In vitro and in vivo evaluation of the effects of demineralized bone matrix or calcium sulfate addition to polycaprolactone–bioglass composites

The objective of this study was to improve the efficacy of polycaprolactone/bioglass (PCL/BG) bone substitute using demineralized bone matrix (DBM) or calcium sulfate (CS) as a third component. Composite discs involving either DBM or CS were prepared by compression moulding. Bioactivity of discs was evaluated by energy dispersive X-ray spectroscopy (ESCA) and scanning electron microscopy (SEM) following simulated body fluid incubation. The closest Calcium/Phosphate ratio to that of hydroxyl carbonate apatite crystals was observed for PCL/BG/DBM group (1.53) after 15 day incubation. Addition of fillers increased microhardness and compressive modulus of discs. However, after 4 and 6-week PBS incubations, PCL/BG/DBM discs showed significant decrease in modulus (from 266.23 to 54.04 and 33.45 MPa, respectively) in parallel with its highest water uptakes (36.3 and 34.7%). Discs preserved their integrity with only considerable weight loss (7.5–14.5%) in PCL/BG/DBM group. In vitro cytotoxicity tests showed that all discs were biocompatible. Composites were implanted to defects on rabbit humeri. After 7 weeks, new tissue formation and mineralization at bone-implant interface were observed for all implants. Bone mineral densities at interface were higher than that of implant site and negative controls (defects left empty) but lower than healthy bone level. However, microhardness of implant sites was higher than in vitro results indicating in vivo mineralization of implants. Addition of DBM or CS resulted with higher microhardness values at interface region (ca. 650 μm from implant) compared to PCL/BG and negative control. Histological studies revealed that addition of DBM enhanced bone formation around and into implant while CS provided cartilage tissue formation around the implant. From these results, addition of DBM or CS could be suggested to improve bone healing efficacy of PCL/BG composites.     

Initial in vitro biocompatibility of a bone cement composite containing a poly-ε-caprolactone microspheres

The biocompatibility of a reinforced calcium phosphate injectable bone substitute (CPC-IBS) containing 30% poly-ε-caprolactone (PCL) microspheres was evaluated. The IBS consisted of a solution of chitosan and citric acid as the liquid phase and tetracalcium phosphate (TTCP) and dicalcium phosphate anhydrous (DCPA) powder as the solid phase with 30% PCL microspheres. The surface of the CPC-IBS was observed by SEM, and analyzed by EDX profiles. The initial setting of the sample was lower in the IBS containing 0% citric acid than in the IBS containing 10 or 20% citric acid. The compressive strength of the PCL-incorporated CPC-IBS was measured using a Universal Testing Machine. The 20% citric acid samples had the highest mechanical strength at day 12, which was dependent on both time and the citric acid concentration. The in vitro bioactivity experiments with simulated body fluid (SBF) confirmed the formation of apatite on the sample surfaces after 2, 7, and 14 days of incubation in SBF. Ca and P ion release profile by ICP method also confirmed apatite nucleation on the CPC-IBS surfaces. The in vitro biocompatibility of the CPC-IBS was evaluated by using MTT, cellular adhesion, and spreading studies. In vitro cytotoxicity tests by MTT assay showed that the 0 and 10% CPC-IBS was cytocompatible for fibroblast L-929 cells. The SEM micrograph confirmed that MG-63 cells maintained their phenotype on all of the CPC-IBS surfaces although cellular attachment was better in 0 and 10% CPC-IBS than 20% samples.     

Engineering cartilage tissue interfaces using a natural glycosaminoglycan hydrogel matrix — An in vitro study

Hydrogels are suitable matrices for cartilage tissue engineering on account of their resemblance to native extracellular matrix of articular cartilage and also considering its ease of application, they can be delivered to the defect site in a minimally invasive manner. In this study, we evaluate the suitability of a fast gelling natural biopolymer hydrogel matrix for articular cartilage tissue engineering. A hydrogel based on two natural polymers, chitosan and hyaluronic acid derivative was prepared and physicochemically characterized. Chondrocytes were then encapsulated within the hydrogel and cultured over a period of one month. Cartilage regeneration was assessed by histological, biochemical and gene expression studies. Chondrocytes maintained typical round morphology throughout the course of this investigation, indicating preservation of their phenotype with sufficient production of extracellular matrix and expression of typical chondrogenic markers Collagen type 2 and aggrecan. The results suggest that the natural polymer hydrogel matrix can be used as an efficient matrix for articular cartilage tissue engineering.     

