Chitosan/poly(dl,lactide-co-glycolide) scaffolds for tissue engineering

Chitosan/poly(dl-lactide-co-glycolide) (Ch/dl PLG) composite scaffolds were fabricated by freeze-drying lyophilization, and were evaluated and compared for use as a bone regeneration scaffold through measurements of the compression mechanical properties of the porous scaffolds. Also, In vitro cell culture of Sprague?CDawley rat??s osteoblasts were used to evaluate the phenotype expression of cells in the scaffolds, characterizing the cellular adhesion, proliferation and alkaline phosphatase activity. The gene expression of osteocalcin, sialoprotein, alkaline phosphatase, Type I collagen and TGF??1 were confirmed in the samples; moreover, it was confirmed, the mineralization by IR spectra and EDS analysis. Our results thus show that Ch/dl PLG scaffolds are suitable for biological applications.     

In vitro response of primary rat osteoblasts to titania/hydroxyapatite coatings compared with transformed human osteoblast-like cells

The biocompatibility of titania/hydroxyapatite (TiO₂HA) composite coatings, at different ratio obtained by sol–gel process, was investigated studying the behavior of primary cultures of rat osteoblastic cells, isolated by femoral trabecular bone tissue. Moreover, the results have been compared with the response of human osteoblast-like MG63 cell line. Cytotoxicity of coatings was assessed by lactate dehydrogenase activity (LDH). The cellular behavior was analyzed by the cell proliferation (MTT test), cell morphology (SEM) and the biochemical markers evaluation of osteoblastic phenotype, such as alkaline phosphatase activity (ALP) and osteocalcin production. The results showed that TiO₂/HA coatings have no toxic effects and seemed to be a good support for cell adhesion and proliferation. Moreover, these materials allowed the differentiation of osteoblasts, stimulating the expression of alkaline phosphatase activity. The responses of the primary rat osteoblasts and human osteoblast-like MG63 cell line grown onto these coatings were similar in terms of proliferation and ALP activity. Differences were found considering the osteocalcin production. The results show that these coatings, thanks to their chemical composition and the deposition technique, are very promising for the potential orthopedic and dental applications.     

Osteopenic bone cell response to strontium-substituted hydroxyapatite

Ionic substitution is a powerful tool to improve the biological performance of calcium phosphate based materials. In this work, we investigated the response of primary cultures of rat osteoblasts derived from osteopenic (O-OB) bone to strontium substituted hydroxyapatite (SrHA), and to hydroxyapatite (HA) as reference material, compared to normal (N-OB) bone cells. Strontium (Sr) and calcium (Ca) cumulative releases in physiological solution are in agreement with the greater solubility of SrHA than HA, whereas the differences between the two materials are levelled off in DMEM, which significantly reduced ion release. O-OB cells grown on SrHA exhibited higher proliferation and increased values of the differentiation parameters. In particular, Sr substitution increased the levels of proliferation, alkaline phosphatase, and collagen type I, and down-regulated the production of interleukin-6 of O-OB cells, demonstrating a promising future of SrHA in the treatment of bone lesions and defects in the presence of osteoporotic bone.     

In vitro biocompatibility assessment of PHBV/Wollastonite composites

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing bone tissue engineering scaffolds, the in vitro biocompatibility of degradable and bioactive composites consisting of polyhydroxybutyrate-co-hydroxyvalerate (PHBV) and wollastonite (W) was studied by culturing osteoblasts on the PHBV/W substrates, and the cell adhesion, morphology, proliferation, and alkaline phosphatase (ALP) activity were evaluated. The results showed that the incorporation of wollastonite benefited osteoblasts adhesion and the osteoblasts cultured on the PHBV/W composite substrates spread better as compared to those on the pure PHBV after culturing for 3 h. In the prolonged incubation time, the osteoblasts cultured on the PHBV/W composite substrates revealed a higher proliferation and differentiation rate than those on the pure PHBV substrates. In addition, an increase of proliferation and differentiation rate was observed when the wollastonite content in the PHBV/W composites increased from 10 to 20 wt%. All of the results showed that the addition of wollastonite into PHBV could stimulate osteoblasts to proliferate and differentiate and the PHBV/W composites with wollastonite up to 20 wt% were more compatible than the pure PHBV materials for bone repair and bone tissue engineering.     

