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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.

The ongoing and unrestrained application of nitrogen fertilizer to agricultural lands has been directly linked to climate change and reductions in biodiversity. The agricultural sector needs a technological upgrade to adopt sustainable methods for maintaining high yield. We report synthesis of zinc and magnesium doped and undoped hydroxyapatite nanoparticles, and their urea nanohybrids, to sustainably deliver nitrogen to wheat. The urea nanohybrids loaded with up to 42% nitrogen were used as a new source of nitrogen and compared with a conventional urea-based fertilizer for efficient and sufficient nitrogen delivery to pot-grown wheat. Doping with zinc and magnesium manipulated the hydroxyapatite crystallinity for smaller size and higher nitrogen loading capacity. Interestingly, 50% and 25% doses of urea nanohybrids significantly boosted the wheat growth and yield compared with 100% doses of urea fertilizer. In addition, the nutritional elements uptake and grain protein and phospholipid levels were significantly enhanced in wheat treated with nanohybrids. These results demonstrate the potential of the multi-nutrient complexes, the zinc and magnesium doped and undoped hydroxyapatite-urea nanoparticles, as nitrogen delivery agents that reduce nitrogen inputs by at least 50% while maintaining wheat plant growth and nitrogen uptake to the same level as full-dose urea treatments.

To supply adequate food, the ongoing and unrestrained administration of nitrogen fertilizer to agricultural fields is polluting the climate and living organisms. On the other hand, the agriculture sector urgently needs a technological upgrade to effectively confront hunger and poverty. Here, we report a rapid synthesis of zinc and magnesium-doped hydroxyapatite-urea nanohybrids for slow release and delivery of nitrogen to wheat and rice crops. Nanohybrids slowly release nitrogen for up to six weeks compared to the burst release of nitrogen from urea, and their use substantially reduces, by at least 3.8 times, ammonia emissions into the environment compared with that of urea fertilizer. A half‑nitrogen dose applied as multi-nutrient complexed nanohybrids maintained crop growth, yield, and nutritional compositions in wheat and subsequent rice crops. Nanohybrids enhanced the wheat crop yield and nitrogen uptake by 22.13% and 58.30%, respectively. The synthesized nitrogen nanohybrids remained in the soil for two continuous crop cycles, reduced ammonia volatilization, and achieved nitrogen delivery to the crops. Additionally, soil dehydrogenase activity (534.55% above control) and urease activities (81.82% above control) suggest that nanohybrids exhibited no adverse impact on soil microorganisms. Our comprehensive study demonstrates the advantages of 'doping' as a method for tailoring hydroxyapatite nanoparticles properties for extended agricultural and environmental applications. The use of nanohybrids substantially reduced greenhouse gas emissions and enabled the reduction, by half, of nitrogen inputs into the agricultural fields. This study, therefore, reports a novel nano-enabled platform of engineered hydroxyapatite-urea nanohybrids as a nitrogen fertilizer for efficient nitrogen delivery that results in improved crop growth while minimizing environmental pollution.

Human enamel is an incredibly resilient biological material, withstanding repeated daily stresses for decades. The mechanisms behind this resilience remain an open question, with recent studies demonstrating a crack-deflection mechanism contributing to enamel toughness and other studies detailing the roles of the organic matrix and remineralization. Here, we focus on the mineral and hypothesize that self-healing of cracks in enamel nanocrystals may be an additional mechanism acting to prevent catastrophic failure. To test this hypothesis, we used a molecular dynamics (MD) approach to compare the fracture behavior of hydroxyapatite (HAP) and calcite, the main minerals in human enamel and sea urchin teeth, respectively. We find that cracks heal under pressures typical of mastication by fusion of crystals in HAP but not in calcite, which is consistent with the resilience of HAP enamel that calcite teeth lack. Scanning transmission electron microscopy (STEM) images of structurally intact ("sound") human enamel show dashed-line nanocracks that resemble and therefore might be the cracks healed by fusion of crystals produced in silico. The fast, self-healing mechanism shown here is common in soft materials and ceramics but has not been observed in single crystalline materials at room temperature. The crack self-healing in sound enamel nanocrystals, therefore, is unique in the human body and unique in materials science, with potential applications in designing bioinspired materials.

To explore the possible emergence and lived consequences of social inequality in the Atacama, we analyzed a large set (n = 288) of incredibly well preserved and contextualized human skeletons from the broad Middle Period (AD 500-1000) of the San Pedro de Atacama (Chile) oases. In this work, we explore model-based paleodietary reconstruction of the results of stable isotope analysis of human bone collagen and hydroxyapatite. The results of this modeling are used to explore local phenomena, the nature of the Middle Period, and the interaction between local situations and the larger world in which the oases were enmeshed by identifying the temporal, spatial, and biocultural correlates and dimensions of dietary difference. Our analyses revealed that: 1) over the 600-year period represented by our sample, there were significant changes in consumption patterns that may evince broad diachronic changes in the structure of Atacameño society, and 2) at/near 600 calAD, there was a possible episode of social discontinuity that manifested in significant changes in consumption practices. Additionally, while there were some differences in the level of internal dietary variability among the ayllus, once time was fully considered, none of the ayllus stood out for having a more (or less) clearly internally differentiated cuisine. Finally, sex does not appear to have been a particularly salient driver of observed dietary differences here. While we do not see any de facto evidence for complete dietary differentiation (as there is always overlap in consumption among individuals, ayllus, and time periods, and as isotopic analysis is not capable of pinpointing different foods items or preparations), there are broad aspects of dietary composition changing over time that are potentially linked to status, and foreignness. Ultimately, these stand as the clearest example of what has been termed "gastro-politics," potentially tied to the emergence of social inequality in the San Pedro oases.

ObjectivesTo characterize the paleodiet of individuals from Formative Period (1500 B.C.-A.D. 400) Atacama Desert sites of Ancachi and Quillagua as a means of understanding the dietary and cultural impacts of regional systems of exchange.Materials and methodsThirty-one bone samples recovered from the cemetery of Ancachi (02QU175) and in/around the nearby town of Quillagua were the subject of carbon and nitrogen stable isotope analysis of bone collagen and hydroxyapatite and multisource mixture modeling (FRUITS, food reconstruction using isotopic transferred signals) of paleodiet. These individuals were compared with nearly 200 other Formative Period individuals from throughout the region to identify differences in dietary behaviors.Results80.6% (25/31) of the samples yielded sufficient well-preserved collagen and were included in the multisource mixture model. The FRUITS model, which compared individuals with a robust database of available foods from the region, identified a wide diversity of diets in the Ancachi/Quillagua area (including both coastal and interior individuals), and, most notably, thirteen individuals who consumed an average of 11.2 ± 1.9% terrestrial animals, 19.8 ± 1.9% legumes, and 22.5 ± 3.1% marine fauna, a balanced pattern of protein consumption distinct from both the coastal and inland individuals in our larger regional sample.ConclusionsThe combination of stable isotope analysis and multisource mixture modeling permitted the characterization of dietary behavior of 25 individuals from nodal sites in the Atacama Desert, thus enhancing our understanding of the economic and social relationships that bound together Formative Period sites, populations, and individuals in this hyperarid region.

