In vitro and in vivo degradation of poly(l-lactide-co-glycolide) films and scaffolds

Poly(l-lactide-co-glycolide) (PLGA) was synthesized using a biocompatible initiator, zirconium acetylacetonate. In vitro and in vivo degradation properties of PLGA films (produced by solvent casting, 180 μm thick) and PLGA scaffolds (produced by an innovated solvent casting and particulate leaching, 3 mm thick) were evaluated. The samples were either submitted for degradation in phosphate buffered saline (PBS) at 37 °C for 30 weeks, or implanted into rat skeletal muscles for 1, 4, 12, 22 and 30 weeks. The degradation was monitored by scanning electron microscopy, atomic force microscopy, weight loss, and molecular weight changes (in vitro), and by microscopic observations of the materials’ morphology after histological staining with May-Grunwald-Giemsa (in vivo). The results show that the films in both conditions degraded much faster than the scaffolds. The scaffolds were dimensionally stable for 23 weeks, while the films lost their integrity after 7 weeks in vitro. The films’ degradation was heterogenous—degradation in their central parts was faster than in the surface and subsurface regions due to the increased concentration of the acidic degradation products inside. In the scaffolds, having much thinner pore walls, heterogenous degradation due to the autocatalytic effect was not observed.     

Osteoblast behaviour on in situ photopolymerizable three-dimensional scaffolds based on d,l-lactide and ε-caprolactone: influence of pore volume, pore size and pore shape

Bone marrow cells were cultured on in situ photopolymerizable scaffolds based on D,L-lactide and epsilon-caprolactone. The influence of pore volume, size and shape were evaluated. Bone formation was demonstrated by ALP activity, osteocalcin secretion and histological analysis. TEM at the polymer interface revealed osteoblasts which secreted an extracellular matrix containing matrix vesicles loaded with apatite. Cellular infiltration was possible for scaffolds with a porosity of 70 and gelatin particle size of 250-355 microm. Scaffolds with a porosity less than 70 had the tendency to form a polymer top layer. Although increasing the gelatin particle size to 355-500 microm, leads to infiltration even in scaffolds with a porosity of 60. No infiltration was possible in scaffolds with sodium chloride as porogen. On the contrary, sucrose and gelatin leads to better interconnected scaffolds at the same porosity. Hence, spherical gelatin particles are suitable to use as porogen in photopolymerizable scaffolds.     

Rhodamine-loaded poly(lactic-co-glycolic acid) nanoparticles for investigation of in vitro interactions with breast cancer cells

Nanoparticle-based drug delivery systems are considered promising for the delivery of imaging agents and drugs for the detection and treatment of illnesses, including cancer. Investigation of nanoparticle interactions with the diseased cells can lead to better designs. In this work, poly(lactic-co-glycolic acid) nanoparticles loaded with rhodamine 6G were prepared by nanoprecipitation with high encapsulation efficiency. In vitro release studies demonstrated that rhodamine escaped from the nanoparticles at a very slow rate at physiological pH, thus making it ideal for imaging studies. At acidic pH this agent was released quickly, suggesting charge interactions between the polymer and rhodamine. Microscopy and flow cytometry studies show higher uptake in MDA-MB-231 breast cancer cells when exposed to rhodamine-loaded nanoparticles than to rhodamine in solution.     

Fabrication of three-dimensional poly(ε-caprolactone) scaffolds with hierarchical pore structures for tissue engineering