Physical and cytocompatibility properties of bioactive glass–polyvinyl alcohol–sodium alginate biocomposite foams prepared via sol–gel processing for trabecular bone regeneration

In the present work, biocomposite foams of bioactive glass along with polyvinyl alcohol and sodium alginate are designed and developed as a potential biomaterial for bone regeneration. These biocomposite foams have a low density of 0.92 g/cm³, providing desired property for bone tissue engineering applications. Biocomposite foams were prepared via surfactant foaming. Scanning electron microscopic characterization revealed pore size of 200–500 μm of the biocomposite foams. When these materials were incubated in simulated body fluid, hydroxyapatite layer formation was observed on the material surface. To confirm the cell viability and proliferation on these materials, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed with NIH 3T3 fibroblast cells and the results revealed good biocompatibility with the biocomposite foams. Cell adhesion studies further confirmed the biocompatibility of the scaffolds via cell attachment and ECM production. The optimally synthesized biocomposite foams had a good combination of physical properties with compressive strength of 1.64 MPa and elastic modulus of 18 MPa. In view of the favorable combination of physical and biological properties, the newly developed materials are considered to be suitable for regeneration of trabecular bone.     

Examining porous bio-active glass as a potential osteo-odonto-keratoprosthetic skirt material

Bio-active glass has been developed for use as a bone substitute with strong osteo-inductive capacity and the ability to form strong bonds with soft and hard tissue. The ability of this material to enhance tissue in-growth suggests its potential use as a substitute for the dental laminate of an osteo-odonto-keratoprosthesis. A preliminary in vitro investigation of porous bio-active glass as an OOKP skirt material was carried out. Porous glass structures were manufactured from bio-active glasses 1-98 and 28-04 containing varying oxide formulation (1-98, 28-04) and particle size range (250–315 μm for 1-98 and 28-04a, 315–500 μm for 28-04b). Dissolution of the porous glass structure and its effect on pH was measured. Structural 2D and 3D analysis of porous structures were performed. Cell culture experiments were carried out to study keratocyte adhesion and the inflammatory response induced by the porous glass materials. The dissolution results suggested that the porous structure made out of 1-98 dissolves faster than the structures made from glass 28-04. pH experiments showed that the dissolution of the porous glass increased the pH of the surrounding solution. The cell culture results showed that keratocytes adhered onto the surface of each of the porous glass structures, but cell adhesion and spreading was greatest for the 98a bio-glass. Cytokine production by all porous glass samples was similar to that of the negative control indicating that the glasses do not induce a cytokine driven inflammatory response. Cell culture results support the potential use of synthetic porous bio-glass as an OOKP skirt material in terms of limited inflammatory potential and capacity to induce and support tissue ingrowth.     

Robotic dispensing of composite scaffolds and in vitro responses of bone marrow stromal cells

The development of bioactive scaffolds with a designed pore configuration is of particular importance in bone tissue engineering. In this study, bone scaffolds with a controlled pore structure and a bioactive composition were produced using a robotic dispensing technique. A poly(ε-caprolactone) (PCL) and hydroxyapatite (HA) composite solution (PCL/HA = 1) was constructed into a 3-dimensional (3D) porous scaffold by fiber deposition and layer-by-layer assembly using a computer-aided robocasting machine. The in vitro tissue cell compatibility was examined using rat bone marrow stromal cells (rBMSCs). The adhesion and growth of cells onto the robotic dispensed scaffolds were observed to be limited by applying the conventional cell seeding technique. However, the initially adhered cells were viable on the scaffold surface. The alkaline phosphatase activity of the cells was significantly higher on the HA–PCL than on the PCL and control culture dish, suggesting that the robotic dispensed HA–PCL scaffold should stimulate the osteogenic differentiation of rBMSCs. Moreover, the expression of a series of bone-associated genes, including alkaline phosphatase and collagen type I, was highly up-regulated on the HA–PCL scaffold as compared to that on the pure PCL scaffold. Overall, the robotic dispensed HA–PCL is considered to find potential use as a bioactive 3D scaffold for bone tissue engineering. Seok-Jung Hong and Ishik Jeong contributed equally.     

