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

OBJECTIVETo evaluate the biological effects of nano-hydroxyapatite/polyamide 66(nHA-PA66) on the growth and activity of osteoblast.

METHODSMTT assay was used to determine the growth of osteoblast, enzymatic measure was used to determine the activity of ALP and quantitative RT-PCR (QRT-PCR) to evaluate the changes of osteoclacin mRNA expression in osteoblasts treated by DMEM eluate of nHA-PA66.

RESULTSOsteoblasts of different test groups demonstrated relative proliferation rate ranging from 98% - 106% without dose-dependent effect. The ALP activity and osteocalcin mRNA expression were similar in test and control groups (P > 0.05).

CONCLUSIONnHA-PA66 has no negative effects on the osteoblast and its osteoblast-compatibility is proved.

Durapatite ; pharmacology ; Nylons ; pharmacology ; Osteoblasts ; drug effects ; Osteocalcin ; metabolism ; RNA, Messenger ; metabolism

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

To modify the surface property of poly lactide-co-glycolide (PLGA) by biomimetic mineralization to construct a new kind of artificial bone. PLGA films and 3-diamensional (3-D) porous scaffolds hydrolyzed in alkaline solution were minerilized in SBF for 14 days. The morphology and composition of the mineral grown on PLGA were analyzed with SEM, FTIR and XRD. The porosity of the scaffolds was detected by using the liquid displacement method. The compressive strength of the scaffolds was detected by using a Shimadzu universal mechanic tester. An obvious mineral coating was detected on the surface of films and scaffolds. The main component of the mineral was carbonated hydroxyapatite (HA) similar to the major mineral component of bone tissues. The porosity of the un-mineralized and mineralized porous scaffolds was (84.86 +/- 8.52) % and (79.70 +/- 7.70) % respectively. The compressive strength was 0.784 +/- 0.156 N/mm2 in un-mineralized 3-D porous PLGA and 0.858 +/- 0.145 N/mm2 in mineralized 3-D porous PLGA. There were no significant differences between the mineralized and un-mineralized scaffolds (P > 0.05) in porosity and biomechanics. Biomimetic mineralization is a suitable method to construct artificial bone.
Biocompatible Materials ; Bone Substitutes ; Calcification, Physiologic ; Durapatite/metabolism ; Lactic Acid/*chemistry ; Polyglycolic Acid/*chemistry ; Polymers/*chemistry ; Porosity ; Tissue Engineering

OBJECTIVETo investigate the effect of nanometer hydroxyapatite on the proliferation and the osteogenetic differentiation of periodontal ligament cells (PDLC).

METHODSNano-hydroxyapatite powders were fabricated with sol-gel method. The fourth passage periodontal ligament cells were cultured with nanometer hydroxyapatite powder (nano-HA), dense hydroxyapatite powder (dense-HA) and only medium as control respectively. On the 5th, 8th day of culture, the osteogenetic differentiation of human periodontal ligament cells was evaluated though alkaline phosphatase (ALP) activity, ALP immunohistochemical stain and ALP positive flow cytometry.

RESULTSThere were significant differences among nano-HA group, dense-HA group and control group on the 5th and 8th day of culture. A majority of nano-HA group and dense-HA group cells sample showed positive ALP stain. But the ALP positive stain of nano-HA group cells sample was denser than that of dense-HA group. In FCM, the distribution of ALP positive cells cultured with nanoparticles were significantly more than that of other groups.

CONCLUSIONSThe nano-HA, as a calcium phosphate biomaterial, has ability to promote the activity of osteogenetic differentiation for periodontal ligament cells compared with dense-HA.

Alkaline Phosphatase ; metabolism ; Cell Differentiation ; drug effects ; Cells, Cultured ; Durapatite ; administration & dosage ; pharmacology ; Humans ; Periodontal Ligament ; cytology ; enzymology

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

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

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

In order to study the cytocompatibility of nanophase hydroxyapatite ceramic in vitro, we prepared hydroxyapatite by use of the wet chemistry techniques. The grain size of hydroxyapatite of interest to the present study was determined by scanning electron microscopy and atomic force microscopy with image analysis software. Primary culture of osteoblast from rat calvaria was established. Protein content, synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase hydroxyapatite ceramics and on conventional hydroxyapatite ceramics for 7, 14, 21 and 28 days were examined. The results showed that the average surface grain size of the nanophase and that of the conventional HA compact formulations was 55 (nanophase) and 780 (conventional) nm, respectively. More importantly, compared to the synthesis of alkaline phosphatase and deposition of calcium-containing mineral by osteoblasts cultured on nanophase was significantly greater than that on conventional ceramics after 21 and 28 days. The cytocompatibility was significantly greater on nanophase HA than on conventional formulations of the same ceramic.
Alkaline Phosphatase ; metabolism ; Animals ; Biocompatible Materials ; chemistry ; Cell Adhesion ; Cells, Cultured ; Ceramics ; chemistry ; Durapatite ; chemistry ; Microscopy, Atomic Force ; Microscopy, Electron, Scanning ; Nanostructures ; chemistry ; Osteoblasts ; cytology ; Rats

OBJECTIVETo study the incorporation rate and release behavior of bovine serum albumin (BSA) incorporated into the calcium phosphate coating by biomimetic deposition, as well as the physical and chemical properties of the hybrid coating, and to provide experimental basis for the fabrication of growth factor/biomimetic calcium phosphate coating and exploration for the loading/release behavior of growth factors.

METHODSPure titanium specimens were immersed into saturated calcium phosphate solutions(SCP) containing no BSA (controlled group) and 3 different concentrations of BSA (experimental groups) : 1, 10 and 100 mg/L. Biomimetic calcium phosphate coating was formed on titanium surface and BSA was incorporated into the coating through co-deposition. The topography of the specimen was observed using scanning electron microscopy (SEM). Chemical structure and phase composition of coatings were detected by Fourier infrared spectroscopy (FTIR) analysis and X-ray diffraction (XRD) respectively. BSA incorporation rate and release profile were determined by bicinchoninic acid protein assay kit.

RESULTSThe biomimetic calcium phosphate coating was mainly composed of hydroxyapatite and octacalcium phosphate. BSA was successfully incorporated into the calcium phosphate coatings in all the 3 experimental groups. With the increase of BSA concentration, plate-like units of the coatings were turned into small grid structure. BSA incorporation rates of the three experimental groups were (72.4 ± 2.4)%, (62.3 ± 0.9)% and (42.2 ± 1.7)% respectively. The in vitro release test showed that all three BSA release profiles could be divided into two significant different stages: early burst release stage and later sustained release stage. The amount of BSA release of the 3 experimental groups in 24 h and 30 d were (1.57 ± 0.09), (8.82 ± 0.93), (140.24 ± 3.12) µg, and (2.39 ± 0.29), (14.39 ± 0.70), (151.06 ± 2.00) µg respectively.

CONCLUSIONSBiomimetic calcium phosphate coating can be used as an effective carrier for protein. BSA concentration has an impact on the incorporation rate and release speed of BSA from the calcium phosphate coating. Favorable BSA incorporation rate and release behavior can be obtained at BSA concentration of 10 mg/L.

Biomimetic Materials ; Calcium Phosphates ; chemistry ; Durapatite ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Serum Albumin, Bovine ; metabolism ; Spectrophotometry, Infrared ; Surface Properties ; Titanium ; X-Ray Diffraction