The physical properties of tissue engineering scaffolds such as microstructures play important roles in controlling cellular behaviors and neotissue formation. Among them, the pore size stands out as a key determinant factor. In the present study, we aimed to fabricate porous scaffolds with pre-defined hierarchical pore sizes, followed by examining cell growth in these scaffolds. This hierarchical porous microstructure was implemented via integrating different pore-generating methodologies, including salt leaching and thermal induced phase separation (TIPS). Specifically, large (L, 200–300 μm), medium (M, 40–50 μm) and small (S, < 10 μm) pores were able to be generated. As such, three kinds of porous scaffolds with a similar porosity of ~ 90% creating pores of either two (LS or MS) or three (LMS) different sizes were successfully prepared. The number fractions of different pores in these scaffolds were determined to confirm the hierarchical organization of pores. It was found that the interconnectivity varied due to the different pore structures. Besides, these scaffolds demonstrated similar compressive moduli under dry and hydrated states. The adhesion, proliferation, and spatial distribution of human fibroblasts within the scaffolds during a 14-day culture were evaluated with MTT assay and fluorescence microscopy. While all three scaffolds well supported the cell attachment and proliferation, the best cell spatial distribution inside scaffolds was achieved with LMS, implicating that such a controlled hierarchical microstructure would be advantageous in tissue engineering applications.     

In vitro behavior of human osteoblast-like cells (SaOS2) cultured on surface modified titanium and titanium–zirconium alloy

In this study, titanium (Ti) and titanium–zirconium (TiZr) alloy samples fabricated through powder metallurgy were surface modified by alkali-heat treatment and calcium (Ca)-ion-deposition. The alteration of the surface morphology and the chemistry of the Ti and TiZr after surface modification were examined. The bioactivity of the Ti and TiZr alloys after the surface modification was demonstrated. Subsequently, the cytocompatibility of the surface modified Ti and TiZr was evaluated via in vitro cell culture using human osteoblast-like cells (SaOS2). The cellular attachment, adhesion and proliferation after cell culture for 14 days were characterized by scanning electron microscopy (SEM) and MTT assay. The relationship between surface morphology and chemical composition of the surface modified Ti and TiZr and cellular responses was investigated. Results indicated that the surface-modified Ti and TiZr alloys exhibited excellent in vitro cytocompatibility together with satisfactory bioactivity. Since osteoblast adhesion and proliferation are essential prerequisites for a successful implant in vivo, these results provide evidence that Ti and TiZr alloys after appropriate surface modification are promising biomaterials for hard tissue replacement.     

In vitro behavior of osteoblast-like cells on PLLA films with a biomimetic apatite or apatite/collagen composite coating

To investigate the methods to improve the cell–material interaction of devices or tissue engineering scaffolds made of poly(l-lactic acid) (PLLA) polymer, apatite and apatite/collagen composite coatings were formed on PLLA films within 24 h through accelerated biomimetic processes. In vitro investigation using Saos-2 osteoblast-like cells through cell culture was conducted to assess the biological performance of these biomimetic coatings. The cell morphology on three types of surfaces, viz., PLLA film, PLLA film with the apatite coating, and PLLA film with the apatite/collagen composite coating, was studied using scanning electron microscopy (SEM). Cell viability was estimated using the MTT assay. The differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. The results obtained indicated that the biomimetic apatite and apatite/collagen composite coatings could significantly enhance the proliferation and differentiation of osteoblast-like cells. The apatite/collagen composite coating appears to be promising for the surface modification of PLLA-based devices with much improved interactions with osteoblastic cells.     

Osteogenic differentiation of rat bone mesenchymal stem cells cultured on poly (hydroxybutyrate-co-hydroxyvalerate), poly (ε-caprolactone) scaffolds

Journal of Materials Science: Materials in Medicine - A thin endocrown restoration was often applied in endodontically treated teeth with vertical bite height loss or inadequate clinical crown...     

Effect of particle size on in vitro cytotoxicity of titania and alumina nanoparticles

Aluminium oxide (Al₂O₃) and titanium dioxide (TiO₂) nanoparticles (NPs) have been widely used in nanotechnology-based products. Recently, researchers and the public have raised concerns about the adverse effects of these NPs in biological systems, particularly in humans. The aim of this study was to investigate the possible adverse effects of these two common metal oxide NPs on human lung epithelium cells (A549) and to investigate NP size-dependent effects on these cells, considering both the primary and hydrodynamic particle size. NPs were found to inhibit cell viability and proliferation at the highest concentration level (10?mg/mL) included in this study, as measured by a clonogenic assay. Moreover, cell viability, proliferation and metabolism were impaired to a greater extent by the smaller NPs (5?nm TiO₂ and 10?nm Al₂O₃) relative to the larger particles (200?nm TiO₂ and 50?nm Al₂O₃) included in this study, as measured by cell proliferation and metabolism. Notably, the observed cytotoxic effects correlated to the primary size, rather than the hydrodynamic size. Similarly, NP cytotoxicity was found to be correlated with the NP surface area. These findings highlight the importance of including primary size and surface area information in NP characterisation in cytotoxicity studies.     

