Physicochemical Properties of Cellulose-Based Hydrogel for Biomedical Applications.

Hydrogels are three-dimensional network structures of hydrophilic polymers, which have the capacity to take up an enormous amount of fluid/water. Carboxymethyl cellulose (CMC) is a commercially available cellulose derivative that can be used for biomedical applications due to its biocompatibility. It has been used as a major component to fabricate hydrogels because of its superabsorbent nature. In this study, we developed carboxylic acid crosslinked carboxymethyl cellulose hydrogels for biomedical applications. The physicochemical, morphological, and thermal properties were analyzed to confirm the crosslinking of carboxymethyl cellulose. Fourier-transform infrared spectra confirmed the crosslinking of carboxymethyl cellulose with the presence of peaks due to an esterification reaction. The distinct peak at 1718 cm-1 in hydrogel samples is due to the carbonyl group vibrations of the ester bond from the crosslinking reaction. The total carboxyl content of the sample was measured with crosslinker immersion time. The swelling of crosslinked hydrogels showed an excellent swelling capacity for CG02 that is much higher than CG01 in water and PBS. Morphological analysis of the hydrogel showed it has a rough surface. The thermal degradation of hydrogel showed stability with respect to temperature. However, the mechanical analysis showed that CG01 has a higher compressive strength than CG01. The optimum swelling ratio and higher compressive strength of CG01 hydrogels could give them the ability to be used in load-bearing tissue regeneration. These results inferred that the carboxylic acid crosslinked CMC hydrogels could be a suitable matrix for biomedical or tissue-engineering applications with improved stability.

Gelatin renaturation and the interfacial role of fillers in bionanocomposites.

This work describes a systematic study of gelatin-sepiolite structural bionanocomposites to show how the renaturation level of the biopolymer is highly dependent on the type of mineral particle used. The aim of the work is to prove that chemical interactions between both components (hydrogen and covalent bonding) determine the organization level of the biopolymer which in turn results in drastic differences in the elastic properties of the prepared bionanocomposites. To assess this, several systematic modifications were introduced into the silicate structure and surface, generating four derivatives. Two derivatives prepared by thermal treatments, monohydrated sepiolite and protoenstatite, and two chemically modified sepiolites, amino and epoxy terminated, were prepared and used as the inorganic (or hybrid) phase in the bionanocomposites. The thermal and chemical modifications performed on the sepiolite surface induced a dramatic decrease in the renaturation level as determined by DSC and FTIR techniques. On the other hand, untreated sepiolite induced a higher renaturation level in the polypeptide, probably due to the alignment of the collagen-like triple helix along sepiolite external surface channels. The measured mechanical properties of the studied compositions confirm that the renaturation level of gelatin is a key factor in understanding the elastic properties of bionanocomposites. These results suggest that mineral particles introduced in the polypeptide matrix provide an effective control over the matrix crystallinity giving rise to tunable mechanical properties of the final bionanocomposite.

Characterization and In Vitro Evaluations of Injectable Calcium Phosphate Cement Doped with Magnesium and Strontium.

Injectable calcium phosphate cement is a promising biomaterial for hard tissue repair due to its osteoinductivity, biocompatibility properties, and its use to correct defect areas involving narrow cavities with limited accessibility by the minimally invasive technique. Microwave-synthesized hydroxyapatite (HA) was used for the preparation of cement. In recent years, both magnesium and strontium calcium phosphate cements have exhibited rapid setting, improved mechanical strength, and a good resorption rate. A big step still remains to develop injectable magnesium and strontium phosphate cements with ideal self-setting properties, adequate mechanical strength, and good biocompatibility for clinical applications. In this study, both magnesium and strontium were doped with synthesized semiamorphous and crystalline hydroxyapatite (HA). The powder mixture was mixed with Na2HPO4, NaH2PO4, and a carboxymethyl cellulose (CMC) solution to develop the novel magnesium and strontium calcium phosphate cement. The setting time, physiochemical properties of hardened cement, microstructure, mechanical strength, and injectability of the prepared cement were studied. The toxicity evaluation and cell adhesion, which are necessary to identify the suitability of the material for different applications, were quantified and investigated using fibroblast cells. The setting time of cement was reduced substantially for magnesium- or strontium-doped cement by 2 min. The phase composition of the hardened cement expresses the semiamorphous or crystalline phase of HA with additives. Smooth and complete injection of cement paste was observed in semiamorphous HA-based cement. The intercellular reactive oxygen stress (ROS) of the Sr2+-doped cement sample showed varied degrees of toxicity to cells in terms of different concentrations. The Mg2+-doped cement showed significant attachment of cells after treatment at varying incubation times.

In Vitro and In Vivo Evaluation of Carboxymethyl Cellulose Scaffolds for Bone Tissue Engineering Applications.