ObjectivesTo characterize the paleodiet of individuals from Formative Period (1500 B.C.-A.D. 400) Atacama Desert sites of Ancachi and Quillagua as a means of understanding the dietary and cultural impacts of regional systems of exchange.Materials and methodsThirty-one bone samples recovered from the cemetery of Ancachi (02QU175) and in/around the nearby town of Quillagua were the subject of carbon and nitrogen stable isotope analysis of bone collagen and hydroxyapatite and multisource mixture modeling (FRUITS, food reconstruction using isotopic transferred signals) of paleodiet. These individuals were compared with nearly 200 other Formative Period individuals from throughout the region to identify differences in dietary behaviors.Results80.6% (25/31) of the samples yielded sufficient well-preserved collagen and were included in the multisource mixture model. The FRUITS model, which compared individuals with a robust database of available foods from the region, identified a wide diversity of diets in the Ancachi/Quillagua area (including both coastal and interior individuals), and, most notably, thirteen individuals who consumed an average of 11.2 ± 1.9% terrestrial animals, 19.8 ± 1.9% legumes, and 22.5 ± 3.1% marine fauna, a balanced pattern of protein consumption distinct from both the coastal and inland individuals in our larger regional sample.ConclusionsThe combination of stable isotope analysis and multisource mixture modeling permitted the characterization of dietary behavior of 25 individuals from nodal sites in the Atacama Desert, thus enhancing our understanding of the economic and social relationships that bound together Formative Period sites, populations, and individuals in this hyperarid region.

Magnesium (Mg) and its alloys have shown attractive biocompatibility and mechanical strength for medical applications, but low corrosion resistance of Mg in physiological environment limits its broad clinical translation. Hydroxyapatite (HA) nanoparticles (nHA) are promising coating materials for decreasing degradation rates and prolonging mechanical strength of Mg-based implants while enhancing bone healing due to their osteoconductivity and osteoinductivity. Conformal HA coatings with nano-to-submicron structures, namely nHA and mHA coatings, were deposited successfully on Mg plates and rods using a transonic particle acceleration (TPA) process under two different conditions, characterized, and investigated for their effects on Mg degradation in vitro. The nHA and mHA coatings enhanced corrosion resistance of Mg and retained 86-90% of ultimate compressive strength after in vitro immersion in rSBF for 6 weeks, much greater than non-coated Mg that only retained 66% of strength. Mg-based rods with or without coatings showed slower degradation than the respective Mg-based plates in rSBF after 6 weeks, likely because of the greater surface-to-volume ratio of Mg plates than Mg rods. This indicates that Mg-based plate and screw devices may undergo different degradation even when they have the same coatings and are implanted at the same or similar anatomical locations. Therefore, in addition to locations of implantation, the geometry, dimension, surface area, volume, and mass of Mg-based implants and devices should be carefully considered in their design and processing to ensure that they not only provide adequate structural and mechanical stability for bone fixation, but also support the functions of bone cells, as clinically required for craniomaxillofacial (CMF) and orthopedic implants. When the nHA and mHA coated Mg and non-coated Mg plates were cultured with bone marrow derived mesenchymal stem cells (BMSCs) using the in vitro direct culture method, greater cell adhe

Delayed vascularization and resultant resorption limits the clinical use of tissue engineered bony constructs. The objective of this study is to develop a strategy to accelerate the neovascularization of tissue-engineered bony constructs using endothelial differentiated adipose-derived stem cells (ASC). The authors harvested ASC from inguinal fat pads of male Lewis rats (n = 5) and induced toward endothelial and osteoblastic lineages. The authors created critical size calvarial defects on male Lewis rats (n = 30) and randomized the animals into 4 groups. For the repair of the defects the authors used hydroxyapatite/poly(lactide-co-glycolide) [HA-PLG] scaffolds in group I, HA-PLG scaffolds seeded with ASC in group II, HA-PLG scaffolds seeded with ASC-derived endothelial cells in group III, and HA-PLG scaffolds seeded with ASC-derived osteoblasts in group IV. The authors evaluated the bone healing histologically and with micro-computed tomography (CT) scans 8 weeks later. Adipose-derived stem cells exhibited the characteristics of endothelial and osteogenic lineages, and attached on HA-PLG scaffolds after differentiation. Micro-CT analysis revealed that highest bone mineral density was in group IV (1.46 ± 0.01 g/cm) followed by groups III (1.43 ± 0.05 g/cm), I (1.42 ± 0.05 g/cm), and II (1.3 ± 0.1 g/cm). Hematoxylin-Eosin and Masson Trichrome staining revealed similar results with the highest bone regeneration in group IV followed by groups II, III, and I. Regenerated bone in group IV also had the highest vascular density, but none of these differences achieved statistical significance (P > 0.05). The ASC-derived endothelial cells and osteoblasts provide a limited increase in calvarial bone healing when combined with HA-PLG scaffolds.

Delayed vascularization and resultant resorption limits the clinical use of tissue engineered bony constructs. The objective of this study is to develop a strategy to accelerate the neovascularization of tissue-engineered bony constructs using endothelial differentiated adipose-derived stem cells (ASC). The authors harvested ASC from inguinal fat pads of male Lewis rats (n = 5) and induced toward endothelial and osteoblastic lineages. The authors created critical size calvarial defects on male Lewis rats (n = 30) and randomized the animals into 4 groups. For the repair of the defects the authors used hydroxyapatite/poly(lactide-co-glycolide) [HA-PLG] scaffolds in group I, HA-PLG scaffolds seeded with ASC in group II, HA-PLG scaffolds seeded with ASC-derived endothelial cells in group III, and HA-PLG scaffolds seeded with ASC-derived osteoblasts in group IV. The authors evaluated the bone healing histologically and with micro-computed tomography (CT) scans 8 weeks later. Adipose-derived stem cells exhibited the characteristics of endothelial and osteogenic lineages, and attached on HA-PLG scaffolds after differentiation. Micro-CT analysis revealed that highest bone mineral density was in group IV (1.46 ± 0.01 g/cm) followed by groups III (1.43 ± 0.05 g/cm), I (1.42 ± 0.05 g/cm), and II (1.3 ± 0.1 g/cm). Hematoxylin-Eosin and Masson Trichrome staining revealed similar results with the highest bone regeneration in group IV followed by groups II, III, and I. Regenerated bone in group IV also had the highest vascular density, but none of these differences achieved statistical significance (P > 0.05). The ASC-derived endothelial cells and osteoblasts provide a limited increase in calvarial bone healing when combined with HA-PLG scaffolds.