Cultivation of human keratinocytes without feeder cells on polymer carriers containing ethoxyethyl methacrylate: in vitro study

The cell/tissue engineering therapy of extensive or chronic skin wounds is a highly topical task of the contemporary medicine. One of possible therapeutic approaches is grafting of in vitro cultured keratinocytes directly to the wound bed, where the cells colonize the wound, proliferate and improve the re-epithelization process. Because the successful cultivation of keratinocytes needs an application of feeder cells, the exclusion of these cells from the cultivation system is highly required. In this study we show a positive influence of 2-ethoxyethyl methacrylate as a component of cultivation support on growth of keratinocytes without feeder cells. Keratinocytes cultured on these surfaces are able to migrate to the model wound bed in vitro, where they form distinct colonies and have a normal differentiation potential.     

The behavior of osteoblast-like cells on various substrates with functional blocking of integrin-β1 and integrin-β3

This study was designed to examine the influence of integrin subunit-β1 and subunit-β3 on the behavior of primary osteoblast-like cells, cultured on calcium phosphate (CaP)-coated and non coated titanium (Ti). Osteoblast-like cells were incubated with specific monoclonal antibodies against integrin-β1 and integrin-β3 to block the integrin function. Subsequently, cells were seeded on Ti discs, either non coated or provided with a 2 μm carbonated hydroxyapatite coating using Electrostatic Spray Deposition. Results showed that on CaP coatings, cellular attachment was decreased after a pre-treatment with either anti-integrin-β1 or anti-integrin-β3 antibodies. On Ti, cell adhesion was only slightly affected after a pre-treatment with anti-integrin-β3 antibodies. Scanning electron microscopy showed that on both types of substrate, cellular morphology was not changed after a pre-treatment with either antibody. With quantitative PCR, it was shown for both substrates that mRNA expression of integrin-β1 was increased after a pre-treatment with either anti-integrin-β1 or anti-integrin-β3 antibodies. Furthermore, after a pre-treatment with either antibody, mRNA expression of integrin-β3 and ALP was decreased, on both types of substrate. In conclusion, osteoblast-like cells have the ability to compensate to great extent for the blocking strategy as applied here. Still, integrin-β1 and β3 seem to play different roles in attachment, proliferation, and differentiation of osteoblast-like cells, and responses on CaP-coated substrates differ to non coated Ti. Furthermore, the influence on ALP expression suggests involvement of both integrin subunits in signal transduction for cellular differentiation.     

Osteoblastic cells culture on electrospun poly(ε-caprolacton) scaffolds incorporating amphiphilic PEG–POSS telechelic

In this work, novel poly(ε-caprolactone) (PCL) fibrous membranes incorporating amphiphilic polyhedral oligosilsesquioxane (POSS) telechelic (PEG–POSS telechelic) were prepared via electrospinning. The unique microstructure, morphology, thermal stability of the resulting PCL/PEG–POSS telechelic electrospun nanowebs were investigated by X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis, respectively. The addition of amphiphilic PEG–POSS telechelic strongly influenced the fiber diameters, microstructures of the resultant PCL/PEG–POSS telechelic nanofibers, compared to pure PCL nanofibers. The potential biomedical applications of such PEG–POSS telechelic nanowebs as a scaffolding material were also evaluated in vitro using mouse osteoblast-like MC3T3-E1 cells. The cell adhesion, spreading, and interaction behavior of pure PCL and PCL/PEG–POSS telechelic fibrous membranes were explored. It was found that electrospun PCL fibrous membranes incorporating amphiphilic PEG–POSS telechelic showed higher initial cell attachment than pure PCL due to the higher surface free energy of POSS siloxanes. Moreover, the obtained PCL/PEG–POSS telechelic fibrous scaffolds were found to be nontoxic and to maintain the good adhesion ratio between cells and surface (about ~93 %) after cell culturing for 24 h.     

