Ameloblasts are specialized cells derived from the dental epithelium that produce enamel, a hierarchically structured tissue comprised of highly elongated hydroxylapatite (OHAp) crystallites. The unique function of the epithelial cells synthesizing crystallites and assembling them in a mechanically robust structure is not fully elucidated yet, partly due to limitations with in vitro experimental models. Herein, we demonstrate the ability to generate mineralizing dental epithelial organoids (DEOs) from adult dental epithelial stem cells (aDESCs) isolated from mouse incisor tissues. DEOs expressed ameloblast markers, could be maintained for more than five months (11 passages) in vitro in media containing modulators of Wnt, Egf, Bmp, Fgf and Notch signaling pathways, and were amenable to cryostorage. When transplanted underneath murine kidney capsules, organoids produced OHAp crystallites similar in composition, size, and shape to mineralized dental tissues, including some enamel-like elongated crystals. DEOs are thus a powerful in vitro model to study mineralization process by dental epithelium, which can pave the way to understanding amelogenesis and developing regenerative therapy of enamel.
Mice ; Animals ; Durapatite/metabolism* ; Dental Enamel/metabolism* ; Ameloblasts/metabolism* ; Amelogenesis ; Stem Cells ; Organoids

Skeletal mineralization is initiated in matrix vesicles (MVs), the small extracellular vesicles derived from osteoblasts and chondrocytes. Calcium and inorganic phosphate (Pi) taken up by MVs form hydroxyapatite crystals, which propagate on collagen fibrils to mineralize the extracellular matrix. Insufficient calcium or phosphate impairs skeletal mineralization. Because active vitamin D is necessary for intestinal calcium absorption, vitamin D deficiency is a significant cause of rickets/osteomalacia. Chronic hypophosphatemia also results in rickets/osteomalacia. Excessive action of fibroblast growth factor 23 (FGF23), a key regulator of Pi metabolism, leads to renal Pi wasting and impairs vitamin D activation. X-linked hypophosphatemic rickets (XLH) is the most common form of hereditary FGF23-related hypophosphatemia, and enhanced FGF receptor (FGFR) signaling in osteocytes may be involved in the pathogenesis of this disease. Increased extracellular Pi triggers signal transduction via FGFR to regulate gene expression, implying a close relationship between Pi metabolism and FGFR. An anti-FGF23 antibody, burosumab, has recently been developed as a new treatment for XLH. In addition to various forms of rickets/osteomalacia, hypophosphatasia (HPP) is characterized by impaired skeletal mineralization. HPP is caused by inactivating mutations in tissue-nonspecific alkaline phosphatase, an enzyme rich in MVs. The recent development of enzyme replacement therapy using bone-targeting recombinant alkaline phosphatase has improved the prognosis, motor function, and quality of life in patients with HPP. This links impaired skeletal mineralization with various conditions, and unraveling its pathogenesis will lead to more precise diagnoses and effective treatments.
Absorption ; Alkaline Phosphatase ; Calcium ; Chondrocytes ; Collagen ; Diagnosis ; Durapatite ; Enzyme Replacement Therapy ; Extracellular Matrix ; Extracellular Vesicles ; Familial Hypophosphatemic Rickets ; Fibroblast Growth Factors ; Gene Expression ; Humans ; Hypophosphatasia ; Hypophosphatemia ; Metabolism ; Miners ; Osteoblasts ; Osteocytes ; Prognosis ; Quality of Life ; Receptors, Fibroblast Growth Factor ; Rickets ; Signal Transduction ; Vitamin D ; Vitamin D Deficiency

BACKGROUNDSystemic administration of bisphosphonates has shown promising results in the treatment of osteonecrosis of the femoral head (ONFH). However, few studies have evaluated the efficacy of local zoledronate (ZOL) administration in the treatment of ONFH. The purpose of this study was to investigate whether local administration of bisphosphonate-soaked hydroxyapatite (HA) could improve bone healing in an experimental rabbit model of ONFH.

