Infrared spectroscopic assessment of the inflammation-mediated osteoporosis (IMO) model applied to rabbit bone

A model of osteoporosis based on induced inflammation (IMO) was applied on rabbit bones. The structural heterogeneity and molecular complexity of bone significantly affect bone mechanical properties. A tool like Fourier transform infrared spectroscopy, able to analyze both the inorganic and organic phase simultaneously, could provide compositional information regarding cortical and trabecular sections under normal and osteoporotic conditions. In this study, we assessed the mineral/matrix ratio, carbonate and phosphate content and labile (i.e., non-apatitic) species contribution to bone mineral and collagen cross-linking patterns. Clear differences were observed between cortical and trabecular bone regarding mineral and carbonate content. Induced inflammation lowers the mineral/matrix ratio and increases the overall carbonate accumulation. Elevated concentrations of labile species were detected in osteoporotic samples, especially in the trabecular sections. Collagen cross-linking patterns were indirectly observed through the 1660/1690 cm^− 1 ratio in the amide I band and a positive correlation was found with the mineralization index. Principal component analysis (PCA) applied to female samples successfully clustered trabecular and osteoporotic cases. The important role played by the phosphate ions was confirmed by corresponding loadings plots. The results suggest that the application of the IMO model to rabbit bones effectively alters bone remodeling and forms an osteoporotic bone matrix with a dissimilar composition compared to the normal one.     

Connective tissue mineralization in Abcc6-/- mice, a model for pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by ectopic mineralization. However, the structure of the mineral deposits, their interactions with the connective tissue matrix, and the details of the progressive maturation of the mineral crystals are currently unknown. In this study, we examined the mineralization processes in Abcc6(-/-) mice, a model system for PXE, by energy dispersive X-ray and Fourier transform infrared imaging spectroscopy (FT-IRIS). The results indicated that the principal components of the mineral deposits were calcium and phosphate which co-localized within the histologically demonstrable lesions determined by topographic mapping. The Ca/P ratio increased in samples with progressive mineralization reaching the value comparable to that in endochondral bone. A progressive increase in mineralization was also reflected by increased mineral-to-matrix ratio determined by FT-IRIS. Determination of the mineral phases by FT-IRIS suggested progressive maturation of the mineral deposits from amorphous calcium phosphate to hydroxyapatite. These results provide critical information of the mechanisms of mineralization in PXE, with potential pharmacologic implications.     

In situ examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy.

At the tissue level it is well established that the rate of remodeling is related to the degree of mineralization. However, it is unknown how long it takes for an individual bone structural unit (BSU) to become fully mineralized during secondary mineralization. Using synchrotron Fourier transform infrared microspectroscopy (FTIRM) we examined the time required for newly formed bone matrix to reach a physiological mineralization limit. Twenty-six, four-month old female New Zealand white rabbits were administered up to four different fluorochrome labels at specific time points to evaluate the chemical composition of labeled osteons from the tibial diaphysis that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from 505 day old rabbits was used as a reference value for the physiological limit to which bone mineralizes. Using synchrotron FTIRM, area integrations were carried out on protein (Amide I: 1688-1623 cm(-1)), carbonate (v(2)CO(3)(2-): 905-825 cm(-1)), and phosphate (v(4)PO(4)(3-): 650-500 cm(-1)) IR bands. IR spectral data are presented as ratios of phosphate/protein (overall matrix mineralization) and carbonate/protein. The rate of mineralization of osteonal bone proceeded rapidly between day 1 and 18, reaching 67% of interstitial bone levels. This was followed by a slower, more progressive accumulation of mineral up to day 350. By 350 days the rate of increase plateaued. The ratio of carbonate/protein also increased rapidly during the first 18 days, reaching 73% of interstitial bone levels. The ratio of carbonate/protein plateaued by day 315, reaching levels not significantly different to interstitial bone levels. In conclusion, our data demonstrate that bone accumulates mineral rapidly during the first 18 days (primary mineralization), followed by a more gradual increase in the accumulation of mineral (secondary mineralization) which we found to be completed in 350 days.     