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.

Aims We report on the outcome of the Synergy cementless femoral stem with a minimum followup of 15 years (15 to 17). Patients and Methods A retrospective review was undertaken of a consecutive series of 112 routine primary cementless total hip arthroplasties (THAs) in 102 patients (112 hips). There were 60 female and 42 male patients with a mean age of 61 years (18 to 82) at the time of surgery. A total of 78 hips in the 69 patients remain in situ; nine hips in eight patients died before 15 years, and 16 hips in 16 patients were revised. Clinical outcome scores and radiographs were available for 94 hips in 85 patients. Results In all, four stems were revised. One stem was revised for aseptic loosening; two stems because of deep infection; and one because of periprosthetic femoral fracture. There was a significant improvement in all components of the Western Ontario and McMaster Universities Osteoarthritis Index score at the final follow-up (total: p < 0.001, pain: p < 0.001, stiffness: p < 0.001, function: p < 0.001). The mean Harris Hip Scores improved from 47 points (27 to 59) pre-operatively to 89 points (65 to 100) at the latest follow-up (p < 0.001). Kaplan-Meier survivorship, with stem revision for aseptic loosening as the endpoint, was 98.9% at 15 years (95% confidence interval (CI) 96.9 to 100, number at risk at 15 years: 90) and with stem revision for any reason was 95.7% (95% CI 91.7 to 99.8, number at risk at 15 years: 90). Conclusion The Synergy cementless femoral stem demonstrates excellent survivorship and functional outcomes at 15 years.

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.

ObjectiveThe intent of the study is to describe an unusual pattern of intramuscular migration of calcific deposits related to hydroxyapatite deposition disease (HADD) involving the rotator cuff, to illustrate the characteristic imaging features of this phenomenon, and to discuss the clinical significance of such migration.Materials and methodsA series of cases of intramuscular accumulation of calcium hydroxyapatite crystals collected over a 7-year period at multiple hospitals within the same academic institution were retrospectively reviewed.ResultsThe patient group was composed of seven men and four women, ranging in age from 51 to 79 years, with a mean age of 63 years. All subjects presented with acute shoulder pain. The majority of subjects reported the spontaneous onset of the symptoms (64%), while others reported weight lifting (27%) and a fall on the arm (9%) as the mechanisms of injury. The right shoulder was affected in 73% of the subjects. The supraspinatus was the most commonly affected muscle (82%), followed by the infraspinatus muscle (36%).ConclusionsKnowledge of the imaging features of intramuscular migration of hydroxyapatite deposits is important in order to avoid the erroneous diagnosis of other causes of muscle edema and inflammation such as myotendinous injury, myositis, subacute denervation, and neoplasm.

UnlabelledPluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo. Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications.Statement of significanceDirecting stem cell differentiation and morphogenesis via biomaterials represents a novel strategy to promote cell fates and tissue formation. Our study demonstrates the ability of calcium phosphate-based mineral particles to promote osteochondrogenic differentiation of embryonic stem cell aggregates as well as modulate teratoma formation in vivo. This hybrid biomaterial-ESC aggregate approach serves as an enabling platform to evaluate the ability of biomaterials to regulate stem cell fate and regenerate functional skeletal tissues for clinical applications.

ObjectiveThe intent of the study is to describe an unusual pattern of intramuscular migration of calcific deposits related to hydroxyapatite deposition disease (HADD) involving the rotator cuff, to illustrate the characteristic imaging features of this phenomenon, and to discuss the clinical significance of such migration.Materials and methodsA series of cases of intramuscular accumulation of calcium hydroxyapatite crystals collected over a 7-year period at multiple hospitals within the same academic institution were retrospectively reviewed.ResultsThe patient group was composed of seven men and four women, ranging in age from 51 to 79 years, with a mean age of 63 years. All subjects presented with acute shoulder pain. The majority of subjects reported the spontaneous onset of the symptoms (64%), while others reported weight lifting (27%) and a fall on the arm (9%) as the mechanisms of injury. The right shoulder was affected in 73% of the subjects. The supraspinatus was the most commonly affected muscle (82%), followed by the infraspinatus muscle (36%).ConclusionsKnowledge of the imaging features of intramuscular migration of hydroxyapatite deposits is important in order to avoid the erroneous diagnosis of other causes of muscle edema and inflammation such as myotendinous injury, myositis, subacute denervation, and neoplasm.

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.

Pluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo. Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications.Statement of significanceDirecting stem cell differentiation and morphogenesis via biomaterials represents a novel strategy to promote cell fates and tissue formation. Our study demonstrates the ability of calcium phosphate-based mineral particles to promote osteochondrogenic differentiation of embryonic stem cell aggregates as well as modulate teratoma formation in vivo. This hybrid biomaterial-ESC aggregate approach serves as an enabling platform to evaluate the ability of biomaterials to regulate stem cell fate and regenerate functional skeletal tissues for clinical applications.

NEL-like molecule-1 (NELL-1) is a novel osteogenic protein that showing high specificity to osteochondral cells. It was widely used in bone regeneration research by loading onto carriers such as tricalcium phosphate (TCP) particles. However, there has been little research on protein controlled release from this material and its potential application. In this study, TCP was first modified with a hydroxyapatite coating followed by a chitosan coating to prepare chitosan/hydroxyapatite-coated TCP particles (Chi/HA-TCP). The preparation was characterized by SEM, EDX, FTIR, XRD, FM and Zeta potential measurements. The NELL-1 loaded Chi/HA-TCP particles and the release kinetics were investigated in vitro. It was observed that the Chi/HA-TCP particles prepared with the 0.3% (wt/wt) chitosan solution were able to successfully control the release of NELL-1 and maintain a slow, steady release for up to 28 days. Furthermore, more than 78% of the loaded protein's bioactivity was preserved in Chi/HA-TCP particles over the period of the investigation, which was significantly higher than that of the protein released from hydroxyapatite coated TCP (HA-TCP) particles. Collectively, this study suggests that the osteogenic protein NELL-1 showed a sustained release pattern after being encapsulated into the modified Chi/HA-TCP particles, and the NELL-1 integrated composite of Chi/HA-TCP showed a potential to function as a protein delivery carrier and as an improved bone matrix for use in bone regeneration research.

Pluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo. Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications.Statement of significanceDirecting stem cell differentiation and morphogenesis via biomaterials represents a novel strategy to promote cell fates and tissue formation. Our study demonstrates the ability of calcium phosphate-based mineral particles to promote osteochondrogenic differentiation of embryonic stem cell aggregates as well as modulate teratoma formation in vivo. This hybrid biomaterial-ESC aggregate approach serves as an enabling platform to evaluate the ability of biomaterials to regulate stem cell fate and regenerate functional skeletal tissues for clinical applications.

Amelogenins are proteins formed by alternative splicing of the amelogenin gene, and are essential for tooth enamel formation. However, the unique functions of various alternatively spliced amelogenins in enamel formation are not well understood. In this study, we determined the spatiotemporal location of amelogenins derived from transcripts containing exon4 (AMG+4) in the enamel matrix, and the relative binding of recombinant AMG+4 to hydroxyapatite (HAP). Immunohistochemistry and mass spectrometry analyses showed that AMG+4 proteins were secreted into the enamel matrix at the early maturation stage. A stage-specific increase in the synthesis of AMG+4 was further supported by our observation that in mice overexpressing leucine-rich amelogenin peptide (TgLRAP), in which ameloblasts differentiate earlier, AMG+4 transcripts were also upregulated earlier. In vitro binding studies, supported by in silico modeling of protein binding to calcium and phosphate, showed that more recombinant AMG+4 bound to hydroxyapatite (HAP) as compared with recombinant AMG-4. The temporal and spatial localization of amelogenins containing exon4 peptide, and their functional differences in HAP binding, suggests that the unique properties of amelogenins containing exon4 cause a specific enhancement of biomineralization related to stabilization of early-formed HAP at the maturation stage.

Amelogenins are proteins formed by alternative splicing of the amelogenin gene, and are essential for tooth enamel formation. However, the unique functions of various alternatively spliced amelogenins in enamel formation are not well understood. In this study, we determined the spatiotemporal location of amelogenins derived from transcripts containing exon4 (AMG+4) in the enamel matrix, and the relative binding of recombinant AMG+4 to hydroxyapatite (HAP). Immunohistochemistry and mass spectrometry analyses showed that AMG+4 proteins were secreted into the enamel matrix at the early maturation stage. A stage-specific increase in the synthesis of AMG+4 was further supported by our observation that in mice overexpressing leucine-rich amelogenin peptide (TgLRAP), in which ameloblasts differentiate earlier, AMG+4 transcripts were also upregulated earlier. In vitro binding studies, supported by in silico modeling of protein binding to calcium and phosphate, showed that more recombinant AMG+4 bound to hydroxyapatite (HAP) as compared with recombinant AMG-4. The temporal and spatial localization of amelogenins containing exon4 peptide, and their functional differences in HAP binding, suggests that the unique properties of amelogenins containing exon4 cause a specific enhancement of biomineralization related to stabilization of early-formed HAP at the maturation stage.

We present an uncommon case of nasal alar and facial necrosis following calcium hydroxylapatite filler injection performed elsewhere without direct physician supervision. The patient developed severe full-thickness necrosis of cheek and nasal alar skin 24 h after injections into the melolabial folds. Management prior to referral included oral antibiotics, prednisone taper, and referral to a dermatologist (day 3) who prescribed valacyclovir for a presumptive herpes zoster reactivation induced by the injection. Referral to our institution was made on day 11, and after herpetic outbreak was ruled out by a negative Tzanck smear, debridement with aggressive local wound care was initiated. After re-epithelialization and the fashioning of a custom intranasal stent to prevent vestibular stenosis, pulsed dye laser therapy was performed for wound modification. The patient healed with an acceptable cosmetic outcome. This report underscores the importance of facial vasculature anatomy, injection techniques, and identification of adverse events when using fillers. A current treatment paradigm for such events is also presented.

Synthetic bone graft substitutes based on composites consisting of a polymer and a calcium-phosphate (CaP) ceramic are developed with the aim to satisfy both mechanical and bioactivity requirements for successful bone regeneration. In the present study, we have employed extrusion to produce a composite consisting of 50 wt.% poly(D,L-lactic acid) (PLA) and 50 wt.% nano-sized hydroxyapatite (HA) powder, achieving homogeneous distribution of the ceramic within the polymeric phase. In vitro, in both a simulated physiological saline (SPS) and a simulated body fluid (SBF), a greater weight loss was observed for PLA/HA than for PLA particles upon 12-week immersion. Furthermore, in SPS, a continuous release of calcium and phosphate from the composite was measured, whereas in SBF, decrease of the amount of the two ions in the solution was observed both for PLA and PLA/HA accompanied with the formation of a CaP layer on the surface. In vitro characterization of the composite bioactivity was performed by culturing human mesenchymal stromal cells (hMSCs) and assessing proliferation and osteogenic differentiation, with PLA as a control. Both PLA/HA composite and PLA were shown to support hMSCs proliferation over a period of two weeks. In addition, the composite significantly enhanced alkaline phosphatase (ALP) activity of hMSCs in osteogenic medium as compared with the polymer control. A novel implant design was employed to develop implants from dense, extruded materials, suitable for testing osteoinductivity in vivo. In a preliminary study in dogs, PLA/HA composite implants induced heterotopic bone formation upon 12-week intramuscular implantation in all animals, in contrast to PLA control, which was not osteoinductive. Unlike in vitro, a more pronounced degradation of PLA was observed in vivo as compared with PLA/HA composite.

Background/Aim. Autogenous bone grafting has been the gold standard in clinical cases when bone grafts are required for bone defects in dentistry. The study was undertaken to evaluate multilevel designed carbonated hydroxyapatite (CHA) obtained by hydrothermal method, as a bone substitute in healing bone defects with or without corticosteroid treatment in rats as assessed by histopathologic methods. Methods. Bone defects were created in the alveolar bone by teeth extraction in 12 rats. The animals were initially divided into two groups. The experimental group was pretreated with corticosteroids: methylprednisolone and dexamethasone, intramuscularly, while the control group was without therapy. Posterior teeth extraction had been performed after the corticosteroid therapy. The extraction defects were fulfilled with hydroxyapatite with bimodal particle sizes in the range of 50-250 mu m and the sample from postextocactional defect of the alveolar bone was analyzed pathohystologically. Results. The histopatological investigations confirmed the biologic properties of the applied material. The evident growth of new bone in the alveolar ridge was clearly noticed in both groups of rats. Carbonated HA obtained by hydrothermal method promoted bone formation in the preformed defects, confirming its efficacy for usage in bone defects. Complete resorption of the materials particles took place after 25 weeks. Conclusion. Hydroxyapatite completely meets the clinical requirements for a bone substitute material. Due to its microstructure, complete resorption took place during the observation period of the study. Corticosteroid treatment did not significantly affect new bone formation in the region of postextractional defects.