METHODSThis experimental study was conducted between October 2014 and June 2015. Forty-five rabbits underwent simulated ONFH surgery. Immediately following surgery, they were divided into three groups: model (untreated, n = 15), HA (treated with HA alone, n = 15), and HA + ZOL (treated with HA soaked in a low-dose ZOL solution, n = 15). Histological, immunohistochemical, and quantitative analyses were performed to evaluate bone formation and resorption 2, 4, and 8 weeks after surgery.

RESULTSGross bone matrix and hematopoietic tissue formation were observed in the HA + ZOL group 4 weeks after surgery. The immunohistochemical staining intensities for 5-bromodeoxyuridine, runt-related transcription factor 2, osteocalcin, osteopontin, and osteoprotegerin were significantly higher in the HA + ZOL group than that in the model (P < 0.001, P< 0.001, P< 0.001, P< 0.001, and P = 0.018, respectively) and HA groups (P = 0.003, P = 0.049, P< 0.001, P = 0.020, and P = 0.019, respectively), whereas receptor activator of the nuclear factor-κB ligand staining intensity was significantly lower in the HA + ZOL group than that in the model and HA groups (P = 0.029 and P = 0.015, respectively) 4 weeks after surgery. No significant differences in bone formation or bone resorption marker expression were found between the three groups 2 or 8 weeks after surgery (P > 0.05).

CONCLUSIONSLocal administration of HA soaked in a low-dose ZOL solution increased new bone formation while inhibiting bone resorption in an animal model of ONFH, which might provide new evidence for joint-preserving surgery in the treatment of ONFH.

Animals ; Diphosphonates ; administration & dosage ; therapeutic use ; Durapatite ; administration & dosage ; therapeutic use ; Female ; Femur Head Necrosis ; drug therapy ; metabolism ; Imidazoles ; administration & dosage ; therapeutic use ; Immunohistochemistry ; Male

OBJECTIVE: To explore the interaction between arginine functionalized hydroxyapatite (HAP/Arg) nanoparticles and endothelial cells, and to investigate mechanisms for endocytosis kinetics and endocytosis.
 METHODS: Human umbilical vein endothelial cells (HUVECs) were selected as the research model.Cellular uptake of HAP/Arg nanoparticles were observed by laser scanning confocal microscopy.Average fluorescence intensity of cells after ingestion with different concentrations of HAP/Arg nanoparticles were determined by flow cytometer and atomic force microscopy.
 RESULTS: The HAP/Arg nanoparticles with doped terbium existed in cytoplasm, and most of them distributed around the nucleus area after cellular uptake by HUVECs. Cellular uptake process of HAP/Arg nanoparticles in HUVECs was in a time and concentration dependent manner. 4 h and 50 mg/L was the best condition for uptake. HAP/Arg nanoparticles were easier to be up-taken into the cells than HAP nanoparticles without arginine functionalized.
 CONCLUSION HAP/Arg nanoparticles are internalized by HUVECs cells through an active transport and energy-dependent endocytosis process, and it is up-taken by cells mainly through caveolin-mediated endocytosis, but the clathrin-dependent endocytic pathway is also involved..
Arginine ; pharmacology ; Biological Transport, Active ; physiology ; Caveolins ; physiology ; Cells, Cultured ; Clathrin ; physiology ; Durapatite ; pharmacokinetics ; Endocytosis ; physiology ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Nanoparticles ; metabolism

The present research was to study the biocompatibility of a composite of hydroxyapatite (HA), carbon fiber (CF) and polyetheretherket-one (PEEK) by co-culturing with the osteoblasts in vitro. Cell relative growth (RGR) was used as a quantitative assessment for cytotoxicity of the biomaterials by CCK-8. The proliferation index of the co-cultured cells and ALP activity was measured to study the effect of PEEK-HA-CF composites. Morphological properties of the osteoblast cells in vitro were observed by scanning electro-microscopy (SEM). The PEEK-HA-CF materials have no cytotoxicity to osteoblasts. The proliferation index of PEEK-HA-CF was higher than that of Ti alloy group, but these was no significant difference compared to that of control group. The ALP activity was the highest on PEEK-HA-CF composites surface after 7 days. The osteoblast cells co-cultured with the PEEK-HA-CF composite were adhered well to the biomaterial as observed under the SEM. The results suggested that the PEEK-HA-CF composites had good biocompatibility in vitro and might be a novel orthopedic implanted material.
Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; Carbon ; chemistry ; Cell Proliferation ; drug effects ; Cells, Cultured ; Durapatite ; chemistry ; Ketones ; chemistry ; Orthopedics ; Osteoblasts ; chemistry ; Polyethylene Glycols ; chemistry ; Prostheses and Implants ; Rats