In vitro models of periodontal cells: a comparative study of long-term gingival,periodontal ligament and alveolar bone cell cultures in the presence of β-glycerophosphate and dexamethasone

Human gingival (HG), periodontal ligament (HPL) and alveolar bone (HAB) cells (first subculture) were cultured (10⁴ cells/cm²) for 35 days in α-Minimal Essential Medium supplemented with 10% fetal bovine serum in the presence of (i) ascorbic acid (AA, 50 μg/mL), (ii) AA + β-glycerophosphate (βGP, 10 mM) and (iii) AA + βGP + dexamethasone (Dex, 10 nM). Cultures were assessed for cell attachment and spreading, cell proliferation, alkaline phosphatase (ALP) and acid phosphatase (ACP) activities and matrix mineralization. HG cell cultures presented a high proliferation rate, a low ability to synthesize ALP and ACP and the formation of a non-mineralized extracellular matrix, regardless the experimental situation. HPL cell cultures were very sensitive to the culture conditions and showed a high proliferation rate, synthesis of moderate levels of ALP and ACP and a modest matrix mineralization in the presence of AA + βGP + Dex. HAB cell cultures presented a growth rate lower than that of HG and HPL cells, a high ALP activity and comparatively low levels of ACP, and the ready formation of a heavy mineralized matrix in the presence of βGP. In the three periodontal cell cultures, Dex enhanced cell proliferation and expression of osteoblastic markers. Results showed that βGP and Dex allowed the modulation of the cell proliferation/differentiation behavior within the proposed physiological and regenerative capabilities of these periodontal cells.     

The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/β-glycerophosphate hydrogel: in vitro and in vivo

Type I collagen was added to the composite chitosan solution in a ratio of 1:2 to build a physical cross-linked self-forming chitosan/collagen/β-GP hydrogel. Osteogenic properties of this novel injectable hydrogel were evaluated. Gelation time was about 8 min which offered enough time for handling a mixture containing cells and the subsequent injection. Scanning electronic microscopy (SEM) observations indicated good spreading of bone marrow mesenchymal stem cells (BMSCs) in this hydrogel scaffold. Mineral nodules were found in the dog-BMSCs inoculated hydrogel by SEM after 28 days. After subcutaneous injection into nude mouse dorsum for 4 weeks, partial bone formation was observed in the chitosan/collagen/β-GP hydrogel loaded with pre-osteodifferentiated dog-BMSCs, which indicated that chitosan/collagen/β-GP hydrogel composite could induce osteodifferentiation in BMSCs without exposure to a continual supply of external osteogenic factors. In conclusion, the novel chitosan/collagen/β-GP hydrogel composite should prove useful as a bone regeneration scaffold.     

Silicone adhesive,a better matrix for tolterodine patches—a research based on in vitro/in vivo studies

Objective: To design and optimize a drug-in-adhesive (DIA) type transdermal patch for tolterodine (TOL) based on acrylic and silicone matrixes.

Methods: Initial in vitro studies were conducted to optimize the formulations. Two types of adhesive matrixes, drug loading, and enhancers were evaluated on the TOL transport across rabbit skin. For in vivo studies, patches were administered to rabbit abdominal skin. Pharmacokinetic assessments were performed based on plasma level of TOL up to 28?h for acrylic patch and 52?h for silicone patch after topical application.

Results: The final formulation of acrylic adhesive type patch consisted of 10% TOL (w/w) and 5.8?×?10^?4 mol isopropyl myristate (IPM) and 2.9?×?10^?4 mol Span 80 in per unit gram (mol/g) of adhesive, while 2.5% TOL (w/w) and 2.9?×?10^?4 mol/g IPM for silicone adhesive type patch. Comparison of the pharmacokinetic parameters between two types of patches showed that the steady-state concentration of silicone type patch was 2-fold higher than that of acrylic type patch being 0.97?mg/L versus 0.49?mg/L, and the absolute bioavailability was 27.5% for silicone type patch and 6.3% for acrylic type patch, respectively. In addition, the prediction of in vivo drug level from the in vitro permeation data of silicone adhesive formulation was in good agreement with actual observed concentration data in rabbits.