Comparative in vitro study of the proliferation and growth of ovine osteoblast-like cells on various alloplastic biomaterials manufactured for augmentation and reconstruction of tissue or bone defects

In this in vitro study ovine osteoblast-like cells were cultured on seven different alloplastic biomaterials used for augmentation and for reconstruction of bone defects in dental and craniomaxillofacial surgery. The aim of this study was to examine the growth behaviour (viability, cell density and morphology) of ovine osteoblast-like cells on the investigated biomaterials to get knowledge which biomaterial is qualified to act as a cell carrier system in further in vivo experiments. The biomaterials were either synthetically manufactured or of natural origin. As synthetically manufactured biomaterials Ethisorb, MakroSorb, PalacosR, and PDS film were used. As biomaterials of natural origin BeriplastP, Bio-Oss and Titanmesh were investigated. The cell proliferation and cell colonization were analyzed by a proliferation assay and scanning electron microscopy. Osteoblast-like cells proliferated and attached on all biomaterials, except on Beriplast. On Ethisorb the highest cell proliferation rate was measured followed by PalacosR. Both biomaterials offer suitable growth and proliferation conditions for ovine osteoblast-like cells. The proliferation rates of Bio-Oss, MakroSorb, PDS-film and Titanmesh were low and SEM examinations of these materials showed less spread osteoblast-like cells. The results showed that ovine osteoblast-like cells appear to be sensitive to substrate composition and topography. This in vitro study provides the basis for further in vivo studies using the sheep model to examine the biocompatibility and the long-term interaction between the test material and tissue (bone regeneration).     

Silk fibroin modified porous poly(ε-caprolactone) scaffold for human fibroblast culture in vitro

In order to develop scaffolds with improved biocompatibility for cell culture, hybrid scaffolds were fabricated by modifying poly(epsilon-caprolactone) (PCL) with silk fibroin (SF) in a porous structure. Scanning electronic microscopy revealed that the morphology of the PCL-SF hybrid scaffold was affected by the concentration of the SF solution. Availability of SF on the surface and the conformational transition induced by methanol treatment were proved by attenuated total reflection Fourier transformed infrared spectroscopy (ATR-FTIR), and wettability of the hybrid scaffold was greatly improved. To evaluate scaffold biocompatibility, human fibroblasts were cultured on the hybrid scaffold with the unmodified PCL scaffold as control. An MTT assay indicated that although fewer cells were initially held on the hybrid scaffold after one day of culture, comparable cell numbers were achieved after four days and significantly more cells proliferated on the hybrid after seven days. The cell morphology also indicated that the PCL-SF hybrid scaffold was favorable for cell culture. This study suggests that surface modification with SF would be an effective way to improve the biocompatibility of PCL, facilitating its application in practical tissue engineering.     

Development,characterization, and in vitro bioactivity studies of sol–gel bioactive glass/poly(l-lactide) nanocomposite scaffolds