Animals ; Bone and Bones ; cytology ; metabolism ; pathology ; Collagen ; metabolism ; Durapatite ; metabolism ; Female ; Osteoporosis ; metabolism ; pathology ; Ovariectomy ; adverse effects ; Rats ; Rats, Sprague-Dawley

Compound membranes of chitosan/hydroxyapatite were prepared by blending. The physical performance showed that the air-water contact angles decreased from chitosan's 103 degrees to chitosan/hydroxyapatite's 57 and the water adsorption rate increased slightly. When immersed into culture medium, the materials adsorbed Ca2+, and low crystalline hydroxyapatite deposited on the surface of the membranes. Chitosan/hydroxyapatite compound membranes could enhance the attachment and proliferation of mescenchymal stem cells (MSCs). After 12 days' induction on the materials, the alkaline phosphatase (ALP) activity value of MSCs on the compound membrane was 10.1, being much higher than 1.6 on chitosan membrane (P<0.01). All these results indicate that chitosan does not have very good affinity for MSCs, but the biocompatibility of chitosan can be apparently enhanced after mixing with hydroxyapatite. The compound membrane stimulates MSCs to differentiate into osteoblasts and it may be a good potential material for bone substitution.
Alkaline Phosphatase ; metabolism ; Animals ; Bone Substitutes ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Chitosan ; chemical synthesis ; pharmacology ; Durapatite ; chemical synthesis ; pharmacology ; Membranes, Artificial ; Mesenchymal Stromal Cells ; cytology ; Rats

OBJECTIVETo study location of MT1-MMP and effect of its change in expression on rabbit VX2 tumor tissues after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol.

METHODSSixty rabbits implanted with tumor tissue of cell line VX2 were divided into three groups (control group, lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group). The transarterial embolization was performed super-selectively via gastro- duodenal artery of rabbits, each rabbit in control group was inserted with 1 ml normal saline,that in lipiodol group was inserted with 0.3 lipiodol ml/kg, also 0.3 ml hydroxyapatite nanoparticles loaded with lipiodol per kg for that in the last group. Results of embolization were detected by using CT scanning 3 days after operation. After two weeks, all tumors were took out as specimens to investigate location of MT1-MMP in VX2 tumor tissues,and also to determine the change of its expression in tumor tissues after embolization with different medicines, with three-step immunohistochemical technique (S-P). MT1-MMP mRNA was measured by RT-PCR to determine whether there were differences in three groups. Western blot technique was performed to determine difference of MT1-MMP protein expression of in three groups.

RESULTSImmunohistochemical results exposed that MT1-MMP was expressed on membrane of tumor cells and in extracellular matrix of tumor cells. Comparison of MT1-MMP expression in control group with that in other two groups, showed a significant lower level in control group (P < 0.05). There was no difference in MT1-MMP expression between lipiodol group, hydroxyapatite nanoparticles loaded with lipiodol group (P > 0.05). Western blot supported this conclusion. RT-PCR detecting MT1-MMP mRNA was found no differences among three groups (P > 0.05).

CONCLUSIONSMT1-MMP was mainly expressed on membrane of tumor cells and in extracellular matrix of tumor cells. There was an increasing tendency on expression of MT1-MMP in tumor tissues and extracellular matrix after transarterial embolization with hydroxyapatite nanoparticles loaded with lipiodol,it might be one of important mechanisms provoking high recurrence rate for hepatocellular carcinoma after treatment embolization.