Conclusion: These results indicate that the silicone type of TOL patch is an appropriate delivery system for the treatment of overactive bladder (OAB).     

Influence of β-radiation sterilisation in properties of new chitosan/soybean protein isolate membranes for guided bone regeneration

Novel chitosan (cts) and soybean protein isolate (SI) blended membranes were prepared. These membranes were produced by solvent casting. Besides combining the advantages of both materials, cts/SI membranes exhibit a biphasic structure that will eventually originate in situ porous formation, through a two-step degradation mechanism. In this particular work the effect of beta-radiation over the properties of these membranes was evaluated. beta-radiation sterilisation was performed at three different doses (25, 50 and 100 kGy) and eventual surface chemical changes were evaluated by Fourier transformed infrared--with attenuated total reflection and contact angle measurements. Moreover, eventual bulk properties changes due to beta-radiation were assessed by means of mechanical tensile tests and water uptake measurements. In general, no substantial changes were detected on the studied properties, with the exception of the surface energy that was found to be slightly increased for higher applied doses.     

Mineralised collagen—an artificial,extracellular bone matrix—improves osteogenic differentiation of bone marrow stromal cells

In the field of bone tissue engineering there is a high demand on bone graft materials which promote bone formation. By combination of collagen type I with nanocrystalline hydroxyapatite (HA) we generated a resorbable material which structure and composition is close to those of the extracellular bone matrix. This nanocomposit material was produced in a biomimetic process in which collagen fibril assembly and mineralisation with hydroxyapatite occur simultaneously. In this study the proliferation and osteogenic differentiation of human bone marrow-derived stromal cells (hBMSC) on membranes of biomimetically mineralised collagen type I was investigated. To this end, we optimised biochemical assays for determination of cell number and alkaline phosphatase activity corresponding to the special properties of this biomaterial. For cell experiments hBMSC were seeded on the mineralised collagen membranes and cultivated over 35 days, both in static and perfusion culture, in the presence and absence of dexamethasone, β-glycerophosphate and ascorbate. Compared to cells grown on tissue culture polystyrene we found attenuated proliferation rates, but markedly increased activity of alkaline phosphatase on the mineralised collagen indicating its promoting effect on the osteogenic differentiation of hBMSC. Therefore this bone-like material may act as a suitable artificial extracellular matrix for bone tissue engineering. Perfusion of the 2D cell matrix constructs with cell culture medium did not improve proliferation and osteogenic differentiation of the hBMSC. Anne Bernhardt and Anja Lode contributed equally to this paper     

The controlling biodegradation of chitosan fibers by N-acetylation in vitro and in vivo

Aim In the present study, we investigated the biodegradation of the fibers of chitosan and its acetylated derivatives in vitro and in vivo. Methods A series of chitosan fibers, with acetylation degrees of 7.7%, 21.6%, 40.9%, 61.2%, 82.5% and 93.4%, were obtained by acetylating chitosan filament with acetic anhydride, and were investigated by FT-IR analysis, elemental analysis and scanning electron microscopy analysis. Results The in vitro experimental data indicated that the degradation rate of chitosan fiber was strongly dependent on the degree of acetylation, and the degradation rate increased with an enhancement of the acetylation degree of chitosan fibers. In vivo degradation experiment evaluated by light microscopy as well as scanning electron microscopy, was studied by implanting the fibers between the two nerve stumps of the rat sciatic nerve gap (6 months). The findings demonstrated that acetylation degree could influence the degradation rate of chitosan fibers in vivo. Conclusion These results suggested that acetylated chitosan (chitin) fibers were more biodegradable than chitosan and the biodegradation rate of chitin fiber can be controlled to desirable extent by the variation of acetylation degree.     