Developing materials combining the advantages of synthetic polymers and bioactive glass nanoparticles can provide an efficient bone engineering scaffold. In this study, sol–gel bioactive glass (SG) nanoparticles were synthesized by quick alkali-mediation; sol–gel derived bioactive glass/poly(l-lactide) nanocomposite scaffolds were then developed. The influence of the glass content on the porosity of nanocomposite scaffolds was evaluated by SEM. The results showed that the neat polymer scaffold (PLA) has a highly interconnected porous structure with a maximum pore size of about 250 μm. For the composite scaffold containing 25 wt.% glass (SGP25), the decrease in the maximum pore size, (to about 200 μm) was not significant while for the SGP50 composite scaffold containing 50 wt.% glass it was a significant decrease (to about 100 μm). The apparent porosity of the scaffolds was 56.56% ± 7.15, 54.14% ± 3.84, and 53.11% ± 3.99 for PLA, SGP25, and, SGP50 respectively. FT-IR, TGA, and XRD results revealed some interaction of the glass filler with the polymeric matrix in the scaffolds. The degradation study showed that, by increasing the glass content in the scaffolds, the water absorption decreased, the weight loss increased, and the cumulative ion concentrations released from them also increased. This indicates the possibility of modulating the degradation rate by varying the glass/polymer ratio. At the end of the incubation period, the weight losses were around 5.44% ± 0.96, 32.50% ± 2.73, and 41.47% ± 3.02 for the PLA, SGP25, and SGP50, respectively. Moreover, the water uptake reached 119.65% ± 18.88 and 93.39% ± 13.01 for SGP25 and SGP50, respectively. The addition of the SG to the scaffolds was found to enhance their in vitro bioactivity. Therefore, these nanocomposite scaffolds have a potential to be applied in bone engineering. All data are expressed as mean ± standard deviation (n = 3).     

Differentiation of human adipose-derived stem cells seeded on mineralized electrospun co-axial poly(ε-caprolactone) (PCL)/gelatin nanofibers

Mineralized poly(ε-caprolactone)/gelatin core–shell nanofibers were prepared via co-axial electrospinning and subsequent incubation in biomimetic simulated body fluid containing ten times the calcium and phosphate ion concentrations found in human blood plasma. The deposition of calcium phosphate on the nanofiber surfaces was investigated through scanning electronic microscopy and X-ray diffraction. Energy dispersive spectroscopy results indicated that calcium-deficient hydroxyapatite had grown on the fibers. Fourier transform infrared spectroscopy analysis suggested the presence of hydroxyl-carbonate-apatite. The results of a viability assay (MTT) and alkaline phosphatase activity analysis suggested that these mineralized matrices promote osteogenic differentiation of human adipose-derived stem cells (hASCs) when cultured in an osteogenic medium and have the potential to be used as a scaffold in bone tissue engineering. hASCs cultured in the presence of nanofibers in endothelial differentiation medium showed lower rates of proliferation than cells cultured without the nanofibers. However, endothelial cell markers were detected in cells cultured in the presence of nanofibers in endothelial differentiation medium.     

The influence of lithium fluoride on in vitro biocompatibility and bioactivity of calcium aluminate–PMMA composite cement

The objective of this study is to assess the influence of lithium fluoride on in vitro biocompatibility and bioactivity of calcium aluminate (CA)-polymethylmethacrylate (PMMA) composite cement exhibiting quick setting time ( < 15 min), low exothermic temperature (< 47 degrees C), and high compressive strength (> 100 MPa). The biocompatibility was measured by examining cytotoxicity tests such as the agar diffusion test with L929 cell line and the hemolysis test with fresh rabbit blood. To estimate the bioactivity of CA-PMMA composite cement, we determined hydroxyapatite (HAp) formation on the surface of composite cement in the simulated body (SBF) solution by using thin-film XRD, XPS, SEM, EPMA and ICP-AES. The results of biocompatibility tests indicated that all experimental compositions of this study had no cytotoxicity and no hemolysis so that there was no cytotoxicity with regard to non-reacted monomers (MMA and TEGDMA) and lithium fluoride. The results of bioactivity tests revealed that CA-PMMA composite cement without lithium fluoride did not form HAp on its surface after 60 days of soaking in the SBF. On the other hand, LiAl2(OH)7 . 2H2O and HAp were formed on the surface of CA-PMMA composite cement including 1.0% by weight of lithium fluoride after 7 and 15 days of soaking in the SBF, respectively. The 5 microm of LiAl2(OH)7 . 2H2O and HAp mixed layers were formed on the surface of specimen after 60 days of soaking in the SBF.     