The present study involves the development of citric acid-cross-linked carboxymethyl cellulose (C3CA) scaffolds by a freeze-drying process. Scaffolds were fabricated at different freezing temperatures of -20, -40, or -80 °C to investigate the influence of scaffold pore size on bone regeneration. All three scaffolds were porous in structure, and the pore size was measured to be 74 ± 4, 55 ± 6, and 46 ± 5 µm for -20, -40, and -80 °C scaffolds. The pores were larger in scaffolds processed at -20 °C compared to -40 and -80 °C, indicating the reduction in pore size of the scaffolds with a decrease in freezing temperature. The cytocompatibility, cell proliferation, and differentiation in C3CA scaffolds were assessed with the Saos-2 osteoblast cell line. These scaffolds supported the proliferation and differentiation of Saos-2 cells with significant matrix mineralization in scaffolds processed at -40 °C. Subcutaneous implantation of C3CA scaffolds in the rat model was investigated for its ability of vascularization and new matrix tissue formation. The matrix formation was observed at the earliest of 14 days in the scaffolds when processed at -40 °C while it was observed only after 28 days of implantation with the scaffolds processed at -20 and -80 °C. These results suggest that the citric acid-cross-linked CMC scaffolds processed at -40 °C can be promising for bone tissue engineering application.

Characterization and Evaluation of Carboxymethyl Cellulose-Based Films for Healing of Full-Thickness Wounds in Normal and Diabetic Rats.

Artificial skin substitute made of polymeric films are of great demand in the field of skin tissue engineering. We report here the fabrication of carboxymethyl cellulose (CMC) and poly(ethylene glycol) (PEG) blend films by solution casting method for wound healing applications. The physicochemical characteristics and the thermal stability of the films were analyzed. The surface morphology shows crystalline structures with large hexagonal-like platelet crystals of CMC on the surface of the films. Pure CMC films exhibited higher tensile strength than the CMC/PEG blend films. The swelling ratio (SR) of the films was influenced by the pH of Tris-HCL buffer (2.0, 5.0, and 7.0), which increased with increase in pH. The hemocompatibility assay and cytotoxicity test using NIH 3T3 fibroblast cells showed that the films were biocompatible. To evaluate the wound healing efficacy, the films were applied in full-thickness wounds created in normal and diabetic Wistar albino rats. The wounds healed faster with pure CMC film compared to blend films in both normal and diabetic rats, evidenced by intensive collagen formation in histopathological analysis. Thus, the films have potential application in skin regeneration, thereby to restore the structural and functional characteristics of the skin.

Self-assembly of amorphous calcium carbonate microlens arrays.

Biological materials are often based on simple constituents and grown by the principle of self-assembly under ambient conditions. In particular, biomineralization approaches exploit efficient pathways of inorganic material synthesis. There is still a large gap between the complexity of natural systems and the practical utilization of bioinspired formation mechanisms. Here we describe a simple self-assembly route leading to a CaCO(3) microlens array, somewhat reminiscent of the brittlestars' microlenses, with uniform size and focal length, by using a minimum number of components and equipment at ambient conditions. The formation mechanism of the amorphous CaCO(3) microlens arrays was elucidated by confocal Raman spectroscopic imaging to be a two-step growth process mediated by the organic surfactant. CaCO(3) microlens arrays are easy to fabricate, biocompatible and functional in amorphous or more stable crystalline forms. This shows that advanced optical materials can be generated by a simple mineral precipitation.

Improving the osteointegration and bone-implant interface by incorporation of bioactive particles in sol-gel coatings of stainless steel implants.

In this study, we report a hybrid organic-inorganic TEOS-MTES (tetraethylorthosilicate-methyltriethoxysilane) sol-gel-made coating as a potential solution to improve the in vivo performance of AISI 316L stainless steel, which is used as permanent bone implant material. These coatings act as barriers for ion migration, promoting the bioactivity of the implant surface. The addition of SiO(2) colloidal particles to the TEOS-MTES sol (10 or 30 mol.%) leads to thicker films and also acts as a film reinforcement. Also, the addition of bioactive glass-ceramic particles is considered responsible for enhancing osseointegration. In vitro assays for bioactivity in simulated body fluid showed the presence of crystalline hydroxyapatite (HA) crystals on the surface of the double coating with 10mol.% SiO(2) samples on stainless steel after 30 days of immersion. The HA crystal lattice parameters are slightly different from stoichiometric HA. In vivo implantation experiments were carried out in a rat model to observe the osteointegration of the coated implants. The coatings promote the development of newly formed bone in the periphery of the implant, in both the remodellation zone and the marrow zone. The quality of the newly formed bone was assessed for mechanical and structural integrity by nanoindentation and small-angle X-ray scattering experiments. The different amount of colloidal silica present in the inner layer of the coating slightly affects the material quality of the newly formed bone but the nanoindentation results reveal that the lower amount of silica in the coating leads to mechanical properties similar to cortical bone.

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep.

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small-angle X-ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Reinforcing of a calcium phosphate cement with hydroxyapatite crystals of various morphologies.