Microwave sintering and in vitro study of defect-free stable porous multilayered HAp–ZrO2 artificial bone scaffold

Abstract

Continuously porous hydroxyapatite (HAp)/t-ZrO₂ composites containing concentric laminated frames and microchanneled bodies were fabricated by an extrusion process. To investigate the mechanical properties of HAp/t-ZrO₂ composites, the porous composites were sintered at different temperatures using a microwave furnace. The microstructure was designed to imitate that of natural bone, particularly small bone, with both cortical and spongy bone sections. Each microchannel was separated by alternating lamina of HAp, HAp–(t-ZrO₂) and t-ZrO₂. HAp and ZrO₂ phases existed on the surface of the microchannel and the core zone to increase the biocompatibility and mechanical properties of the HAp-ZrO₂ artificial bone. The sintering behavior was evaluated and the optimum sintering temperature was found to be 1400 °C, which produced a stable scaffold. The material characteristics, such as the microstructure, crystal structure and compressive strength, were evaluated in detail for different sintering temperatures. A detailed in vitro study was carried out using MTT assay, western blot analysis, gene expression by polymerase chain reaction and laser confocal image analysis of cell proliferation. The results confirmed that HAp-ZrO₂ performs as an artificial bone, showing excellent cell growth, attachment and proliferation behavior using osteoblast-like MG63 cells.     

Fourier-transform infrared spectroscopy study of an organic–mineral composite for bone and dental substitute materials

A new injectable biomaterial for bone and dental surgery is a composite consisting of a polymer as a matrix and bioactive calcium phosphate (CaP) ceramics as fillers. The stability of the polymer is essential in the production of a ready-to-use injectable sterilized biomaterial. The purpose of this study was to detect possible polymer degradation which may have been caused by the interaction with the fillers using Fourier transform infrared spectroscopy. Composites containing CaP fillers (biphasic calcium phosphate, hydroxyapatite and peroxidized hydroxyapatite) and polymer (hydroxypropyl methyl cellulose) were prepared. To investigate the properties of the polymer, the inorganic and organic phases of the composite were separated using several extraction methods. The difficulty in separating the organic (polymer) from the mineral (CaP fillers) phases in the composite investigated in this study suggested the presence of strong interactions between the two phases. Spectra of extracted polymers showed new absorption bands of low intensities and indications that some chemical modifications of the original polymers have occurred. Results also indicated that the filler composition has an effect on the integrity of the polymer.     

Central composite rotatable design for optimization of budesonide-loaded cross-linked chitosan–dextran sulfate nanodispersion: characterization,in vitro diffusion and aerodynamic study

Budesonide is a BCS class II drug with low water solubility (0.045?mg/mL) and low oral bioavailability (6–8%) due to high first pass effect. The aim is to prepare cross-linked chitosan–dextran sulfate nanoparticles and/or nanodispersion. Nebulizable cross-linked nanodispersion was prepared by the solvent evaporation technique and characterized through XRPD, FTIR, mean particle size (MPS), polydispersity index (PDI), zeta potential (ZP), drug loading, entrapment efficiency, SEM, % production yield, in vitro diffusion, aerodynamic and stability study. The optimization of formulation was done by using central composite rotatable design to study the effect of independent variables, concentration of chitosan (X1) and concentration dextran sulfate (X2) on the dependent variables, MPS (Y1), drug loading (Y2) and % CDR (% cumulative drug release) (Y3). The MPS, PDI, and ZP of budesonide-loaded nanoparticles were 160.8?±?0.27?nm, 0.36?±?0.04, and 13?±?0.894?mV, respectively. The percent drug loading of all the batches was found in range of 10–16%. The emitted drug in target region (alveoli) was measured by using HPLC and it was found to be 18.26%. It was found that, nanodispersion had the optimum in vitro aerodynamic behavior. Stability study results showed no significant change in MPS, PDI, ZP, and % CDR after three month storage. In conclusion, cross-linked chitosan–dextran sulfate nanoparticles had properties suitable for nebulizable dispersion of increased drug loading, in vitro drug release and avoiding the first pass effect.     

