Gelatin nanofibrous membrane fabricated by electrospinning of aqueous gelatin solution for guided tissue regeneration

The electrospinning of gelatin aqueous solution was successfully carried out by elevating the spinning temperature. The effects of spinning temperature and solution concentration were investigated on the morphology of gelatin nanofibers in the current study. To improve the stability and mechanical properties in moist state, the gelatin nanofibrous membrane was chemically crosslinked by 1-ethyl-3-dimethyl-aminopropyl carbodiimide hydrochloride and N-hydroxyl succinimide. The concentration of crosslinker was optimized by measuring the swelling degree and weight loss. Nanofibrous structure of the membrane was retained after lyophilization, although the fibers were curled and conglutinated. Tensile test revealed that the hydrated membrane becomes pliable and provides predetermined mechanical properties. Periodontal ligament cells cultured on the membrane in vitro exhibited good cell attachment, growth, and proliferation. Gelatin nanofibrous membrane can be one of promising biomaterials for the regeneration of damaged periodontal tissues.     

Production of microspheres based on hydrophobically associating alginate derivatives by dispersion/gelation in aqueous sodium chloride solutions

A new "all aqueous" procedure for the preparation of stable polysaccharide microparticles was developed. The method consists of dispersing a water solution of an amphiphilic alginate derivative (in the current work, alginate substituted with low amounts of dodecyl chains) first fluidified under mechanical stress, into an NaCl solution. The procedure exploits the ability of amphiphilic associative derivatives to form strong hydrogels in the presence of nonchaotropic salts and their shear-thinning/thixotropic properties. Depending on the experimental conditions, the size of the microparticles can be varied from 10 microm to several hundred micrometers. Their mechanical properties can eventually be reinforced by addition of low concentrations of calcium chloride. The resulting microparticles exhibit a better stability than that of plain Ca(2+)-alginate particles, as they are not disrupted when nongelling cations or calcium-sequestering agents are added to the solution. In addition, the particles can be easily redispersed after being centrifuged or freeze-dried.     

Haversian remodeling in guided bone regeneration with calcium alginate film in circular bone defect model of rabbit

The objective of this study is to investigate the effect of bioabsorbable Calcium alginate film in guided bone regeneration by the study of Haversian remodeling. Circular bone defects of 5 mm diameter were created in the corners of mandibles in 35 rabbits. The defects were covered with calcium alginate film (CAF) served as the experimental group, or collagen membrane (CM) as the control group, respectively. Healing condition was analyzed with gross, histological and immunohistochemical studies after 1, 2, 4, 6 and 8 weeks. The experimental group appeared more and earlier Haversian remodeling and osteoinductive factors leading to better bone regeneration. The control group showed more macrophages, less and later Haversian remodeling, absorbed slowly, while collected fewer osteoinductive factors in the early stage. Calcium alginate film, which is a relatively cheaper material, provides better effect than the collagen membrane in bone regeneration, Haversian remodeling and quantity of osteoinductive factors.     

Enhanced osteogenesis and angiogenesis by PCL/chitosan/Sr-doped calcium phosphate electrospun nanocomposite membrane for guided bone regeneration

Abstract

Membranes play pivotal role in guided bone regeneration (GBR) technique for reconstruction alveolar bone. GBR membrane that is able to stimulate both osteogenic and angiogenic differentiation of cells may be more effective in clinic practice. Herein, we fabricated the Sr-doped calcium phosphate/polycaprolactone/chitosan (Sr-CaP/PCL/CS) nanohybrid fibrous membrane by incorporating 20?wt% bioactive Sr-CaP nanoparticles into PCL/CS matrix via one-step electrospinning method, in order to endow the membrane with stimulation of osteogenesis and angiogenesis. The physicochemical properties, mechanical properties, Sr²⁺ release behavior, and the membrane stimulate bone mesenchymal stem cell (BMSCs) differentiation were evaluated in comparison with PCL/CS and CaP/PCL/CS membranes. The SEM images revealed that the nanocomposite membrane mimicked the extracellular matrix structure. The release curve presented a 28-day long continuous release of Sr²⁺ and concentration which was certified in an optimal range for positive biological effects at each timepoint. The in vitro cell culture experiments certified that the Sr-CaP/PCL/CS membrane enjoyed excellent biocompatibility and remarkably promoted rat bone mesenchymal stem cell (BMSCs) adhesion and proliferation. In terms of osteogenic differentiation, BMSCs seeded on the Sr-CaP/PCL/CS membrane showed a higher ALP activity level and a better matrix mineralization. What’s more, the synergism of the Sr²⁺ and CaP from the Sr-CaP/PCL/CS membrane enhanced BMSCs angiogenic differentiation, herein resulting in the largest VEGF secretion amount. Consequently, the Sr-CaP/PCL/CS nanohybrid electrospun membrane has promising applications in GBR.     