We present an uncommon case of nasal alar and facial necrosis following calcium hydroxylapatite filler injection performed elsewhere without direct physician supervision. The patient developed severe full-thickness necrosis of cheek and nasal alar skin 24 h after injections into the melolabial folds. Management prior to referral included oral antibiotics, prednisone taper, and referral to a dermatologist (day 3) who prescribed valacyclovir for a presumptive herpes zoster reactivation induced by the injection. Referral to our institution was made on day 11, and after herpetic outbreak was ruled out by a negative Tzanck smear, debridement with aggressive local wound care was initiated. After re-epithelialization and the fashioning of a custom intranasal stent to prevent vestibular stenosis, pulsed dye laser therapy was performed for wound modification. The patient healed with an acceptable cosmetic outcome. This report underscores the importance of facial vasculature anatomy, injection techniques, and identification of adverse events when using fillers. A current treatment paradigm for such events is also presented.

Synthetic bone graft substitutes based on composites consisting of a polymer and a calcium-phosphate (CaP) ceramic are developed with the aim to satisfy both mechanical and bioactivity requirements for successful bone regeneration. In the present study, we have employed extrusion to produce a composite consisting of 50 wt.% poly(D,L-lactic acid) (PLA) and 50 wt.% nano-sized hydroxyapatite (HA) powder, achieving homogeneous distribution of the ceramic within the polymeric phase. In vitro, in both a simulated physiological saline (SPS) and a simulated body fluid (SBF), a greater weight loss was observed for PLA/HA than for PLA particles upon 12-week immersion. Furthermore, in SPS, a continuous release of calcium and phosphate from the composite was measured, whereas in SBF, decrease of the amount of the two ions in the solution was observed both for PLA and PLA/HA accompanied with the formation of a CaP layer on the surface. In vitro characterization of the composite bioactivity was performed by culturing human mesenchymal stromal cells (hMSCs) and assessing proliferation and osteogenic differentiation, with PLA as a control. Both PLA/HA composite and PLA were shown to support hMSCs proliferation over a period of two weeks. In addition, the composite significantly enhanced alkaline phosphatase (ALP) activity of hMSCs in osteogenic medium as compared with the polymer control. A novel implant design was employed to develop implants from dense, extruded materials, suitable for testing osteoinductivity in vivo. In a preliminary study in dogs, PLA/HA composite implants induced heterotopic bone formation upon 12-week intramuscular implantation in all animals, in contrast to PLA control, which was not osteoinductive. Unlike in vitro, a more pronounced degradation of PLA was observed in vivo as compared with PLA/HA composite.

Background/Aim. Autogenous bone grafting has been the gold standard in clinical cases when bone grafts are required for bone defects in dentistry. The study was undertaken to evaluate multilevel designed carbonated hydroxyapatite (CHA) obtained by hydrothermal method, as a bone substitute in healing bone defects with or without corticosteroid treatment in rats as assessed by histopathologic methods. Methods. Bone defects were created in the alveolar bone by teeth extraction in 12 rats. The animals were initially divided into two groups. The experimental group was pretreated with corticosteroids: methylprednisolone and dexamethasone, intramuscularly, while the control group was without therapy. Posterior teeth extraction had been performed after the corticosteroid therapy. The extraction defects were fulfilled with hydroxyapatite with bimodal particle sizes in the range of 50-250 mu m and the sample from postextocactional defect of the alveolar bone was analyzed pathohystologically. Results. The histopatological investigations confirmed the biologic properties of the applied material. The evident growth of new bone in the alveolar ridge was clearly noticed in both groups of rats. Carbonated HA obtained by hydrothermal method promoted bone formation in the preformed defects, confirming its efficacy for usage in bone defects. Complete resorption of the materials particles took place after 25 weeks. Conclusion. Hydroxyapatite completely meets the clinical requirements for a bone substitute material. Due to its microstructure, complete resorption took place during the observation period of the study. Corticosteroid treatment did not significantly affect new bone formation in the region of postextractional defects.

We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

With an increasingly active and aging population, a growing number of orthopedic procedures are performed annually. However, traditional orthopedic implants face many complications such as infection, implant loosening, and poor host tissue integration leading to implant failure. Metal implant materials such as titanium and its alloys are widely used in orthopedic applications mainly based on their excellent mechanical properties and biological inertness. Since human bone extracellular matrix is nanometer in dimension comprised of rich nanostructured hydroxyapatite particles and collagen nanofibers, it is highly desirable to design a biologically-inspired nanostructured coating which renders the biocompatible titanium surface into a biomimetic and bioactive interface, thus enhancing osteoblast adhesion and promoting osseointegration. For this purpose, a biomimetic nanostructured coating based on nanocrystalline hydroxyapatite and single wall carbon nanotubes was designed. Specifically, nano hydroxyapatites with good crystallinity and biomimetic dimensions were prepared via a wet chemistry method and hydrothermal treatment. Microcrystalline hydroxyapatite with larger grain sizes can be obtained without hydrothermal treatment. The carbon nanotubes with different diameter and length were synthesized via an arc plasma method in the presence or absence of a magnetic field. Transmission electron microscopy images illustrate the regular, rod-like nanocrystalline and biomimetic nanostructure of hydrothermally treated nano hydroxyapatite. In addition, the length of carbon nanotubes can be significantly increased under external magnetic fields when compared to nanotubes produced without a magnetic field. More importantly, the in vitro study demonstrated for the first time that osteoblast and mesenchymal stem cell adhesion and proliferation were greater on titanium with hydrothermally treated nanocrystalline hydroxyapatites/magnetically treated carbon nanotubes, which suggests

We have previously identified amelotin (AMTN) as a novel protein expressed predominantly during the late stages of dental enamel formation, but its role during amelogenesis remains to be determined. In this study we generated transgenic mice that produce AMTN under the amelogenin (Amel) gene promoter to study the effect of AMTN overexpression on enamel formation in vivo. The specific overexpression of AMTN in secretory stage ameloblasts was confirmed by Western blot and immunohistochemistry. The gross histological appearance of ameloblasts or supporting cellular structures as well as the expression of the enamel proteins amelogenin (AMEL) and ameloblastin (AMBN) was not altered by AMTN overexpression, suggesting that protein production, processing and secretion occurred normally in transgenic mice. The expression of Odontogenic, Ameloblast-Associated (ODAM) was slightly increased in secretory stage ameloblasts of transgenic animals. The enamel in AMTN-overexpressing mice was much thinner and displayed a highly irregular surface structure compared to wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. Tomes' process, the hallmark of secretory stage ameloblasts, did not form in transgenic mice. Collectively our data demonstrate that the overexpression of amelotin has a profound effect on enamel structure by disrupting the formation of Tomes' process and the orderly growth of enamel prisms.