A series of biocomposite materials was successfully prepared by reinforcing advanced calcium phosphate cement with hydroxyapatite fibrous and elongated plate-like particles. Powder X-ray diffraction showed that ball-milled biocomposite precursors (dicalcium and tetracalcium phosphates) entirely transform to a single phase hydroxyapatite end product within 7 h at 37 °C. Electron microscopy showed that the resultant biocomposites are constituted of nanoscaled cement particles intimately associated with the reinforcement crystals. The influence of shape, size, and concentration of the hydroxyapatite filler on the compression strength of reinforced cements is discussed. The best compression strength of 37 ± 3 MPa (enhancement of ∼50% compared to pure cement) was achieved using submicrometer-sized hydroxyapatite crystals with complementary shapes. Nanoindentation revealed that averaged elastic modulus and hardness values of the cements are consistent with those reported for trabecular and cortical human bones, indicating a good match of the micromechanical properties for their potential use for bone repair. The stiffness of the biocomposites was confirmed to gradate-compliant cement matrix, cement-filler interface, and stiff filler-as a result of the structuring at the nanometer-micrometer level. This architecture is critical in conditioning the final mechanical properties of the functional composite biomaterial. In vitro cell culture experiments showed that the developed biomaterial system is noncytotoxic.

Two stages in three-dimensional in vitro growth of tissue generated by osteoblastlike cells.

Bone regeneration is controlled by a variety of biochemical, biomechanical, cellular, and hormonal mechanisms. In particular, physical properties of the substrate such as stiffness and architecture highly influence the proliferation and differentiation of cells. The aim of this work is to understand the influence of scaffold stiffness and cell seeding densities on the formation of tissue by osteoblast cells within polyether urethane scaffolds containing pores of different sizes. MC3T3-E1 preosteoblast cells were seeded on the scaffold, and the amount of tissue formed within the pores was analyzed for culture times up to 49 days by phase contrast microscopy. The authors show that the kinetics of three-dimensional tissue growth in these scaffolds follows two stages and can be described by a universal growth law. The first stage is dominated by cell-material interactions with cell adherence and differentiation being strongly dependent on the polymer material. After a delay time of a few weeks, cells begin to grow within their own matrix, the delay being strongly dependent on substrate stiffness and seeding protocols. In this later stage of growth, three-dimensional tissue amplification is controlled rather by the pore geometry than the scaffold material properties. This emphasizes how geometric constraints may guide tissue formation in vitro and shows that optimizing scaffold architectures may improve tissue formation independent of the scaffold material used.

Bone material quality in transiliac bone biopsies of postmenopausal osteoporotic women after 3 years of strontium ranelate treatment.

Strontium ranelate (SrR) is a relatively new treatment for osteoporosis. In this study we investigated its potential impact on human bone material quality in transiliac bone biopsies from postmenopausal osteoporotic women treated 3 years with calcium and vitamin D plus either 2 g SrR per day or placebo. Bone mineralization density distribution (BMDD), strontium (Sr) concentration, collagen cross-link ratio, and indentation modulus were analyzed by quantitative backscattered electron imaging, electron-induced X-ray fluorescence analysis, synchrotron radiation induced micro X-ray fluorescence elemental mapping, Fourier transform infrared imaging, and nanoindentation, respectively. The BMDD of SrR-treated patients was shifted to higher atomic numbers (Z(mean) +1.5%, p < .05 versus placebo). We observed Sr being preferentially incorporated in bone packets formed during SrR treatment up to 6% atom fraction [Sr/(Sr + Ca)] depending on the SrR serum levels of the individuals (correlation r = 0.84, p = .018). Collagen cross-link ratio was preserved in SR-treated bone. The indentation modulus was significantly decreased in younger versus older bone packets for both placebo- (-20.5%, p < .0001) and SrR-treated individuals (-24.3%, p < .001), whereas no differences were found between the treatment groups. In conclusion, our findings indicate that after SrR treatment, Sr is heterogeneously distributed in bone and preferentially present in bone packets formed during treatment. The effect of SrR on BMDD seems to be due mainly to the uptake of Sr and not to changes in bone calcium content. Taken together, these data provide evidence that the investigated bone quality determinants at tissue level were preserved in postmenopausal osteoporotic women after 3-year treatment with 2 g SrR per day plus calcium and vitamin D.

Multiple roles for neurofibromin in skeletal development and growth.

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder primarily characterized by the formation of neurofibromas, café-au-lait spots and freckling. Skeletal abnormalities such as short stature or bowing/pseudarthrosis of the tibia are relatively common. To investigate the role of the neurofibromin in skeletal development, we crossed Nf1flox mice with Prx1Cre mice to inactivate Nf1 in undifferentiated mesenchymal cells of the developing limbs. Similar to NF1 affected individuals, Nf1(Prx1) mice show bowing of the tibia and diminished growth. Tibial bowing is caused by decreased stability of the cortical bone due to a high degree of porosity, decreased stiffness and reduction in the mineral content as well as hyperosteoidosis. Accordingly, osteoblasts show an increase in proliferation and a decreased ability to differentiate and mineralize in vitro. The reduction in growth is due to lower proliferation rates and a differentiation defect of chondrocytes. Abnormal vascularization of skeletal tissues is likely to contribute to this pathology as it exerts a negative effect on cortical bone stability. Furthermore, Nf1 has an important role in the development of joints, as shown by fusion of the hip joints and other joint abnormalities, which are not observed in neurofibromatosis type I. Thus, neurofibromin has multiple essential roles in skeletal development and growth.