Analysis of drug release models from biodegradable nanomodified chitosan based materials

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

Objects with complex shape and functions have always attracted attention and interest. The morphological diversity and complexity of naturally occurring forms and patterns have been a motivation for humans to copy and adopt ideas from Nature to achieve functional, aesthetic and social value. Biomimetics is addressed to the design and development of new synthetic materials using strategies adopted by living organisms to produce biological materials. In particular, biomineralized tissues are often sophisticate composite materials, in which the components and the interfaces between them have been defined and optimized, and that present unusual and optimal chemical-physical, morphological and mechanical properties. Moreover, biominerals are generally produced by easily traceable raw materials, in aqueous media and at room pressure and temperature, that is through cheap process and materials. Thus, it is not surprising that the idea to mimic those strategies proper of Nature has been employed in several areas of applied sciences, such as for the preparation of liquid crystals, ceramic thin films computer switches and many other advanced materials. On this basis, this PhD thesis is focused on the investigation of the interaction of biologically active ions and molecules with calcium phosphates with the aim to develop new materials for the substitution and repair of skeletal tissue, according to the following lines: I. Modified calcium phosphates. A relevant part of this PhD thesis has been addressed to study the interaction of Strontium with calcium phosphates. It was demonstrated that strontium ion can substitute for calcium into hydroxyapatite, causing appreciable structural and morphological modifications. The detailed structural analysis carried out on the nanocrystals at different strontium content provided new insight into its interaction with the structure of hydroxyapatite. At variance with the behaviour of Sr towards HA, it was found that this ion inhibits the synthesis of octacalcium phosphate. However, it can substitute for calcium in this structure up to 15 atom %, in agreement with the increase of the cell parameters observed on increasing ion concentration. A similar behaviour was found for Magnesium ion, whereas Manganese inhibits the synthesis of octacalcium phosphate and it promotes the precipitation of dicalcium phosphate dehydrate. It was also found that Strontium affects the kinetics of the reaction of hydrolysis of α-TCP. It inhibits the conversion from α-TCP to hydroxyapatite. However, the resulting apatitic phase contains significant amounts of Sr2+ suggesting that the addition of Sr2+ to the composition of α-TCP bone cements could be successfully exploited for its local delivery in bone defects. The hydrolysis of α-TCP has been investigated also in the presence of increasing amounts of gelatin: the results indicated that this biopolymer accelerates the hydrolysis reaction and promotes the conversion of α-TCP into OCP, suggesting that its addition in the composition of calcium phosphate cements can be employed to modulate the OCP/HA ratio, and as a consequence the solubility, of the set cement. II. Deposition of modified calcium phosphates on metallic substrates. Coating with a thin film of calcium phosphates is frequently applied on the surface of metallic implants in order to combine the high mechanical strength of the metal with the excellent bioactivity of the calcium phosphates surface layers. During this PhD thesis, thank to the collaboration with prof. I.N. Mihailescu, head of the Laser-Surface-Plasma Interactions Laboratory (National Institute for Lasers, Plasma and Radiation Physics – Laser Department, Bucharest) Pulsed Laser Deposition has been successfully applied to deposit thin films of Sr substituted HA on Titanium substrates. The synthesized coatings displayed a uniform Sr distribution, a granular surface and a good degree of crystallinity which slightly decreased on increasing Sr content. The results of in vitro tests carried out on osteoblast-like and osteoclast cells suggested that the presence of Sr in HA thin films can enhance the positive effect of HA coatings on osteointegration and bone regeneration, and prevent undesirable bone resorption. The possibility to introduce an active molecule in the implant site was explored using Matrix Assisted Pulsed Laser Evaporation to deposit hydroxyapatite nanocrystals at different content of alendronate, a bisphosphonate widely employed in the treatments of pathological diseases associated to bone loss. The coatings displayed a good degree of crystallinity, and the results of in vitro tests indicated that alendronate promotes proliferation and differentiation of osteoblasts even when incorporated into hydroxyapatite. III. Synthesis of drug carriers with a delayed release modulated by a calcium phosphate coating. A core-shell system for modulated drug delivery and release has been developed through optimization of the experimental conditions to cover gelatin microspheres with a uniform layer of calcium phosphate. The kinetics of the release from uncoated and coated microspheres was investigated using aspirin as a model drug. It was shown that the presence of the calcium phosphate shell delays the release of aspirin and allows to modulate its action.