Animals ; Durapatite ; Embolization, Therapeutic ; Iodized Oil ; Liver Neoplasms, Experimental ; enzymology ; pathology ; therapy ; Matrix Metalloproteinase 14 ; genetics ; metabolism ; Nanoparticles ; RNA, Messenger ; genetics ; Rabbits

with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 3T3-E1 the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTZ assay. After being Abstract Nanohydroxyapatite/chitosan composite scaffolds were fabricated and the proliferation and differentiation of preosteoblast MC 3T3-E1 on them were examined for the assessment of their biocompatibility. Nanohydroxyapatite was combined with chitosan in situ using a chemical method and a porous structure obtained was then lyophilized. Preosteoblast MC 333-E1 cells were inoculated into the porous composite scaffolds and chitosan scaffolds, respectively. The morphology of cells cultured on the scaffolds was examined after staining it with Wright's stain. Their proliferation was assessed using MTT assay. After being cultured in conditioned medium for 30 days, the cells' alkaline phosphatase activities on the scaffolds were studied in situ to compare their differentiation levelabout. Moreover, the alkaline phosphatase activities were assessed with a kit. The expression level of characteristic osteogenic gene was evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). The results indicated that MC 3T3-E1 cells grown on the composite scaffolds showed a higher proliferation rate and spread better than that on chitosan scaffolds. The alkaline phosphatase stain results showed that the alkaline phosphatase activity of cells on composite scaffolds was significantly higher than that on the chitosan scaffolds. In addition, the quantitative examination of alkaline phosphatase activity indicated that the cells cultured on the composite scaffolds expressed an activity level about 8 times higher than that on chitosan scaffolds. Simultaneously, the osteogenic gene osteopontin (OPN) of cells cultured on composite scaffolds showed a higher expression level than that on chitosan scaffolds. Another osteogenic gene osteocalcin (OC) was expressed in cells cultured on composite scaffolds, whereas it was not detected in cells on chitosan scaffolds. The addition of nanohydroxyapatite in the scaffolds improved not only the proliferation but also the differentiation of preosteoblast cultured on them. The composite scaffolds showed good biocompatibility and bioactivity. These scaffolds would be promising in bone tissue engineering.
Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; chemistry ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Chitosan ; chemistry ; Durapatite ; chemistry ; Gene Expression ; Mice ; Nanostructures ; Osteoblasts ; cytology ; metabolism ; Osteocalcin ; genetics ; Osteopontin ; genetics ; Porosity ; Reverse Transcriptase Polymerase Chain Reaction ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

OBJECTIVE: To transfect the recombinant plasmid enhancement type green fluorescent protein C2- neurotrophic factor-3 (pEGFPC2-NT3) into the spinal ganglion cells(SGCs) of guinea pigs' cochlea injured by the excitotoxicity of hydroxyapatite particle (HAT), to inject the recombinant plasmid pEGFPC2-NT3 into the guinea pigs' cochlea, and to observe the expression of pEGFPC2-NT3 and the protective effect of pEGFPC2-NT3 on SGCs of the cochlea in guinea pigs. METHODS: The recombinant plasmid pEGFPC2-NT3 with gene-green fluorescent protein was established. Kanic acid (KA) was injected into guinea pigs'cochleae and the excitotoxicity model was established. After a week the recombinant plasmid was transferred into SGCs of guinea pigs'cochlea treated with HAT. The following week the expression of NT-3 was examined by the immunohistochemical method, and the morphology of SGNs was observed under the electronic microscope after 4 weeks, in the mean time the changes of auditory brain-stem response (ABR) were examined. RESULTS: The excitotoxicity models were established successfully. NT-3 expression in the intracytoplasm of SGNs was observed by the immunohistochemical method 1 week after the injection, the morphologic damages of SGNs lessened under the electronic microscope after 4 weeks. ABR was partly restored, compared with ABR after the injury of the excitotoxicity. CONCLUSION On the 7th day, NT3 gene transferred by HAT through the scala tympani can lessen the excitotoxicity of SGCs after KA was injected into the guinea pigs cochlea.
Animals ; Cochlea ; drug effects ; Durapatite ; administration & dosage ; pharmacology ; Evoked Potentials, Auditory, Brain Stem ; Ganglia, Spinal ; drug effects ; metabolism ; Genetic Therapy ; Guinea Pigs ; Kainic Acid ; adverse effects ; Nanocomposites ; Neurotoxins ; adverse effects ; Neurotrophin 3 ; genetics ; Plasmids