renal phosphate wasting that is most commonly due to X-linked dominant mutations in PHEX. PHEX mutations cause hypophosphatemia indirectly, through the increased expression of fibroblast growth factor 23 (FGF23) by osteocytes. FGF23 decreases renal phosphate reabsorption and thereby increases phosphate excretion. The lack of phosphate leads to a mineralization defect at the level of growth plates (rickets), bone tissue (osteomalacia), and teeth, where the defect facilitates the formation of abscesses. The bone tissue immediately adjacent to osteocytes often remains unmineralized ("periosteocytic lesions"), highlighting the osteocyte defect in this disorder. Common clinical features of XLH include deformities of the lower extremities, short stature, enthesopathies, dental abscesses, as well as skull abnormalities such as craniosynostosis and Chiari I malformation. For the past four decades, XLH has been treated by oral phosphate supplementation and calcitriol, which improves rickets and osteomalacia and the dental manifestations, but often does not resolve all aspects of the mineralization defects. A newer treatment approach using inactivating FGF23 antibodies leads to more stable control of serum inorganic phosphorus levels and seems to heal rickets more reliably. However, the long-term benefits of FGF23 antibody treatment remain to be elucidated.

Arachidonic acid exacerbates diet-induced obesity and reduces bone mineral content without impacting bone strength in growing male rats.

Long-chain polyunsaturated fatty acids modulate bone mass and adipocyte metabolism. Arachidonic acid (AA, C20:4 n-6) is elevated in obesity and postulated to stimulate bone resorption. This study aimed to determine the effect of AA on bone mass, quality, and adiposity in diet-induced obesity during growth. Male Sprague-Dawley rats (n=42, 4-week) were randomized into groups fed a control diet (CTRL, AIN-93G), high-fat diet (HFD, 35% kcal fat) or HFD + AA (1% w/w diet) for 6 weeks. Body composition, bone mineral density and microarchitecture were measured using dual-energy X-ray absorptiometry and micro-computed tomography. Red blood cell fatty acid profile was measured with gas chromatography. Group differences were evaluated using repeated measures two-way analysis of variance with Tukey-Kramer post hoc testing. Total energy intake did not differ among diet groups. At week 6, HFD + AA had significantly greater body fat % (12%), body weight (6%) and serum leptin concentrations (125%) than CTRL, whereas visceral fat (mass and %, assessed with micro-computed tomography) was increased in both HFD and HFD + AA groups. HFD + AA showed reduced whole body bone mineral content and femur mid-diaphyseal cortical bone cross-sectional area than HFD and CTRL, without impairment in bone strength. Contrarily, HFD + AA had greater femur metaphyseal trabecular vBMD (35%) and bone volume fraction (5%) compared to controls. Inclusion of AA elevated leptin concentrations in male rats. The early manifestations of diet-induced obesity on bone mass were accelerated with AA. Studies of longer duration are needed to clarify the effect of AA on peak bone mass following growth cessation.

Combination of polyetherketoneketone scaffold and human mesenchymal stem cells from temporomandibular joint synovial fluid enhances bone regeneration.

Therapies using human mesenchymal stem cells (MSCs) combined with three-dimensional (3D) printed scaffolds are a promising strategy for bone grafting. But the harvest of MSCs still remains invasive for patients. Human synovial fluid MSCs (hSF-MSCs), which can be obtained by a minimally invasive needle-aspiration procedure, have been used for cartilage repair. However, little is known of hSF-MSCs in bone regeneration. Polyetherketoneketone (PEKK) is an attractive bone scaffold due to its mechanical properties comparable to bone. In this study, 3D-printed PEKK scaffolds were fabricated using laser sintering technique. hSF-MSCs were characterized and cultured on PEKK to evaluate their cell attachment, proliferation, and osteogenic potential. Rabbit calvarial critical-sized bone defects were created to test the bone regenerative effect of PEKK with hSF-MSCs. In vitro results showed that hSF-MSCs attached, proliferated, and were osteogenic on PEKK. In vivo results indicated that PEKK seeded with hSF-MSCs regenerated twice the amount of newly formed bone when compared to PEKK seeded with osteogenically-induced hSF-MSCs or PEKK scaffolds alone. These results suggested that there was no need to induce hSF-MSCs into osteoblasts prior to their transplantations in vivo. In conclusion, the combined use of PEKK and hSF-MSCs was effective in regenerating critical-sized bone defects.

Role of SMPD3 during Bone Fracture Healing and Regulation of Its Expression.

Sphingomyelin phosphodiesterase 3 (SMPD3), a lipid-metabolizing enzyme present in bone and cartilage, has important roles in the developing skeleton. We previously showed that SMPD3 deficiency results in delayed extracellular matrix (ECM) mineralization and severe skeletal deformities in an inducible knockout mouse model, Smpd3flox/flox ; Osx-Cre mice, in which Smpd3 was ablated in Osx-expressing chondrocytes and osteoblasts during early skeletogenesis. However, as shown in the current study, ablation of Smpd3 postnatally in 3-month-old Smpd3flox/flox ; Osx-Cre mice resulted in only a mild bone mineralization defect. Interestingly, though, there was a marked increase of unmineralized osteoid in the fractured tibiae of 3-month-old Smpd3flox/flox ; Osx-Cre mice. As was the case in the embryonic bones, we also observed impaired chondrocyte apoptosis at the fracture sites of Smpd3flox/flox ; Osx-Cre mice. We further examined how Smpd3 expression is regulated in ATDC5 chondrogenic cells by two major regulators of chondrogenesis, bone morphogenetic protein 2 (BMP-2) and PTHrP. Our data show that BMP-2 positively regulates Smpd3 expression via p38 mitogen-activated protein kinase. Taken together, our findings show that SMPD3 plays a significant role in ECM mineralization and chondrocyte apoptosis during fracture healing. Furthermore, our gene expression analyses suggest that BMP-2 and PTHrP exert opposing effects on the regulation of Smpd3 expression in chondrocytes.

The antidepressant drug, sertraline, hinders bone healing and osseointegration in rats' tibiae.

AIM: Selective serotonin reuptake inhibitors (SSRIs) are one of the most common antidepressant drugs. SSRI use is associated with increased risk of bone fracture and titanium implant failure. The aim of this in vivo study was to investigate the effect of SSRIs on osseointegration and bone healing. MATERIALS AND METHODS: On a total of 24 Sprague-Dawley rats, a custom-made titanium implant was placed in the left tibia, while a unicortical defect was created in the right tibia. Rats were assigned randomly into two groups and received a daily dose of either sertraline (5 mg/kg) or saline. After two weeks, they were euthanized and bone healing and osseointegration were assessed by micro-CT and histology. RESULTS: Bone formation in bone defects was significantly lower (p < 0.05) in sertraline-treated rats (BV/TV = 20.67 ± 11.98%) compared to the controls (BV/TV = 37.87 ± 9.56%). Furthermore, the percentage of osseointegration was significantly lower (p < 0.05) in sertraline-treated rats (34.40 ± 7.17%) compared to the controls (54.37 ± 8.58%). CONCLUSION: Sertraline hinders bone healing and implant osseointegration.