Multiphasic calcium phosphate (Ca-P) has widely been explored for bone graft replacement. This study represents a simple method of developing osteoinductive scaffolds by direct printing of seashell resources. The process demonstrates a coagulation-assisted extrusion-based three-dimensional (3D) printing process for rapid fabrication of multiphasic calcium phosphate-incorporated 3D scaffolds. These scaffolds demonstrated an interconnected open porous architecture with improved compressive strength and higher surface area. Multiphasic calcium phosphate (Ca-P) and hydroxyapatite present in the multi-scalar naturally resourced scaffold displayed differential protein adsorption, thus facilitating cell adhesion, migration, and differentiation, resulting in enhanced deposition of the extracellular matrix. The microstructural and physicochemical attributes of the scaffolds also lead to enhanced stem cell differentiation as witnessed from gene and protein expression analysis. Furthermore, the histological study of subcutaneous implantation evidently portrays promising biocompatibility without foreign body reaction. Neo-tissue in-growth was manifested with abundant blood vessels, thus indicative of excellent vascularization. Notably, cartilaginous and proteoglycan-rich tissue deposition indicated ectopic bone formation via an endochondral ossification pathway. The hierarchical interconnected porous architectural tribology accompanied with multiphasic calcium phosphate composition manifests its successful implication in enhancing stem cell differentiation and promoting excellent tissue in-growth, thus making it a plausible alternative in bone tissue engineering applications.

Effects of polycaprolactone on alendronate drug release from Mg-doped hydroxyapatite coating on titanium

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.

Objective To test the hypothesis that the carboxymethyl chitosan (CMC) stabilized liquid precursors can induce biomimetic mineralization of collagen fibrils. Methods CMC-calcium phosphate solution turbidity assessment was used to determine the working concentration of CMC. Reconstituted 2-D typeⅠ collagen model and 3-D collagen membranes were treated with saturated calcium phosphate solution containing the above working concentration of CMC. Samples treated with traditional mineralization liquids (without CMC) were acted as control. The ultrastructural changes of the 2-D collagen were observed by transmission electron microscopy (TEM) and the mineral phase was determined by selected area electron diffraction (SAED) /energy dispersive X-ray analysis (EDXA) . The mineralization composition and degree of 3-D collagen film were analyzed by thermogravimetric (TG) and X-ray diffraction (XRD) . Results According to turbidity assessment of CMC, 200 μg/ml CMC was able to stabilizing calcium phosphate liquid for 7 days and selected for further experiments. Through TEM observation and SAED analysis, needle-like crystals along the long axis of the fibrils were observed in the intrafibrillar spaces of collagen fibrils on the 3rd day. SAED analysis showed the typical diffraction ring of hydroxyapatite. EDXA revealed that the Ca/P of minerals in the collagen fibrils was 1.56. As for 3-D collagen model, the mineral content of collagen membrane treated with the biomimetic mineralization liquid for 14 days was 18.39% via TG analysis and the mineral phase was confirmed to be hydroxyapatite according to XRD curves. Conclusions Carboxymethyl chitosan can induce the biomimetic mineralization of collagen fibrils through stabilizing of liquid precursor and synthesize biomimetic collagen-hydroxyapatite complex with relatively higher degree of mineralization. Key words: Carboxymethyl chitosan; Collagen; Biomimetic mineralization; Polymer-induced liquid precursor

Calcium orthophosphates: crystallization and dissolution.