Material matters: a mechanostat-based perspective on bone development in osteogenesis imperfecta and hypophosphatemic rickets

This perspective paper presents a hypothesis that links abnormalities of bone material with densitometric findings in two congenital metabolic bone disorders, osteogenesis imperfecta type I (OI) and X-linked hypophosphatemic rickets (XLH). Analyses of iliac bone samples from OI patients have shown that material bone density is elevated and that the bone material is abnormally stiff in this disorder. Therefore, a given mechanical load on an OI bone will generate a smaller than normal deformation. This in turn should lead osteocytes, the putative mechanosensing cells, to systematically underestimate the prevailing mechanical forces. According to the mechanostat model, bone strength should then be adapted to the underestimated mechanical loads, which means that bone architecture and mass remain below requirements. Available densitometric studies are in accordance with this hypothesis. In XLH, a mild mineralization defect persists despite treatment. This mineralization defect should lead to soft bone material. In analogy to the above model for OI, mechanical loads should be overestimated, resulting in increased densitometric parameters of bone strength. Indeed, lumbar spine areal bone mineral density is usually elevated in such patients.     

The bone behind a low areal bone mineral density: peripheral quantitative computed tomographic analysis in a woman with osteogenesis imperfecta

Areal bone mineral density (BMD) is the most widely used densitometric parameter. However, this approach makes it difficult to understand the structural basis of bone diseases, because a large number of bone properties are integrated into a single number. This is exemplified in the present case of a 27-year-old woman with osteogenesis imperfecta type I. Peripheral quantitative computed tomographic analysis at the radial metaphysis and at the radial diaphysis revealed a decreased areal BMD at both sites (z score -3.9 and -3.4, respectively). Yet, the structural basis for this decrease was different for the two locations: At the distal radius areal BMD was decreased because volumetric BMD was very low, whereas bone size was above the mean of the reference range. At the proximal radius areal BMD was decreased, because bone size was very low but volumetric BMD was above average. Bone mineral content of the radial diaphysis was very low for forearm muscle size, a finding which is compatible with Frost's hypothesis that the mechanostat setpoint is increased in osteogenesis imperfecta.     

The effect of calcium and vitamin D supplementation on osteoporotic rabbit bones studied by vibrational spectroscopy

Fourier transform infrared spectroscopy is utilized to examine the effects of increased calcium, vitamin D, and combined calcium-vitamin D supplementation on osteoporotic rabbit bones with induced inflammation. The study includes different bone sites (femur, tibia, humerus, vertebral rib) in an effort to explore possible differences among the sites. We evaluate the following parameters: mineral-to-matrix ratio, carbonate content, and non-apatitic species (labile acid phosphate and labile carbonate) contribution to bone mineral. Results show that a relatively high dose of calcium or calcium with vitamin D supplementation increases the bone mineralization index significantly. On the other hand, vitamin D alone is not as effective in promoting mineralization even with high intake. Mature B-type apatite was detected for the group with calcium supplementation similar to that of aged bone. High vitamin D intake led to increased labile species concentration revealing bone formation. This is directly associated with the suppression of pro-inflammatory cytokines linked to induced inflammation. The latter is known to adversely alter bone metabolism, contributing to the aetiopathogenesis of osteoporosis. Thus, a high intake of vitamin D under inflammation-induced osteoporosis does not promote mineralization but suppresses bone resorption and restores metabolic balance.     

