Physicochemical aspects of structuring calcium phosphate on fibrillary collagen scaffolds

Thermal characterization of hydroxyapatite or carbonated hydroxyapatite hybrid composites with distinguished collagens for bone graft

Synthesis of carbonated calcium phosphate ceramics using microwave irradiation

The influence of carbonate substitution (4.4 wt%, mixed A/B type) in hydroxyapatite ceramics for bone remodeling scaffolds was investigated by separately analyzing the response of pre-osteoblasts and osteoclast-like cells. Carbonated hydroxyapatite (CHA) (Ca9.5(PO4)5.5(CO3)0.5(OH)(CO3)0.25-CHA), mimicking the chemical composition of natural bone mineral, and pure hydroxyapatite (HA) (Ca10(PO4)6(OH)2-HA) porous ceramics were processed to obtain a similar microstructure and surface physico-chemical properties (grain size, porosity ratio and pore size, surface roughness and zeta potential). The biological behavior was studied using MC3T3-E1 pre-osteoblastic and RAW 264.7 monocyte/macrophage cell lines. Chemical dissolution in the culture media and resorption lacunae produced by osteoclasts occur with both HA and CHA ceramics, but CHA exhibits much higher dissolution and greater bioresorption ability. CHA ceramics promoted a significantly higher level of pre-osteoblast proliferation. Osteoblastic differentiation, assessed by qRT-PCR of RUNX2 and COLIA2, and pre-osteoclastic proliferation and differentiation were not significantly different on CHA or HA ceramics but cell viability and metabolism were significantly greater on CHA ceramics. Thus, the activity of both osteoclast-like and osteoblastic cells was influenced by the carbonate substitution in the apatite structure. Furthermore, CHA showed a particularly interesting balance between biodegradation, by osteoclasts and chemical dissolution, and osteogenesis through osteoblasts’ activity, to stimulate bone regeneration. It is hypothesized that this amount of 4.4 wt% carbonate substitution leads to an adapted concentration of calcium in the fluid surrounding the ceramic to stimulate the activity of cells. These results highlight the superior biological behavior of microporous 4.4 wt% A/B CHA ceramics that could beneficially replace the commonly used HA of biphasic calcium phosphates for future applications in bone tissue engineering.

The structure and physicochemical properties of scaffolds obtained from collagen gel using connective tissue sheaths of paravertebral tendons of Wistar rats were studied. The scaffolds were obtained at 37 °C (filmy) and 6 °C (volumetric). During hardening, the scaffolds form globular and extraglomerular fractions, which is typical for collagen gels obtained from tendon membranes. The ratio of the fraction volumes is determined by the pore structure and kinking of collagen fibrils. In the filmy scaffold, closed-type pores with weakened kinking are formed, which leads to the dominance of the extraglomerular scaffold. In the volumetric scaffold, kinking is intensified, open-type pores are formed, which determines the dominance of the globular scaffold. The morphogenetic factors of dominant fraction formation are ordering and increased rigidity, while the subdominant fractions are chaotization and elasticization of collagen frameworks. Fibrillar collagen undergoes extra- and intrafibrillar mineralization in situ with structuring of calcium phosphates along the apatite direction. The micromechanical properties of scaffolds induce extrafibrillar synthesis and determine the direction of apatitogenesis: stoichiometric hydroxyapatite is synthesized on rigid matrices, while carbonate-hydroxyapatites are synthesized on loose ones. Intrafibrillar synthesis in combination with temperature determines the degree of crystallinity and the composition of cationic and anionic sublattices of hydroxyapatites. On matrices of fibrillar collagen with strengthened bonds of peptide and carbonate groups, stoichiometric hydroxyapatite is formed, the degree of crystallinity of which is moderated by temperature − the higher the synthesis temperature, the higher the degree of crystallinity and saturation with calcium would be. On matrices with weakened peptide and C−O bonds, carbonate-hydroxyapatites are formed, in which substitutions in the anionic sublattice are regulated by temperature: at elevated temperatures, CO32– groups predominantly replace OH–, and at lower temperatures, PO43– groups.