Development of a new calcium sulphate-based composite using alginate and chemically modified chitosan for bone regeneration

In this work we developed a novel calcium sulphate-based composite in which the hemihydrate calcium sulphate (CHS) can be encapsulated in a polymeric biodegradable and biocompatible matrix, in order to retain the structural integrity and decrease the bioresorption rate in bone regeneration applications. Two polymers were employed to realize this system: chitosan (Ch) and sodium alginate (Alg), both already widely used in biotechnological and biomedical applications. Chitosan was modified in order to obtain a water soluble polymer, the N-succinylchitosan (sCh). The reaction was performed with succinic anhydride in presence of pyridine and confirmed by FT-IR and NMR analyses. Finely ground Alg and sCh powders were mixed in different compositions with CHS and by adding water to the powder mixture it was obtained a mouldable paste that sets in few hours. Thermogravimetric analyses coupled with solvent extraction performed on the composite proved the alginate crosslinking in the presence of CHS. Mechanical studies carried out on composites of different compositions demonstrated that the blend of the two polymeric components causes a substantial synergistic reinforcement of composites. The presence of carboxylic groups on sCh chain in addition to those of alginate could enhance the chelating power of polysaccharide mixture. The results obtained with morphological analyses (SEM) further confirmed the hypotesis of the synergistic effect between alginate and N-succinylchitosan in presence of calcium sulphate. In vitro cytotoxicity tests proved that the developed system was not cytotoxic.     

Direct deposited porous scaffolds of calcium phosphate cement with alginate for drug delivery and bone tissue engineering

This study reports the preparation of novel porous scaffolds of calcium phosphate cement (CPC) combined with alginate, and their potential usefulness as a three-dimensional (3-D) matrix for drug delivery and tissue engineering of bone. An α-tricalcium phosphate-based powder was mixed with sodium alginate solution and then directly injected into a fibrous structure in a Ca-containing bath. A rapid hardening reaction of the alginate with Ca(2+) helps to shape the composite into a fibrous form with diameters of hundreds of micrometers, and subsequent pressing in a mold allows the formation of 3-D porous scaffolds with different porosity levels. After transformation of the CPC into a calcium-deficient hydroxyapatite phase in simulated biological fluid the scaffold was shown to retain its mechanical stability. During the process biological proteins, such as bovine serum albumin and lysozyme, used as model proteins, were observed to be effectively loaded onto and released from the scaffolds for up to more than a month, proving the efficacy of the scaffolds as a drug delivering matrix. Mesenchymal stem cells (MSCs) were isolated from rat bone marrow and then cultured on the CPC-alginate porous scaffolds to investigate the ability to support proliferation of cells and their subsequent differentiation along the osteogenic lineage. It was shown that MSCs increasingly actively populated and also permeated into the porous network with time of culture. In particular, cells cultured within a scaffold with a relatively high porosity level showed favorable proliferation and osteogenic differentiation. An in vivo pilot study of the CPC-alginate porous scaffolds after implantation into the rat calvarium for 6 weeks revealed the formation of new bone tissue within the scaffold, closing the defect almost completely. Based on these results, the newly developed CPC-alginate porous scaffolds could be potentially useful as a 3-D matrix for drug delivery and tissue engineering of bone.     

Compressive and shear properties of alginate gel: effects of sodium ions and alginate concentration.