The effects of strontium on skeletal development in zebrafish embryo

The strontium is an alkaline earth metal found in nature as trace element. Chemically similar to calcium, it is known to be involved in the human bone mineral metabolism. The strontium ranelate has been approved in therapy as drug with both anti-resorption and anabolic effects on bone tissues. Since few data in vivo are available, we used Danio rerio as animal model to evaluate the effects of strontium on skeletal development. First, toxicity assay performed on zebrafish embryos estimated the LC50 around 6 mM. Since several zebrafish bones are formed from cartilage mineralization, we evaluated whether strontium affects cartilage development during embryogenesis. Strontium does not perturb the development of the cartilage tissues before the endochondral osteogenesis takes place. About the mineralization process, we evidentiated an increase of vertebral mineralization respect to controls at lower strontium concentrations whereas higher concentration inhibited mineral deposition in dose dependent fashion. Our results evidentiated, in addition, that the calcium/strontium rate but not the absolute level of strontium modulates the mineralization process during embryonic osteogenesis.Zebrafish represents an excellent animal model to study the role of micronutrients in the development of the tissues/organs because the ions are not absorbed by intestine but assumed by skin diffusion.     

Dental Phenotype in Jalili Syndrome Due to a c.1312 dupC Homozygous Mutation in the CNNM4 Gene

Jalili syndrome denotes a recessively inherited combination of an eye disease (cone-rod dystrophy) and a dental disorder (amelogenesis imperfecta), which is caused by mutations in the CNNM4 gene. Whereas the ophthalmic consequences of these mutations have been studied comprehensively, the dental phenotype has obtained less attention. A defective transport of magnesium ions by the photoreceptors of the retina is assumed to account for the progressive visual impairment. Since magnesium is also incorporated in the mineral of dental hard tissues, we hypothesized that magnesium concentrations in defective enamel resulting from mutations in CNNM4 would be abnormal, if a similar deficiency of magnesium transport also accounted for the amelogenesis imperfecta. Thus, a detailed analysis of the dental hard tissues was performed in two boys of Kosovan origin affected by Jalili syndrome. Retinal dystrophy of the patients was diagnosed by a comprehensive eye examination and full-field electroretinography. A mutational analysis revealed a c.1312 dupC homozygous mutation in CNNM4, a genetic defect which had already been identified in other Kosovan families and putatively results in loss-of-function of the protein. The evaluation of six primary teeth using light and scanning electron microscopy as well as energy-dispersive X-ray spectroscopy showed that dental enamel was thin and deficient in mineral, suggesting a hypoplastic/hypomineralized type of amelogenesis imperfecta. The reduced mineral density of enamel was accompanied by decreased amounts of calcium, but significantly elevated levels of magnesium. In dentin, however, a similar mineral deficiency was associated with reduced magnesium and normal calcium levels. It is concluded that the c.1312 dupC mutation of CNNM4 results in mineralization defects of both enamel and dentin, which are associated with significantly abnormal magnesium concentrations. Thus, we could not disprove the hypothesis that a disrupted magnesium transport is involved in the development of the dental abnormalities observed in Jalili syndrome.     

Scanning electron microscopy and calcification in amelogenesis imperfecta in anterior and posterior human teeth

Teeth fragments from members of a family clinically and genetically diagnosed as having amelogenesis imperfecta were studied by scanning electron microscopy and X-ray microprobe analysis to establish the morphological patterns and the quantitative concentration of calcium in the enamel of anterior (canine, incisor) and posterior (premolar and molar) teeth. The prism patterns in the enamel of teeth from both regions were parallel or irregularly decussate, with occasional filamentous prisms accompanied by small, irregularly rounded formations. Prismless enamel showed the R- and P-type patterns. Calcium levels in enamel of amelogenesis imperfecta and control teeth differed significantly between anterior and posterior teeth, indicating that the factors that influence normal mineralization in different regions of the dental arch are not altered in the process of amelogenesis imperfecta.     