Purpose: To investigate the risks of implant exposure after a combined explant-implant procedure in patients with an exposed porous orbital implant. Methods: Twenty-four consecutive patients who had a combined explant-implant procedure of an exposed hydroxyapatite (21) or porous polyethylene (3) orbital implant from January 2000 to February 2009 were included. The patient records were reviewed; patients were interviewed by telephone and invited for a clinical examination. Histopathological examination was carried out on the removed implants. Main outcome measures were: presence of exposure of the new implant or not, patient graded satisfaction with the cosmetic result, and presence of poor motility. Results: None of the new implants became exposed or infected in the follow-up period of 25 [3-94] months (median [range]). The patients scored their satisfaction with the cosmetic result to a median score of 9 (range 5-10). Poor motility was present in six of 17 patients. Micro-organisms were identified in three removed implants and signs of inflammation were present in 20 removed implants. Conclusions: If a decision of implant removal has been made, it is safe to replace the implant at the same procedure in sockets without profound signs of infection. The procedure carries a possible risk of poor motility.

BackgroundNanomaterials have unique advantages in controlling stem cell function due to their biomimetic characteristics and special biological and mechanical properties. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration.MethodsThis in vitro study investigated the effects of nano-hydroxyapatite, nano-hydroxyapatite-polylactide- co-glycolide (PLGA) composites, and a bone morphogenetic protein (BMP-7)- derived short peptide (DIF-7c) on osteogenic differentiation of human mesenchymal stem cells (MSC). The peptide was chemically functionalized onto nano-hydroxyapatite, incorporated into a nanophase hydroxyapatite-PLGA composite or PLGA control, or directly injected into culture media.ResultsUnlike the PLGA control, the nano-hydroxyapatite-PLGA composites promoted adhesion of human MSC. Importantly, nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites promoted osteogenic differentiation of human MSCs, comparable with direct injection of the DIF-7c peptide into culture media.ConclusionNano-hydroxyapatite and nano-hydroxyapatite-PLGA composites provide a promising alternative in directing the adhesion and differentiation of human MSC. These nanocomposites should be studied further to clarify their effects on MSC functions and bone remodeling in vivo, eventually translating to clinical applications.

BackgroundNanomaterials have unique advantages in controlling stem cell function due to their biomimetic characteristics and special biological and mechanical properties. Controlling adhesion and differentiation of stem cells is critical for tissue regeneration.MethodsThis in vitro study investigated the effects of nano-hydroxyapatite, nano-hydroxyapatite-polylactide- co-glycolide (PLGA) composites, and a bone morphogenetic protein (BMP-7)- derived short peptide (DIF-7c) on osteogenic differentiation of human mesenchymal stem cells (MSC). The peptide was chemically functionalized onto nano-hydroxyapatite, incorporated into a nanophase hydroxyapatite-PLGA composite or PLGA control, or directly injected into culture media.ResultsUnlike the PLGA control, the nano-hydroxyapatite-PLGA composites promoted adhesion of human MSC. Importantly, nano-hydroxyapatite and nano-hydroxyapatite-PLGA composites promoted osteogenic differentiation of human MSCs, comparable with direct injection of the DIF-7c peptide into culture media.ConclusionNano-hydroxyapatite and nano-hydroxyapatite-PLGA composites provide a promising alternative in directing the adhesion and differentiation of human MSC. These nanocomposites should be studied further to clarify their effects on MSC functions and bone remodeling in vivo, eventually translating to clinical applications.

Purpose: To investigate the risks of implant exposure after a combined explant-implant procedure in patients with an exposed porous orbital implant. Methods: Twenty-four consecutive patients who had a combined explant-implant procedure of an exposed hydroxyapatite (21) or porous polyethylene (3) orbital implant from January 2000 to February 2009 were included. The patient records were reviewed; patients were interviewed by telephone and invited for a clinical examination. Histopathological examination was carried out on the removed implants. Main outcome measures were: presence of exposure of the new implant or not, patient graded satisfaction with the cosmetic result, and presence of poor motility. Results: None of the new implants became exposed or infected in the follow-up period of 25 [3-94] months (median [range]). The patients scored their satisfaction with the cosmetic result to a median score of 9 (range 5-10). Poor motility was present in six of 17 patients. Micro-organisms were identified in three removed implants and signs of inflammation were present in 20 removed implants. Conclusions: If a decision of implant removal has been made, it is safe to replace the implant at the same procedure in sockets without profound signs of infection. The procedure carries a possible risk of poor motility.

Purpose: To investigate the risks of implant exposure after a combined explant-implant procedure in patients with an exposed porous orbital implant. Methods: Twenty-four consecutive patients who had a combined explant-implant procedure of an exposed hydroxyapatite (21) or porous polyethylene (3) orbital implant from January 2000 to February 2009 were included. The patient records were reviewed; patients were interviewed by telephone and invited for a clinical examination. Histopathological examination was carried out on the removed implants. Main outcome measures were: presence of exposure of the new implant or not, patient graded satisfaction with the cosmetic result, and presence of poor motility. Results: None of the new implants became exposed or infected in the follow-up period of 25 [3-94] months (median [range]). The patients scored their satisfaction with the cosmetic result to a median score of 9 (range 5-10). Poor motility was present in six of 17 patients. Micro-organisms were identified in three removed implants and signs of inflammation were present in 20 removed implants. Conclusions: If a decision of implant removal has been made, it is safe to replace the implant at the same procedure in sockets without profound signs of infection. The procedure carries a possible risk of poor motility.

Purpose: To investigate the risks of implant exposure after a combined explant-implant procedure in patients with an exposed porous orbital implant. Methods: Twenty-four consecutive patients who had a combined explant-implant procedure of an exposed hydroxyapatite (21) or porous polyethylene (3) orbital implant from January 2000 to February 2009 were included. The patient records were reviewed; patients were interviewed by telephone and invited for a clinical examination. Histopathological examination was carried out on the removed implants. Main outcome measures were: presence of exposure of the new implant or not, patient graded satisfaction with the cosmetic result, and presence of poor motility. Results: None of the new implants became exposed or infected in the follow-up period of 25 [3-94] months (median [range]). The patients scored their satisfaction with the cosmetic result to a median score of 9 (range 5-10). Poor motility was present in six of 17 patients. Micro-organisms were identified in three removed implants and signs of inflammation were present in 20 removed implants. Conclusions: If a decision of implant removal has been made, it is safe to replace the implant at the same procedure in sockets without profound signs of infection. The procedure carries a possible risk of poor motility.