The equilibrium and viscoelastic properties of alginate gel crosslinked with Ca2+ were determined as a function of alginate concentration and duration of exposure to physiologic concentrations of NaCl. Compressive and shear stress relaxation tests and oscillatory shear tests were performed to measure the material properties at two time periods after storage in NaCl compared to no NaCl exposure. The effect of concentration was determined by testing 1-3% alginate gel in a bath of physiological NaCl and CaCl2. After 15 h of exposure to NaCl, the compressive, equilibrium shear, and dynamic shear moduli decreased by 63, 84, and 90% of control values, respectively. The material properties exhibited no further changes after 7 days of exposure to NaCl. The loss angle and amplitude of the relaxation function in the shear also decreased, indicating less viscous behaviors in both dynamic and transient configurations. All moduli, but not the loss angle, significantly increased with increasing alginate concentration. The observed decrease in compressive and shear stiffness for alginate gel after exposure to Na+ was significant and indicated that physiological conditions will soften the gel over a time period of up to 7 days after gelation. The alginate gel retains significant solid-like behaviors, however, as measured by a loss angle of approximately 3 degrees. This study provides the first available data for material properties of alginate gel tested in physiological saline.     

In vitro and in vivo evaluation of alginate/sol-gel synthesized aminopropyl-silicate/alginate membrane for bioartificial pancreas

Alginate/aminopropyl-silicate/alginate (Alg/AS/Alg) membrane was prepared on Ca-alginate gel beads by a sol-gel process. The membrane has identical to Si-O-Si identical to bonds as well as electrostatic bonds between amino groups of AS and carboxyl groups of alginate. Permeability and stability were investigated for the membrane. Furthermore, rat islets encapsulated in the membrane (499 +/- 32 microns in diameter, 1000 islets/recipient) were transplanted to the peritoneal cavities of the mice with streptozotocin-induced diabetes. Our data show that the membrane had the molecular weight cut-off point of between 70 and 150 kDa, and hardly inhibited the permeation of glucose and insulin. The Alg/AS/Alg microcapsule was more stable than the well-known Alg/poly-L-lysine (PLL)/Alg microcapsule. After 30 days of soaking in stimulated body fluid, the percentages of intact microcapsule were 98.4 +/- 0.5 (mean +/- SEM)% and 88.0 +/- 1.5% (p < 0.001) for the Alg/AS/Alg and Alg/PLL/Alg microcapsules, respectively. The maximum maintenance period of normoglycemia was 105 days without administration of immunosuppressive drugs.     

Polyanionic collagen membranes for guided tissue regeneration: Effect of progressive glutaraldehyde cross-linking on biocompatibility and degradation

The ultimate goal of periodontal therapy is to control periodontal tissue inflammation and to produce predictable regeneration of that part of the periodontium which has been lost as a result of periodontal disease. In guided tissue regeneration membranes function as mechanical barriers, excluding the epithelium and gingival corium from the root surface and allowing regeneration by periodontal ligament cells. This report aims to study the effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen (PAC) membranes by conducting a histological evaluation of the tissue response (biocompatibility) and by assessing the biodegradation of subcutaneous membrane implants in rats. We studied six different samples: a PAC, a PAC mineralized by alternate soaking processes for either 25 or 75 cycles (PAC 25 and PAC 75, respectively) and these films cross-linked by GA. Inflammatory infiltrate, cytokine dosage, fibrosis capsule thickness, metalloproteinase immunohistochemistry and membrane biodegradation after 1, 7, 15 and 30 days were measured. The inflammatory response was found to be more intense in membranes without cross-linking, while the fibrosis capsules became thicker in cross-linked membranes after 30 days. The membranes without cross-linking suffered intense biodegradation, while the membranes with cross-linking remained intact after 30 days. The cross-linking with GA reduced the inflammatory response and prevented degradation of the membranes over the entire course of the observation period. These membranes are thus an attractive option when the production of new bone depends on the prolonged presence of a mechanical barrier.     

Surface treatments of titanium in aqueous solutions containing calcium and phosphate ions.

Immersion treatment of titanium in aqueous solutions containing various kinds of ion concentrations of calcium and phosphate (pH 5.8, 7.0, and 8.0) were attempted to accelerate calcium phosphate precipitation on titanium in body fluid. The performance was confirmed using scanning electron microscopy, X-ray diffractometry, and Fourier transformed infrared absorption spectrometry with a reflection absorption spectrometer of the specimen immersed in Hanks' solution. Calcium phosphate precipitation on titanium in Hanks' solution is accelerated by the immersion treatment in aqueous solutions containing calcium and phosphate ions. The amount, composition, and shape of calcium phosphate precipitate vary according to the pH and ion concentrations of the solutions in which titanium is immersed. This method is effective for the surface treatment of inside pore narrow space of titanium materials.     