The influence of starch gelatinization on the rheological,thermal, and morphological properties of poly(ɛ-caprolactone) with corn starch blends

Blends of poly(ɛ-caprolactone) (PCL) with gelatinized and nongelatinized corn starch (PCL/starch ratios of 75/25, 50/50, and 25/75 wt.%) were prepared by mechanical processing and characterized by their melt flow index (MFI), water absorption (WA), differential scanning calorimetry (DSC), and light microscopy (LM). The MFI showed that the viscosity of the blends increased with increasing starch content and that gelatinized starch made the blends less viscous. Starch made the blends more susceptible to WA. The rupture of the starch granules caused by gelatinization increased the WA and the loss of soluble matter. DSC showed that starch reduced the crystallinity of PCL in the blends. LM showed good dispersion of the starch in the PCL matrix and also showed that the blends with nongelatinized starch had poor interfacial adhesion.     

Evaluation of water sorption property and in vitro blood compatibility of poly(2-hydroxyethyl methacrylate) (PHEMA) based semi interpenetrating polymer networks (IPNs)

pH responsive smart biomaterials of gelatin and poly(2-hydroxyethyl methacrylate-co-acrylic acid) were synthesized by redox polymerization and characterized by FTIR, Environmental Scanning Electron Microscopy (ESEM). The prepared environmental responsive biomaterials containing polyelectrolyte segments were assessed for their water sorption potential under varying experimental conditions. The diffusion mechanism of transport of water molecules arising due to solvent-polymer interaction was also analysed to predict the behaviour of continuously relaxing macromolecular chains. The in vitro blood compatibility of the prepared polymeric hydrophilic materials was evaluated by methods such as blood clot formation, platelet adhesion, percent haemolysis and protein-adsorption study on the surface of the prepared biomaterials.     

The influence of a PHI-5-loaded silicone membrane,on cutaneous wound healing in vivo

This study investigated whether a novel ionogenic substance, containing amongst others zinc and rubidium (PHI-5; Dermagenics Inc, Memphis, TN, USA), could improve the healing of full-thickness skin wounds. Uniform wounds were created on the right flank of guinea pigs. Micro-grooved silicone rubber membranes, containing 0 (controls), 1.25, 5.00, or 10.00 μg PHI-5, were sutured onto this wound. Standardized digital wound photographs were made after 1, 3, and 6 weeks. Also, wound biopsies were taken after 3 and 6 weeks for histological and histomorphometrical evaluation. For all study groups, 6 animals were used. Analysis of the 1-week digital photographs showed that the surface area of the wounds decreased significantly, with an increasing PHI-5 concentration. No other differences were found in the wound photographs. Also, no differences were measured in histomorphometry at 3 and 6 weeks. Concluding, in our study model a single application of PHI-5 did have a significant positive influence on initial wound healing.     

In vitro evaluation of freeze-dried bone allografts combined with platelet rich plasma and human bone marrow stromal cells for tissue engineering

Freeze-dried bone allograft (FDBA) might be more effective in combination with platelet rich plasma (PRP) and bone marrow stromal cells (BMSC) in accelerating bone healing. The isolation of BMSC through density gradient (pBMSC) is not extensively applicable in clinical practice, because it increases the risk of infection. Alternatively, BMSC can be concentrated by simple centrifugation (wBMSC) directly in the operating room. However, we do not know if wBMSC act in the same way as pBMSC. BMSC from 10 donors were tested whether, in the presence of a combination of FDBA and autologous PRP, the osteogenic differentiation of the cells concentrated by simple centrifugation (wBMSC + FDBA + PRP) was similar to that of pBMSC. Cell-associated alkaline phosphatase, osterix and fibroblast growth factor-2 were higher in wBMSC + FDBA + PRP. In conclusion, the combination of FDBA and PRP had a favouring effect on the differentiation towards osteoblasts and allowed BMSC concentrated by simple centrifugation to differentiate as fast as BMSC purified by density gradient.