Implantation of autologous skin fibroblasts transduced ex vivo with a replication-defective adenoviral vector, carrying the LIM mineralization protein-3 (Ad-LMP-3), and adsorbed on a hydroxyapatite/collagen (HA/COL) scaffold.

Amelogenins, the chief proteins in enamel matrix, undergo progressive degradation by matrix metalloproteinase-20 (MMP-20) to facilitate crystal growth. Proline is the most abundant residue in amelogenin and is located upstream to all MMP-20 cleavage sites in the amelogenin sequence. Pro(41) is critical for amelogenin N-terminal processing, while the role of prolines at the amelogenin C terminus have not been determined. This study sought to elucidate the effect of the C-terminal prolines on apatite binding and MMP-20 hydrolysis. To compare apatite affinity, recombinant full-length human amelogenin (rh174) and mutated variants (P156T and P164T) were incubated with hydroxyapatite (HAP), and the unbound proteins were quantified by the Bradford assay. rh174 and mutants, as well as 3 oligopeptides, including wild-type peptide and peptides containing 2 mutations, were incubated with MMP-20 both in solution and on HAP. The digested products were analyzed by SDS-PAGE, reverse-phase high-performance liquid chromatography and mass spectrometry. Mutated amelogenins displayed a significantly lower affinity to HAP than the wild type (P156T < P164T < rH174). The proline mutation at amino acid location 164 significantly reduced the initial hydrolysis of either the amelogenins in solution or the proteins bound on HAP, which was confirmed by amelogenin oligopeptide assays. It was concluded that prolines at the amelogenin C terminus are essential for the initial processing of amelogenin and amelogenin-mineral interactions.

An electric-current-assisted method was used to mineralize dense hydrogels and create hydroxyapatite/hydrogel composites with unique hierarchical structures. The microstructure of the final material can be controlled by the mineralization technique and the chemistry of the organic matrix. A hydroxyapatite/hydrogel composite was obtained with a large inorganic content (approximately 60% of the weight of the organics). After being heated to 1050 degrees C, the sintered inorganic phase has a very uniformly distributed porosity and its Brunauer-Emmett-Teller (BET) surface area is 0.68 m(2)/g.

PURPOSE: Various tools are currently available for dose reconstruction in individuals after accidental exposure to ionizing radiation. Among the available biological analyses, Monte Carlo simulations, and biophysical methods, such as electron paramagnetic resonance (EPR), the latter has proved its usefulness for retrospective dosimetry. Although EPR spectroscopy is probably the most sensitive technique, it does not provide spatial dosimetric data. This information is, however, highly desirable when steep dose gradient irradiations are involved. The purpose of this work was to explore the possibilities of EPR imaging (EPRI) for spatial dose reconstruction in irradiated biological material. METHODS: X-band EPRI was used to reconstruct ex vivo the relative dose distribution in human bone samples and hydroxyapatite phantoms after irradiation with brachytherapy seeds or x rays. Three situations were investigated: Homogeneous, stepwise gradient, and continuous gradient irradiation. RESULTS: EPRI gave a faithful relative spin density distribution in bone samples and in hydroxyapatite phantoms. Measured dose ratios were in close agreement with the actual delivered dose ratios. EPRI was able to distinguish the dose gradients induced by two different sources (125I and 192Ir). However, the measured spatial resolution of the system was 1.9 mm and this appeared to be a limiting factor. The method could be improved by using new signal postprocessing strategies. CONCLUSIONS: This study demonstrates that EPRI can be used to assess the regional relative dose distribution in irradiated bone samples. The method is currently applicable to ex vivo measurements of small size samples with low variation in tissue density but is likely to be adapted for in vivo application using L-band EPRI.

An electric-current-assisted method was used to mineralize dense hydrogels and create hydroxyapatite/hydrogel composites with unique hierarchical structures. The microstructure of the final material can be controlled by the mineralization technique and the chemistry of the organic matrix. A hydroxyapatite/hydrogel composite was obtained with a large inorganic content (approximately 60% of the weight of the organics). After being heated to 1050 degrees C, the sintered inorganic phase has a very uniformly distributed porosity and its Brunauer-Emmett-Teller (BET) surface area is 0.68 m(2)/g.

Amelogenins, the chief proteins in enamel matrix, undergo progressive degradation by matrix metalloproteinase-20 (MMP-20) to facilitate crystal growth. Proline is the most abundant residue in amelogenin and is located upstream to all MMP-20 cleavage sites in the amelogenin sequence. Pro(41) is critical for amelogenin N-terminal processing, while the role of prolines at the amelogenin C terminus have not been determined. This study sought to elucidate the effect of the C-terminal prolines on apatite binding and MMP-20 hydrolysis. To compare apatite affinity, recombinant full-length human amelogenin (rh174) and mutated variants (P156T and P164T) were incubated with hydroxyapatite (HAP), and the unbound proteins were quantified by the Bradford assay. rh174 and mutants, as well as 3 oligopeptides, including wild-type peptide and peptides containing 2 mutations, were incubated with MMP-20 both in solution and on HAP. The digested products were analyzed by SDS-PAGE, reverse-phase high-performance liquid chromatography and mass spectrometry. Mutated amelogenins displayed a significantly lower affinity to HAP than the wild type (P156T < P164T < rH174). The proline mutation at amino acid location 164 significantly reduced the initial hydrolysis of either the amelogenins in solution or the proteins bound on HAP, which was confirmed by amelogenin oligopeptide assays. It was concluded that prolines at the amelogenin C terminus are essential for the initial processing of amelogenin and amelogenin-mineral interactions.

Fluorosed enamel is more porous and less mineralized, possibly related to altered amelogenin-modulated crystal growth. The purpose of this study was to examine the role of fluoride in interactions between amelogenin and apatite crystals. Recombinant human amelogenin (rh174) was bound to carbonated hydroxyapatite containing various amounts of fluoride, and analyzed by protein assay, SDS PAGE, and AFM. Interactions between rh174 and fluoride were assayed by isothermal titration calorimetry (ITC). The initial binding rate of rh174, as well as total amount of rh174 bound to fluoride-containing carbonated hydroxyapatite, was greater than that in the control carbonated hydroxyapatite. Fluoride in solution at physiologic (5.3 micromolar, or 0.1 ppm) concentrations showed no significant effect on binding, but higher fluoride levels significantly decreased protein binding. ITC showed no interactions between fluoride and rh174. These results suggest that fluoride incorporation into the crystal lattice alters the crystal surface to enhance amelogenin binding, with no direct interactions between fluoride and amelogenin.