Carbonated hydroxyapatite (CHA), with a chemical composition close to the mineral found in human bone, represented higher solubility than stoichiometric hydroxyapatite (HA). Therefore, the B-type CHA is commonly used for bone tissue engineering. This study fabricated B-type CHA using Indonesian eggshells from chicken, organic chicken, and duck because of the high calcium carbonate (CaCO3) content (94%). A co-precipitation method was used for synthesizing CHA. The physicochemical properties of the CHA were characterized using Scanning Electron Microscopy-Energy Dispersive X-Ray Spectroscopy (SEM-EDS), X-Ray Diffractometer (XRD), and Fourier Transform Infrared Spectroscopy (FTIR). Based on FTIR results for CHA, the stretching functional groups of B-type CO3 were detected at 1452-1453 cm-1, 1417-1418 cm-1, and 873-874 cm-1, which indicated the formation of B-type CHA. Meanwhile, CHA from organic chicken eggshells had low crystalline properties and the best morphology due to a more homogeneous and uniform agglomeration. More specifically, CHA based on organic chicken eggshells has a Ca/P molar ratio following natural human bone, which is 1.71. Therefore, all B-type CHA samples are candidates in bioceramic materials for bone tissue engineering applications.

Biomimetic apatite coatings—Carbonate substitution and preferred growth orientation

Synthesis of B-type carbonated hydroxyapatite by a new dissolution-precipitation method

The study is devoted to the preparation of antimicrobial Ag-containing bioactive calcium phosphate ceramics based on carbonated hydroxyapatite and to the determination of the dependence of its phase composition and microstructure on the synthesis temperature. Composite ceramics was obtained by sintering powders of carbonated hydroxyapatite (CHA), synthesized as a result of a reaction between calcium carbonate and phosphoric acid, with the addition of silver nitrate. Ceramics were sintered at 900 and 1000 °C temperatures, i.e., temperatures below and above the melting point of silver. X-ray analysis, electron microscopy, and infrared spectroscopy showed that synthesis at a 900 °C temperature (below the melting point of metallic silver) produces a two-phase composite based on CHA with inclusions of silver nanoparticles smaller than 50 nm in size. From X-ray analysis, with an increase in silver concentration, the lattice constant a remains practically unchanged, while the constant c ‒ increases. This behavior, due to the significant difference in the ionic radii of calcium and silver (Ca2+ ‒ 0.99 Å, Ag+ ‒ 1.28 Å), usually leads to the preferential substitution of Ca(1) sites in the CHA and a linear increasing in the lattice parameters of the CHA with the Ag concentration. That is, even at relatively low temperatures, as a result of the solid-phase reaction in CHA, partial replacement of calcium ions by silver ions occurs and Ag-substituted ceramics are formed. At temperatures above 1000 °C, a single-phase silver-substituted product is synthesized where part of the Ca2+ ions is replaced by Ag+ ions. At the same time, the lattice constant c continues to increase, and in the electron microscopic images only the apatite grain structure is visible without any inclusions. Sintering of composite ceramics at a temperature when silver is in the liquid phase and more easily dissociates into ions compared to the solid phase, results in a single-phase silver-substituted ceramic.

Production of ultra-fine bioresorbable carbonated hydroxyapatite

Mechanochemical-hydrothermal synthesis of calcium phosphate powders with coupled magnesium and carbonate substitution

Carbonated hydroxyapatite (CHA) was synthesized by solid-ion-exchange reaction of hydroxyapatite (HA) powders in CO2 atmosphere. The effects of the treatment atmosphere, temperature and time on carbonate substitution were investigated. The phase composition of the powders were characterized by X-ray diffraction and the content and type of the carbonate substitution were studied using Carbon-sulfur elemental analysis and Fourier transform infrared spectroscopy, respectively. The results show that the carbonate ion can enter the HA crystal lattice to form CHA giving priority to A-type by controlling the treatment temperature, atmosphere and time. Wet atmosphere treatment favors the reconstitution of hydroxyl and treatment in dry CO2 atmosphere favors the formation of A-Type substitution. The appropriate reaction temperature is 900 . Carbonate content increases with the treatment time and a further extension of treatment time tends to a slower increase of carbonate content.