Occurrence of osteoblast necroses during ossification of long bone cortices in mouse fetuses

Previous investigations concerned with in vitro osteogenesis and mineralization have revealed some indication of a participation of cell necroses in the course of calcification. These observations were confirmed by in vivo investigations on desmoid ossification in fetal mouse calvariae, where abundant necrotic osteoblasts were found at the mineralization border and in the osteoid. In the present study, ossification of long bone cortices from fetal mice was investigated by use of electron microscopy. Specimens obtained from the collection of the Institute of Anatomy, Free University of Berlin (mouse fetuses, forearm; rat fetuses, forearm) were reinvestigated for control purposes. In all cases, mineralization of osteoid was accompanied by cell necroses. Cell degeneration was characterized by swelling of the endoplasmic reticulum and loss of the plasma membrane resulting in freely distributed vesicular structures. Cell debris was incorporated within the mineral. Initially, cell necroses in the perichondrium occurred in the region surrounding the hypertrophic cartilage and the matrix of which showed spots of endochondral mineralization. Necrotic osteoblasts occurred simultaneously with mineralization of the osteoid. During further ossification of the long bone cortices, the number of necrotic cells increased markedly. In addition to necrotic cells, healthy osteoblasts, osteocytes and perichondral tissue were present, indicating that an artifact can be excluded. The importance of cell necroses in the process of mineralization is as yet unclear. Possibly, the cells act as calcium and/or phosphate stores, which are liberated by cell death to increase the amount of mineral constituents at sites of mineralization.     

A continuous culture biofilm model of cariogenic responses

AIMS: To validate an in vitro model for the analysis of physiological and ecological responses to sugar challenge in bacterial populations, and subsequent changes in enamel mineralization. METHODS AND RESULTS: A seven-organism bacterial consortium was grown in a biofilm mode on enamel and hydroxyapatite (HA) surfaces in a continuous culture system and exposed to repeated sucrose challenges. This produced 'pH-cycling' conditions within the system. Populations on HA surfaces were enumerated. Changes in relative proportions of the different populations, and in the total viable count, were observed, between different treatments. Microradiography of the enamel sections showed increasing demineralization with increasing sucrose concentration. The lesions formed were similar to 'white-spot' lesions found in vivo. Differences in the quality of biofilms formed were also observed using Confocal Laser Scanning Microscopy. CONCLUSION: An in vitro model has been validated for the analysis of both physiological and ecological responses to sucrose challenges in bacterial populations, and subsequent changes in enamel mineralization. SIGNIFICANCE AND IMPACT OF THE STUDY: This model should facilitate the study of changes in bacterial populations in response to application of putative anticaries agents and concomitant changes in enamel mineralization.     

Loss of PiT-2 results in abnormal bone development and decreased bone mineral density and length in mice

Normal bone mineralization requires phosphate oversaturation in bone matrix vesicles, as well as normal regulation of phosphate metabolism via the interplay among bone, intestine, and kidney. In turn, derangement of phosphate metabolism greatly affects bone function and structure. The type III sodium-dependent phosphate transporters, PiT-1 and PiT-2, are believed to be important in tissue phosphate metabolism and physiological bone formation, but their requirement and molecular roles in bone remain poorly investigated. In order to decipher the role of PiT-2 in bone, we examined normal bone development, growth, and mineralization in global PiT-2 homozygous knockout mice. PiT-2 deficiency resulted in reduced vertebral column, femur, and tibia length as well as mandibular dimensions. Micro-computed tomography analysis revealed that bone mineral density in the mandible, femur, and tibia were decreased, indicating that maintenance of bone function and structure is impaired in both craniofacial and long bones of PiT-2 deficient mice. Both cortical and trabecular thickness and mineral density were reduced in PiT-2 homozygous knockout mice compared with wild-type mice. These results suggest that PiT-2 is involved in normal bone development and growth and plays roles in cortical and trabecular bone metabolism feasibly by regulating local phosphate transport and mineralization processes in the bone. Further studies that evaluate bone cell-specific loss of PiT-2 are now warranted and may yield insight into complex mechanisms of bone development and growth, leading to identification of new therapeutic options for patients with bone diseases.     