Fluorosed enamel is more porous and less mineralized, possibly related to altered amelogenin-modulated crystal growth. The purpose of this study was to examine the role of fluoride in interactions between amelogenin and apatite crystals. Recombinant human amelogenin (rh174) was bound to carbonated hydroxyapatite containing various amounts of fluoride, and analyzed by protein assay, SDS PAGE, and AFM. Interactions between rh174 and fluoride were assayed by isothermal titration calorimetry (ITC). The initial binding rate of rh174, as well as total amount of rh174 bound to fluoride-containing carbonated hydroxyapatite, was greater than that in the control carbonated hydroxyapatite. Fluoride in solution at physiologic (5.3 micromolar, or 0.1 ppm) concentrations showed no significant effect on binding, but higher fluoride levels significantly decreased protein binding. ITC showed no interactions between fluoride and rh174. These results suggest that fluoride incorporation into the crystal lattice alters the crystal surface to enhance amelogenin binding, with no direct interactions between fluoride and amelogenin.

Background/Aim. There are very few data about the effects of endogenous growth factors in vital pulp therapy, and still they are often controversial. The aim of the study was to evaluate the effects of platelet rich plasma (PRP) in conjugation with hydroxyapatite (HAP), as pulp capping materials, to root and periodontium formation. Methods. Eight young monkeys (Cercopithecus Aethiops) with permanent dentition and incomplete root formation were involved in this study. After pulpotomy, the pulp lesion was capped with calcium hydroxide (control), hydroxyapatite (experimental group I) or hydroxyapatite in conjugation with PRP (experimental group II). Six months later, the animals were sacrificed, the tissue was removed en block, and prepared for the histological analysis in a routine way. Results. The results of the histological analysis revealed that healing process was characterised by dentin bridge formation, maintained morphological and functional integrity of dental pulp and complete formation of dental root and surrounding periodontium. The inflammatory reaction was scored as mild to moderate, in almost all the samples in all groups, suggesting the biocompatibility of the used materials. Conclusion. Materials used in this study are convenient as capping agents, contributing maintaining the integrity of the pulp tissue and facilitating root and periodontium formation. According to histological data it could be suggested that hydroxyapatite in conjugation with endogenous growth factors, represents superior alternative to other materials used in this study., Uvod/Cilj. Podaci o primeni endogenih faktora rasta u terapiji vitalne pulpe zuba sa nezavršenim rastom korena su veoma oskudni i često kontraverzni. Cilj ovog rada bio je da se ispita uticaj plazme bogate trombocitima (PRP) u kombinaciji sa hidroksiapatitom (HAP), kao materijala za direktno prekrivanje pulpe, na završetak rasta korena i formiranje okolnog parodoncijuma. Metode. Istraživanje je obavljeno na osam mladih majmuna Cercopithecus Aethiops. Sve životinje imale su zube stalne denticije, sa nezavršenim rastom korena. Nakon trepanacije pulpnog prostora, pulpa je uklonjena do nivoa gleđnocementnog spoja, a lezija u pulpi prekrivena je kalcijum hidroksidom (kontrolna grupa), hidroksiapatitom (eksperimentna grupa I) i hidroksiapatitom u kombinaciji sa autogenim PRP faktorom (eksperimentna grupa II). Šest meseci nakon amputacije pulpe, životinje su žrtvovane, a tkivo za histološku analizu uzeto je u obliku blok sekcije i pripremljeno za mikroskopsku analizu. Rezultati. Zarastanje rane u pulpno-dentinskom kompleksu u obe eksperimentne i u kontrolnoj grupi bilo je karakterisano stvaranjem dentinskog mostića procesom reparativne dentinogeneze, očuvanjem morfološkog i funkcijskog integriteta pulpe, kao i završetkom rasta korena i stvaranjem okolnog parodoncijuma. U većini uzoraka obe grupe, zapaljenska reakcija ocenjena je kao blaga do umerena, što govori u prilog biokompatibilnosti primenjenih materijala. Zaključak. Svi primenjeni materijali pogodni su za direktno prekrivanje pulpe, doprinose očuvanju njenog morfološkog i funkcionalnog integriteta i omogućavaju završetak rasta korena i formiranje okolnog parodoncijuma. Ipak, povoljniji rezultati dobijeni su u grupi koja je tretirana HAP/PRP, pa se može reći da HAP obogaćen endogenim faktorima rasta predstavlja superiorniju alternativu u odnosu na ostale materijale primenjene u ovoj studiji.

The incorporation of magnesium ions (in the range 5-10 mol% in respect to Ca) into the hydroxyapatite structure, which is of great interest for the developing of artificial bone, was performed using magnesium chloride, calcium hydroxide and phosphoric acid, as reactants. Among the synthesized powders, the synthetic HA powder containing 5.7% Mg substituting for calcium was selected, due to its better chemico-physical features, and transformed into granules of 400-600 microm, for biocompatibility tests (genotoxicity, carcinogenicity, toxicity, in vitro cytotoxicity and in vivo skin irritation-sensitization tests). In vivo tests were carried out on New Zealand White rabbits using the granulate as filling for a femoral bone defect: osteoconductivity and resorption were found to be enhanced compared to commercial stoichiometric HA granulate, taken as control.

OBJECTIVES: Evaluate the effectiveness of CaHA injection for patients with glottal incompetence. METHODS: Multi-center, open-label, prospective clinical study with each patient serving as his/her own control. Voice-related outcome measures were collected for pre-injection and 1, 3, and 6 months. RESULTS: Sixty-eight patients were available for evaluation. Fifty percent of the injection procedures were done in office. Fifty-seven percent were diagnosed with unilateral paralysis and 42% with glottal incompetence with mobile vocal folds. Patient satisfaction 6 months post showed 56% had significantly improved voice and 38% reported moderately improved voice. Paired t tests from baseline to 6 months showed significant improvements on the VHI and VAS (vocal effort), CAPE-V judgments of voice severity and videoendostroboscopy ratings of glottal closure, and objective voice measures of glottal closure (MPT and S:Z ratio). CONCLUSIONS: Preliminary results in this large cohort of patients demonstrate excellent clinical results. (C) 2007 American Academy of Otolarynaology-Head and Neck Surgery Foundation. All rights reserved.