Background: Collagen has attracted great interest as a biomaterial for various dental and medical uses. Objective: Investigate the characteristics and biocompatibility of type I collagen extracted from rat-tail tendon and bovine Achilles tendon for dental application. Materials and methods: Type-I collagen was extracted from rat-tail and bovine Achilles tendon using pepsin. The purity of collagen extracts was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The biocompatibility with human gingival fibroblasts (HGFs) and human oral keratinocytes (HOKs) was examined using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Scanning electron microscope (SEM) illustrations of purified collagen alone and collagen with HGFs and HOKs were presented. A three-dimensional wound-healing model of fibroblast populated collagen lattice (FPCL) was used to determine the capability of both sources of collagen to induce wound healing in vitro. Cellular collagen lattices were fabricated to examine the contraction rate of these collagens. Results: The average yield of collagen extracted from rat-tail and bovine Achilles tendon were 21.8±14.9% and 5.4±0.4%, respectively. The SDS-PAGE analysis showed that the extracts were composed of alpha 1, alpha 2 and beta chains with little contamination of other small proteins. The MTT assay showed good proliferation of cells cultured with each collagen extract, indicating that collagen extracts were non-toxic to the cells. SEM and the FPCL analysis showed that both types of collagen were biocompatible with both HGFs and HOKs, inducing good contraction in the in vitro model. Conclusion: Type-I collagen extracted from rat-tail and bovine Achilles tendon appeared to be biocompatible with HGFs and HOKs. Both biomaterials may be of use in dental practice.

Background and objectives: Calcium phosphates have been widely used as bone substitutes, but their properties are limited to osteoconduction. The association of calcium phosphates with osteoinductive bioactive molecules has been used as a strategy in regenerative medicine. Melatonin has been studied due to its cell protection and antioxidant functions, reducing osteoclastic activity and stimulating newly formed bone. This study aimed to evaluate the effect of topical application of melatonin associated with nanostructured carbonated hydroxyapatite microspheres in the alveolar bone repair of Wistar rats through histological and histomorphometric analysis. Materials and Methods: Thirty female Wistar rats (300 g) were used, divided randomly into three experimental groups (n = 10), G1: nanostructured carbonated hydroxyapatite microspheres associated with melatonin gel (CHA-M); G2: nanostructured carbonated hydroxyapatite (CHA); G3: blood clot (without alveolar filling). The animals were euthanized after 7 and 42 days of the postoperative period and processed for histological and histomorphometric evaluation. Kruskal-Wallis and Dunn's post-test were applied to investigate statistical differences between the groups at the same time point for new bone and connective tissue variables. Mann-Whitney was used to assess statistical differences between different time points and in the biomaterial variable. Results: Results showed a greater volume of residual biomaterial in the CHA-M than the CHA group (p = 0.007), and there were no significant differences in terms of newly formed bone and connective tissue between CHA and CHA-M after 42 days. Conclusions: This study concluded that both biomaterials improved alveolar bone repair from 7 to 42 days after surgery, and the association of CHA with melatonin gel reduced the biomaterial's biodegradation at the implanted site but did not improve the alveolar bone repair.

Calcium phosphate ceramics have been widely considered as scaffolds for bone tissue engineering. Selection of the best support for cultured cells, crucial for tissue engineered systems, is still required. We examined three types of calcium phosphate compounds: α-tricalcium phosphate - the most soluble one, carbonate hydroxyapatite - chemically the most similar to the bone mineral and biphasic calcium phosphate - with the best in vivo biocompatibility in order to select the best support for osteoblastic cells for tissue engineered systems. Human osteoblasts were tested in direct contact with both dense samples and 3D scaffolds in either static or dynamic culture. Cell viability, cell spreading, osteogenic cell capacity, and extracellular matrix production were examined. The obtained data indicate that biphasic calcium phosphate is the optimal cell-supporting material. In addition, dynamic culture improved cell distribution in the scaffolds, enhanced production of the extracellular matrix and promoted cells osteogenic capacity. Biphasic calcium phosphate should be recommended as the most suitable matrix for osteogenic cells expansion and differentiation in tissue engineered systems.

Effect of employing ultrasonic waves during pulse electrochemical deposition on the characteristics and biocompatibility of calcium phosphate coatings