Periodontal regeneration in intrabony defects after application of enamel matrix proteins with guided tissue regeneration: an experimental study in dogs

The aim of this study was to evaluate the effects of enamel matrix proteins (EMP) at the early stage of wound healing in the periodontal tissues by a combination treatment with guided tissue regeneration (GTR). Intrabony defects were produced surgically at the distal aspects of both mandibles in six beagle dogs. At 12 weeks following the surgery, the defects were exposed using a full thickness mucoperiosteal flap procedure. Subsequently, the defects were treated by the following treatments: a control group treated with GTR alone, and an experimental group treated with a combination of GTR and EMP. After one, two, four and eight weeks of the treatment, the animals were sacrificed, and sections of the tissue were stained and evaluated microscopically. After one and two weeks, the proliferating cell nuclear antigen (PCNA)-positive cell ratios of the experimental group were significantly greater than that of the control group. After 2 and 4 weeks, new bone and new cementum formation in the experimental group were significantly greater than those in the control group (P < 0.05). However, after 8 weeks, no statistical difference was found between the two groups in new bone or cementum formation. The study results suggest that a maturation of periodontal ligament cells might contribute, during the early stage of periodontal healing, to stimulate a proliferation of periodontal ligament cells.     

The role of barrier membranes for guided bone regeneration and restoration of large bone defects: current experimental and clinical evidence

Treatment of large bone defects represents a great challenge in orthopedic and craniomaxillofacial surgery. Although there are several methods for bone reconstruction, they all have specific indications and limitations. The concept of using barrier membranes for restoration of bone defects has been developed in an effort to simplify their treatment by offering a sinlge-staged procedure. Research on this field of bone regeneration is ongoing, with evidence being mainly attained from preclinical studies. The purpose of this review is to summarize the current experimental and clinical evidence on the use of barrier membranes for restoration of bone defects in maxillofacial and orthopedic surgery. Although there are a few promising preliminary human studies, before clinical applications can be recommended, future research should aim to establish the 'ideal' barrier membrane and delineate the need for additional bone grafting materials aiming to 'mimic' or even accelerate the normal process of bone formation. Reproducible results and long-term observations with barrier membranes in animal studies, and particularly in large animal models, are required as well as well-designed clinical studies to evaluate their safety, efficacy and cost-effectiveness.     

The evaluation of clinical predictions. A method and initial application.

Clinical predictions are never certain but are inherently probablisitc. The accuracy coefficient, a measure of probabilistic accuracy based on probability assigned to outcomes that occur, was used to assess the skill of clinical rheumatologists in predicting patient outcomes. Physicians' scores correlated well with degree of clinical experience. An approach to evaluation based on the measure provides a sensitive assessment of marginal benefit of technologies such as laboratory tests, diagnostic procedures or computer consultations. Most currently used methods of computer prediction were not as accurate as the best physicians tested. By allowing measurement of ability to individualize predictions to each patient's unique characteristics, the accuracy-coefficient approach has potential use in physician assessment.     

Polyelectroylte complex composed of chitosan and sodium alginate for wound dressing application.

Drug-impregnated polyelectrolyte complex (PEC) sponge composed of chitosan and sodium alginate was prepared for wound dressing application. The morphological structure of this wound dressing was observed to be composed of a dense skin outer layer and a porous cross-section layer by scanning electron microscopy (SEM). Equilibrium water content and release of silver sulfadiazine (AgSD) could be controlled by the number of repeated in situ PEC reactions between chitosan and sodium alginate. The release of AgSD from AgSD-impregnated PEC wound dressing in PBS buffer (PH = 7.4) was dependent on the number of repeated in situ complex formations for the wound dressing. The antibacterial capacity of AgSD-impregnated wound dressing was examined in agar plate against Pseudomonas aeruginosa and Staphylococcus aureus. From the behavior of antimicrobial release and the suppression of bacterial proliferation, it is thought that the PEC wound dressing containing antimicrobial agents could protect the wound surfaces from bacterial invasion and effectively suppress bacterial proliferation. In the cytotoxicity test, cellular damage was reduced by the controlled released of AgSD from the sponge matrix of AgSD-medicated wound dressing. In vivo tests showed that granulation tissue formation and wound contraction for the AgSD plus dihydroepiandrosterone (DHEA) impregnated PEC wound dressing were faster than any other groups.     