Combined modeling and experimental approach for the development of dual-release polymer millirods

Evaluation of physicochemical, mechanical and biological properties of chitosan/carboxymethyl cellulose reinforced with multiphasic calcium phosphate whisker-like fibers for bone tissue engineering

Reinforcement of freeze-dried chitosan scaffolds with multiphasic calcium phosphate short fibers

In situ forming implants are an attractive choice for controlled drug release into a fixed location. Currently, rapidly solidifying solvent exchange systems suffer from a high initial burst, and sustained release behavior is tied to polymer precipitation and degradation rate. The present studies investigated addition of hydroxyapatite (HA) and drug-loaded poly(β-amino ester) (PBAE) microparticles to in situ forming poly(lactic-co-glycolic acid) (PLGA)-based systems to prolong release and reduce burst. PBAEs were synthesized, imbibed with simvastatin (osteogenic) or clodronate (anti-resorptive), and then ground into microparticles. Microparticles were mixed with or without HA into a PLGA solution, and the mixture was injected into buffer, leading to precipitation and creating solid scaffolds with embedded HA and PBAE microparticles. Simvastatin release was prolonged through 30 days, and burst release was reduced from 81 to 39% when loaded into PBAE microparticles. Clodronate burst was reduced from 49 to 32% after addition of HA filler, but release kinetics were unaffected after loading into PBAE microparticles. Scaffold dry mass remained unchanged through day 15, with a pronounced increase in degradation rate after day 30, while wet scaffolds experienced a mass increase through day 25 due to swelling. Porosity and pore size changed throughout degradation, likely due to a combination of swelling and degradation. The system offers improved release kinetics, multiple release profiles, and rapid solidification compared to traditional in situ forming implants.

Growth of calcium hydroxyapatite (Ca-HAp) on cholesterol and cholestanol crystals from a simulated body fluid: A possible insight into the pathological calcifications associated with atherosclerosis

In recent years, apatite-based materials have garnered significant interest, particularly for applications in tissue engineering. Apatite is most commonly employed as a coating for metallic implants, as a component in composite materials, and as scaffolds for bone and dental tissue regeneration. Among its various forms, hydroxyapatite (HAP) is the most widely used, owing to its natural occurrence in human and animal hard tissues. An emerging area of research involves the use of fluoride-substituted apatite, particularly fluorapatite (FAP), which can serve as a direct fluoride source at the implant site, potentially offering several biological and therapeutic advantages. However, substituting HAP with FAP may lead to unforeseen changes in material behavior due to the differing physicochemical properties of these two calcium phosphate phases. This study investigates the effects of replacing hydroxyapatite with fluorapatite in ceramic-polymer composite materials incorporating β-1,3-glucan as a bioactive polymeric binder. The β-1,3-glucan polysaccharide was selected for its proven biocompatibility, biodegradability, and ability to form stable hydrogels that promote cellular interactions. Nitrogen adsorption analysis revealed that FAP/glucan composites had a significantly lower specific surface area (0.5 m2/g) and total pore volume (0.002 cm3/g) compared to HAP/glucan composites (14.15 m2/g and 0.03 cm3/g, respectively), indicating enhanced ceramic-polymer interactions in fluoride-containing systems. Optical profilometry measurements showed statistically significant differences in profile parameters (e.g., Rp: 134 μm for HAP/glucan vs. 352 μm for FAP/glucan), although average roughness (Ra) remained similar (34.1 vs. 27.6 μm, respectively). Microscopic evaluation showed that FAP/glucan composites had smaller particle sizes (1 μm) than their HAP counterparts (2 μm), despite larger primary crystal sizes in FAP, as confirmed by TEM. XRD analysis indicated structural differences between the apatites, with FAP exhibiting a reduced unit cell volume (524.6 Å3) compared to HAP (528.2 Å3), due to substitution of hydroxyl groups with fluoride ions. Spectroscopic analyses (FTIR, Raman, 31P NMR) confirmed chemical shifts associated with fluorine incorporation and revealed distinct ceramic-polymer interfacial behaviors, including an upfield shift of PO43- bands (964 cm-1 in FAP vs. 961 cm-1 in HAP) and OH vibration shifts (3537 cm-1 in FAP vs. 3573 cm-1 in HAP). The glucan polymer showed different hydrogen bonding patterns when combined with FAP versus HAP, as evidenced by shifts in polymer-specific bands at 888 cm-1 and 1157 cm-1, demonstrating that fluoride substitution significantly influences ceramic-polymer interactions in these bioactive composite systems.

Biodegradable gelatin microparticles as delivery systems for the controlled release of bone morphogenetic protein-2

A new simplified calcifying solution to synthesize calcium phosphate coatings