Mutations in c4orf26, encoding a Peptide with in vitro hydroxyapatite crystal nucleation and growth activity, cause amelogenesis imperfecta

Autozygosity mapping and clonal sequencing of an Omani family identified mutations in the uncharacterized gene, C4orf26, as a cause of recessive hypomineralized amelogenesis imperfecta (AI), a disease in which the formation of tooth enamel fails. Screening of a panel of 57 autosomal-recessive AI-affected families identified eight further families with loss-of-function mutations in C4orf26. C4orf26 encodes a putative extracellular matrix acidic phosphoprotein expressed in the enamel organ. A mineral nucleation assay showed that the protein's phosphorylated C terminus has the capacity to promote nucleation of hydroxyapatite, suggesting a possible function in enamel mineralization during amelogenesis.     

Effects of fibronectin on hydroxyapatite formation.

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.     

Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study

Bone is the most widespread mineralized tissue in vertebrates and its formation is orchestrated by specialized cells - the osteoblasts. Crystalline carbonated hydroxyapatite, an inorganic calcium phosphate mineral, constitutes a substantial fraction of mature bone tissue. Yet key aspects of the mineral formation mechanism, transport pathways and deposition in the extracellular matrix remain unidentified. Using cryo-electron microscopy on native frozen-hydrated tissues we show that during mineralization of developing mouse calvaria and long bones, bone-lining cells concentrate membrane-bound mineral granules within intracellular vesicles. Elemental analysis and electron diffraction show that the intracellular mineral granules consist of disordered calcium phosphate, a highly metastable phase and a potential precursor of carbonated hydroxyapatite. The intracellular mineral contains considerably less calcium than expected for synthetic amorphous calcium phosphate, suggesting the presence of a cellular mechanism by which phosphate entities are first formed and thereafter gradually sequester calcium within the vesicles. We thus demonstrate that in vivo osteoblasts actively produce disordered mineral packets within intracellular vesicles for mineralization of the extracellular developing bone tissue. The use of a highly disordered precursor mineral phase that later crystallizes within an extracellular matrix is a strategy employed in the formation of fish fin bones and by various invertebrate phyla. This therefore appears to be a widespread strategy used by many animal phyla, including vertebrates.     

Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam(+/-) mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam(-/-) mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam(-/-) mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.     

Loss of biological control of enamel mineralization in amelogenin-phosphorylation-deficient mice

Amelogenin, the most abundant enamel matrix protein, plays several critical roles in enamel formation. Importantly, we previously found that the singular phosphorylation site at Ser16 in amelogenin plays an essential role in amelogenesis. Studies of genetically knock-in (KI) modified mice in which Ser16 in amelogenin is substituted with Ala that prevents amelogenin phosphorylation, and in vitro mineralization experiments, have shown that phosphorylated amelogenin transiently stabilizes amorphous calcium phosphate (ACP), the initial mineral phase in forming enamel. Furthermore, KI mice exhibit dramatic differences in the enamel structure compared with wild type (WT) mice, including thinner enamel lacking enamel rods and ectopic surface calcifications. Here, we now demonstrate that amelogenin phosphorylation also affects the organization and composition of mature enamel mineral. We compared WT, KI, and heterozygous (HET) enamel and found that in the WT elongated crystals are co-oriented within each rod, however, their c-axes are not aligned with the rods’ axes. In contrast, in rod-less KI enamel, crystalline c-axes are less co-oriented, with misorientation progressively increasing toward the enamel surface, which contains spherulites, with a morphology consistent with abiotic formation. Furthermore, we found significant differences in enamel hardness and carbonate content between the genotypes. ACP was also observed in the interrod of WT and HET enamel, and throughout aprismatic KI enamel. In conclusion, amelogenin phosphorylation plays crucial roles in controlling structural, crystallographic, mechanical, and compositional characteristics of dental enamel. Thus, loss of amelogenin phosphorylation leads to a reduction in the biological control over the enamel mineralization process.     