Bovine serum albumin partitioning in an aqueous two-phase system: effect of pH and sodium chloride concentration

The partitioning of bovine serum albumin (BSA) in a polyethylene glycol 3350 (8% w/w)-dextran 37 500 (6% w/w)-0.05 M phosphate aqueous two-phase was investigated at different pHs, at varying concentrations of sodium chloride at 20 degrees C. The effect of NaCl concentration on the partition coefficient of BSA was studied for the PEG-dx systems with initial pH values of 4.2, 5.0, 7.0, 9.0, and 9.8. The NaCl concentrations in the phase systems with constant pH value were 0.06, 0.1, 0.2, 0.3, and 0.34 M. It was observed that the BSA partition coefficient decreased at concentrations smaller than 0.2 M NaCl and increased at concentrations greater than 0.2 M NaCl for all systems with initial pHs of 4.2, 5.0, 7.0, 9.0, and 9.8. It was also seen that the partition coefficient of BSA decreased as the pH of the aqueous two-phase systems increased at any NaCl salt concentration studied.     

The calcium silicate/alginate composite: Preparation and evaluation of its behavior as bioactive injectable hydrogels

In this study, an injectable calcium silicate (CS)/sodium alginate (SA) hybrid hydrogel was prepared using a novel material composition design. CS was incorporated into an alginate solution and internal in situ gelling was induced by the calcium ions directly released from CS with the addition of d-gluconic acid δ-lactone (GDL). The gelling time could be controlled, from about 30 s to 10 min, by varying the amounts of CS and GDL added. The mechanical properties of the hydrogels with different amounts of CS and GDL were systematically analyzed. The compressive strength of 5% CS/SA hydrogels was higher than that of 10% CS/SA for the same amount of GDL. The swelling behaviors of 5% CS/SA hydrogels with different contents of GDL were therefore investigated. The swelling ratios of the hydrogels decreased with increasing GDL, and 5% CS/SA hydrogel with 1% GDL swelled by only less than 5%. Scanning electron microscopy (SEM) observation of the scaffolds showed an optimal interconnected porous structure, with the pore size ranging between 50 and 200 μm. Fourier transform infrared spectroscopy and SEM showed that the CS/SA composite hydrogel induced the formation of hydroxyapatite on the surface of the materials in simulated body fluid. In addition, rat bone mesenchymal stem cells (rtBMSCs) cultured in the presence of hydrogels and their ionic extracts were able to maintain the viability and proliferation. Furthermore, the CS/SA composite hydrogel and its ionic extracts stimulated rtBMSCs to produce alkaline phosphatase, and its ionic extracts could also promote angiogenesis of human umbilical vein endothelial cells. Overall, all these results indicate that the CS/SA composite hydrogel efficiently supported the adhesion, proliferation and differentiation of osteogenic and angiogenic cells. Together with its porous three-dimensional structure and injectable properties, CS/SA composite hydrogel possesses great potential for bone regeneration and tissue engineering applications.     

Amphiphilic derivatives of sodium alginate and hyaluronate for cartilage repair: rheological properties

Various amphiphilic derivatives of sodium alginate and hyaluronate were prepared by covalent fixation of long alkyl chains (dodecyl and octadecyl) with various ratios on the polysaccharide backbones via ester functions. In the semidilute regime, aqueous solutions of the resulting compounds exhibited the typical rheological properties of hydrophobically associating polymers: tremendous enhancement of zero shear rate Newtonian viscosity, steep shear-thinning behavior, and formation of physically cross-linked gel-like networks. The influence of the alkyl chain length, its content on the polysaccharide and of the polymer concentration in the solution was well identified. All obtained results are discussed with respect to the schedule of conditions related to materials, which could be used for cartilage repair, such as in synovial fluid viscosupplementation as well as in cartilage replacement. In particular, it is seen that HA-C(12)-5 (hyaluronate substituted with 5% of dodecyl chains) and HA-C(18)-1 (hyaluronate substituted with 1% of octadecyl chains) in a 0.15N NaCl solution at 8 g/L have rheological properties quite similar to those of healthy synovial fluid. On the other hand, the rheological parameters of solutions at 8 g/L in 0.15N NaCl of some of derivatives, such as, for example, AA-C(12)-8 (alginate substituted with 8% of dodecyl chains) or HA-C(18)-2, are well fitted for a use in cartilage repair.