Hydration characteristics of tetracalcium aluminoferrite phase in mixes containing β-hemihydate and phosphogypsum

Tetracalcium aluminoferrite phase was prepared from pure chemicals on a laboratory scale. Five mixes were prepared from the prepared C₄AF phase, -hemihydate, phosphogypsum and calcium hydroxide. The mixes were hydrated at various intervals namely, 24, 72, 168 and 336 h. The kinetics of hydration was mentioned by measuring the chemically combined water and combined lime contents. The phase composition and microstructure were studied by FT-IR Spectroscopy, XRD, DTA and SEM techniques. This work aimed to study the effect of partial to fully substitution of -hemihydrate for phosphogypsum on the hydration characteristics and microstructures of tetracalcium aluminoferrite phase. The mechanism of the hydration of tetracalcium aluminoferrite phase in the presence of phosphogypsum proceeds through a similar manner as in the case of -hemihydrate and affects to a slight extent the rate of hydration reaction.     

In vitro study of the SBF and osteoblast-like cells on hydroxyapatite/chitosan–silica nanocomposite

Hydroxyapatite/chitosan–silica (HApCSi) nanocomposites were synthesized by co-precipitated method and their potential application as filler materials for bone regeneration were investigated in simulated body fluid (SBF). To study their biocompatibility, they were cultured with rat osteoblast-like UMR-106 cells for 3, 7, 14, and 21 days. Studies of the silica contents in chitosan matrix showed the presence of silinol (Si–OH) groups in CSi hybrid and their decrease after being composited with calcium phosphate (CaP) which is desirable for the formation of the apatite. XRD and TEM studies showed that the HAp formed in the CSi matrix were nanometer (20–40 nm) in size. Nanocomposites of HApCSi20 processed with 20%v/v silica whisker showed a micro hardness of 84.7 ± 3.3 MPa. Mineralization study in SBF showed the formation of apatite crystals on the HApCSi surface after being incubated for 7 days. In vitro biocompatibility, cell morphology, proliferation, and cell adhesion tests confirmed the osteoblast attachment and growth on the HApCSi20 surface. The density of cells and the production of calcium nodules on the substrate were seen to increase with increasing cultured time. The mechanical evaluation and in vitro experiment suggested that the use of HApCSi composite will lead to the formation of new apatite particles and thus be a potential implant material.     

Properties and in vitro characterization of polyhydroxybutyrate–chitosan scaffolds prepared by modified precipitation method

Porous polyhydroxybutyrate (PHB)–chitosan biopolymer scaffolds were prepared by co-precipitation from biopolymer solutions with propylene carbonate and acetic acid as solvents. A change of the fibrous character of chitosan precipitates to globular shaped forms with a polyhydroxybutyrate addition was found in suspensions. Scaffolds differ by porosity and morphology of polymers in microstructures, while chitosan represented more compact plate-like fibers and PHB characterized mainly fine fibrous globular agglomerates. Two structurally dissimilar phase regions were verified in blended scaffolds. A rise in the number of smaller pores, and fine structured polymer forms with PHB content were observed in the scaffolds. A significant reduction in the average molecular weight of biopolymers was found in pure chitosan scaffold, this after precipitation of the chitosan in the presence of propylene carbonate and in blends after mutual biopolymer mixing. Interactions between shortened chitosan chains, PHB and chitosan biopolymers in the blends were observed. An excellent fibroblast proliferation was found in scaffolds prepared from biopolymer blends.     

In vitro degradability,bioactivity and primary cell responses to bone cements containing mesoporous magnesium–calcium silicate and calcium sulfate for bone regeneration

Mesoporous calcium sulfate-based bone cements (m-CSBC) were prepared by introducing mesoporous magnesium–calcium silicate (m-MCS) with specific surface area (410.9 m² g⁻¹) and pore volume (0.8 cm³ g⁻¹) into calcium sulfate hemihydrate (CSH). The setting time of the m-CSBC was longer with the increase of m-MCS content while compressive strength decreased. The degradation ratio of m-CSBC increased from 48.6 w% to 63.5 w% with an increase of m-MCS content after soaking in Tris–HCl solution for 84 days. Moreover, the m-CSBC containing m-MCS showed the ability to neutralize the acidic degradation products of calcium sulfate and prevent the pH from dropping. The apatite could be induced on m-CSBC surfaces after soaking in SBF for 7 days, indicating good bioactivity. The effects of the m-CSBC on vitamin D₃ sustained release behaviours were investigated. It was found that the cumulative release ratio of vitamin D₃ from the m-CSBC significantly increased with the increase of m-MCS content after soaking in PBS (pH = 7.4) for 25 days. The m-CSBC markedly improved the cell-positive responses, including the attachment, proliferation and differentiation of MC3T3-E1 cells, suggesting good cytocompatibility. Briefly, m-CSBC with good bioactivity, degradability and cytocompatibility might be an excellent biocement for bone regeneration.     