Evidence of in vivo osteoinduction in adult rat bone by adeno-Runx2 intra-femoral delivery

The bone marrow microenvironment provides a unique opportunity in vivo to assess the role of genes in bone remodeling. The objective of this study was to determine whether Runx2 expression is regulated by rhBMP-2 in vivo and to examine the effect of Runx2 overexpression on bone in vivo. In the in vivo calvaria model we used, rhBMP-2 induced Runx2 protein expression in periosteal cells while in vitro, adenovirus-mediated Runx2 overexpression induced mineralization in mesenchymal stem cells. A single injection of adeno-Runx2 directly into the bone marrow of the right femur in mature rats, and subsequent analysis after 3 weeks, showed a significant bone mineral density (BMD) increase ( approximately 15%) as compared to the controls. The whole-femur mean BMD of the active virus-injected group was 0.193 (g/cm(2)) while that of the control virus-injected group was 0.175 (g/cm(2)) (P < 0.05). In addition, a significant increase (36%) in trabecular BMD at the distal end of the femur was observed. These data demonstrate that directly delivering adeno-Runx2 into bone marrow of adult rats induces osteogenesis and illustrates potential advantages of such approaches over ex vivo gene therapy protocols involving marrow cell isolation, gene transduction, and subsequent in vivo transfer.     

Mineral content of calcified tissues in cystic fibrosis mice

Although abnormal hard tissue mineralization is a recognized complication of cystic fibrosis (CF), the pathogenesis leading from the defective cystic fibrosis transmembrane conductance regulator (CFTR) protein is poorly understood. We hypothesized that CFTR plays a direct role in the mineralization of bone and teeth and tested the hypothesis using CF mouse models [CFTR(−) mice]. In vivo measurements by dual-emission X-ray absorpitometry (DEXA) indicated that bone mineral density (BMD) was reduced in CF mice as compared to gender-matched littermates. However, no change was evident after correction of BMD for the covariant of body weight. The latter finding was confirmed in isolated femurs and nasal bones by standard dry-ashing and instrumental neutron activation analysis (INAA). INAA of the continuously growing hypsodont incisor teeth from CFTR(−) mice revealed reduced Ca and normal P in the enamel layer—a finding consistent with changes in the deciduous teeth of CF children. Interestingly, enamel fluoride was increased in the CFTR(−) incisors and may associate with abnormal enamel crystallite formation. The iron content of the incisor enamel was reduced, explaining the loss of yellow pigmentation in CFTR(−) incisors. In contrast to the incisors, the mineral content of the slow-growing brachydont molar teeth was not different between CFTR(−) and CFTR(+) mice. It was concluded that CFTR does not play a direct role in the mineralization of bones or brachydont teeth in mice. Functional CFTR is apparently required for normal mineralization of the hypsodont incisors. However, multiple changes in the mineral composition of the CF incisors suggest an indirect role for CFTR, perhaps by maintaining a normal salivary environment for continuous tooth eruption. Preliminary reports published in Pediatric Pulmonology, 14, 253A (1997) and 15, 253A (1998).     

Osteogenesis promoted by calcium phosphate N,N-dicarboxymethyl chitosan

The effects of N,N-dicarboxymethyl chitosan (DCMC) on the precipitation of insoluble calcium salts, namely phosphate, sulfate, oxalate, carbonate, bicarbonate and fluoride, and magnesium salts, namely phosphate and carbonate, were studied. Results indicated that the chelating ability of DCMC interfered effectively with the well-known physico-chemical behaviour of magnesium and calcium salts. Dicarboxymethyl chitosan formed self-sustaining gels upon mixing with calcium acetate, as a consequence of calcium chelation. DCMC mixed with calcium acetate and with disodium hydrogen phosphate in appropriate ratios (molar ratio Ca/DCMC close to 2.4) yielded a clear solution, from which, after dialysis and freeze-drying, an amorphous material was isolated containing an inorganic component about one half its weight. This compound was used for the treatment of bone lesions in experimental surgery and in dentistry. Bone tissue regeneration was promoted in sheep, leading to complete healing of otherwise non-healing surgical defects. Radiographic evidence of bone regeneration was observed in human patients undergoing apicectomies and avulsions. The DCMC–CaP chelate favoured osteogenesis while promoting bone mineralization.