Identification of the metastable phase α′m in a Zn/Al alloy containing Cu and Mg

Samples of the commercial pressure-diecast alloy ZA8 containing 8.1% Al, 1.1% Cu and 0.024% Mg have been examined by transmission electron microscopy (TEM). During decomposition of the high temperature f c c phase, on cooling after casting, a transitional phase was formed within the phase in both dendrites and eutectic. This phase was identified as the metastable phase _m containing 11% Al or 23% Al, with a f c c crystal structure and lattice parameter (at 11%) of about 0.394 nm. It had adopted a symmetrical cube/cube orientation relationship with the surrounding f c c phase. The stability of this metastable phase at ambient temperatures was greatly increased by the presence of copper.     

In vitro and in vivo evaluation of chitosan–gelatin scaffolds for cartilage tissue engineering

Chitosan–gelatin polyelectrolyte complexes were fabricated and evaluated as tissue engineering scaffolds for cartilage regeneration in vitro and in vivo. The crosslinker for the gelatin component was selected among glutaraldehyde, bisepoxy, and a water-soluble carbodiimide (WSC) based upon the proliferation of chondrocytes on the crosslinked gelatin. WSC was found to be the most suitable crosslinker. Complex scaffolds made from chitosan and gelatin with a component ratio equal to one possessed the proper degradation rate and mechanical stability in vitro. Chondrocytes were able to proliferate well and secrete abundant extracellular matrix in the chitosan–gelatin (1:1) complex scaffolds crosslinked by WSC (C1G1_WSC) compared to the non-crosslinked scaffolds. Implantation of chondrocytes-seeded scaffolds in the defects of rabbit articular cartilage confirmed that C1G1_WSC promoted the cartilage regeneration. The neotissue formed the histological feature of tide line and lacunae in 6.5 months. The amount of glycosaminoglycans in C1G1_WSC constructs (0.187 ± 0.095 μg/mg tissue) harvested from the animals after 6.5 months was 14 wt.% of that in normal cartilage (1.329 ± 0.660 μg/mg tissue). The average compressive modulus of regenerated tissue at 6.5 months was about 0.539 MPa, which approached to that of normal cartilage (0.735 MPa), while that in the blank control (3.881 MPa) was much higher and typical for fibrous tissue. Type II collagen expression in C1G1_WSC constructs was similarly intense as that in the normal hyaline cartilage. According to the above results, the use of C1G1_WSC scaffolds may enhance the cartilage regeneration in vitro and in vivo.     

Effects of heat treatment of wood on hydroxylapatite type mineral precipitation and biomechanical properties in vitro

Wood is a natural fiber reinforced composite. It structurally resembles bone tissue to some extent. Specially heat-treated birch wood has been used as a model material for further development of synthetic fiber reinforced composites (FRC) for medical and dental use. In previous studies it has been shown, that heat treatment has a positive effect on the osteoconductivity of an implanted wood. In this study the effects of two different heat treatment temperatures (140 and 200°C) on wood were studied in vitro. Untreated wood was used as a control material. Heat treatment induced biomechanical changes were studied with flexural and compressive tests on dry birch wood as well as on wood after 63 days of simulated body fluid (SBF) immersion. Dimensional changes, SBF sorption and hydroxylapatite type mineral formation were also assessed. The results showed that SBF immersion decreases the biomechanical performance of wood and that the heat treatment diminishes the effect of SBF immersion on biomechanical properties. With scanning electron microscopy and energy dispersive X-ray analysis it was shown that hydroxylapatite type mineral precipitation formed on the 200°C heat-treated wood. An increased weight gain of the same material during SBF immersion supported this finding. The results of this study give more detailed insight of the biologically relevant changes that heat treatment induces in wood material. Furthermore the findings in this study are in line with previous in vivo studies.