Effect of hydroxyapatite porosity on growth and differentiation of human osteoblast-like cells

To study whether hydroxyapatite (HA) porosity can influence its osteoconductive properties, cell adhesion, proliferation and differentiation were compared in human osteoblast-like cells grown on HA disks of different porosity (A = 20%, B = 40%, C = 60%). Human osteoblast-like cells were isolated and characterized. Proliferation rate and alkaline phosphatase (ALP) activity were assessed at 3, 7, 15, 21, and 28 days. Type I collagen and osteonectin production were demonstrated with fluorescence microscopy and osteoblast adhesion studied at 7 and 21 days by scanning electron microscopic analysis. Cell growth on HA was three- to six-fold lower than on polystyrene control disks. At 28 days, 2141 (±350) cells/well grew on the most porous disks (Group C), with highly significant differences from controls (p < 0.005). The ALP production was 2–3 fold lower on HA than on plastic. In the Group C the mean ALP activity was of 2.95 (±0.07) UI/well after 28 days, higher than in the other two groups. At 21 and 28 days, proliferation rate and ALP activity on the three HA cultures were significantly different (p < 0.05). A decrease in cell population and increased ALP activity were observed on the most porous material, and high proliferation and poor differentiation rates on the less porous disks.     

Increased osteoblast functions on nanophase titania dispersed in poly-lactic-co-glycolic acid composites

The design of nanophase titania/poly-lactic-co-glycolic acid (PLGA) composites offers an exciting approach to combine the advantages of a degradable polymer with nano-size ceramic grains to optimize physical and biological properties for bone regeneration. Importantly, nanophase titania mimics the size scale of constituent components of bone since it is a nanostructured composite composed of nanometre dimensioned hydroxyapatite well dispersed in a mostly collagen matrix. For these reasons, the objective of the present in vitro study was to investigate osteoblast (bone-forming cell) adhesion and long-term functions on nanophase titania/PLGA composites. Since nanophase titania tended to significantly agglomerate when added to polymers, different sonication output powers were applied in this study to improve titania dispersion. Results demonstrated that the dispersion of titania in PLGA was enhanced by increasing the intensity of sonication and that greater osteoblast adhesion correlated with improved nanophase titania dispersion in PLGA. Moreover, results correlated better osteoblast long-term functions, such as alkaline phosphatase activity and calcium-containing mineral deposition, on nanophase titania/PLGA composites compared to plain PLGA. In fact, the greatest collagen production by osteoblasts occurred when cultured on nanophase titania sonicated in PLGA at the highest powers. In this manner, the present study demonstrates that PLGA composites with well dispersed nanophase titania can enhance osteoblast functions necessary for improved bone tissue engineering applications.     

The fundamental parameters of chitosan in polymer scaffolds affecting osteoblasts (MC3T3-E1)

The aim of this study was to investigate the degree of deacetylation (DD) and molecular weight (MW) of chitosan within chitosan–collagen scaffolds on mouse osteoblasts (MC3T3-E1). The chitosan–collagen scaffolds were fabricated by freeze-drying technique. The studies on cell attachment and proliferation, alkaline phosphatase (ALP) activity, cell morphology, and mineralized nodule formation by osteoblasts on scaffolds were investigated. No statistically significant difference was found on cell attachment, but the chitosan–collagen scaffolds with low-DD chitosan had a statistically significantly (P < 0.05) higher proliferative effect and ALP activity than those scaffolds with high-DD chitosan, regardless of molecular weight. Scanning electron images demonstrated that MC3T3-E1 cells grew well on all test scaffolds; on the contrary, mineralized nodule formation was not found. In conclusion, the DD of chitosan is a crucial factor for MC3T3-E1 cells and it should be considered in further applications for bone tissue engineering.     

Functionalisation of PLLA nanofiber scaffolds using a possible cooperative effect between collagen type I and BMP-2: impact on growth and osteogenic differentiation of human mesenchymal stem cells

Mesenchymal stem cell differentiation of osteoblasts is triggered by a series of signaling processes including integrin and bone morphogenetic protein (BMP), which therefore act in a cooperative manner. The aim of this study was to analyze whether these processes can be remodeled in an artificial poly-(l)-lactide acid (PLLA) based nanofiber scaffold. Matrices composed of PLLA-collagen type I or BMP-2 incorporated PLLA-collagen type I were seeded with human mesenchymal stem cells (hMSC) and cultivated over a period of 22 days, either under growth or osteoinductive conditions. During the course of culture, gene expression of alkaline phosphatase (ALP), osteocalcin (OC) and collagen I (COL-I) as well as Smad5 and focal adhesion kinase (FAK), two signal transduction molecules involved in BMP-2 or integrin signaling were analyzed. Furthermore, calcium and collagen I deposition, as well as cell densities and proliferation, were determined using fluorescence microscopy. The incorporation of BMP-2 into PLLA-collagen type I nanofibers resulted in a decrease in diameter as well as pore sizes of the scaffold. Mesenchymal stem cells showed better adherence and a reduced proliferation on BMP-containing scaffolds. This was accompanied by an increase in gene expression of ALP, OC and COL-I. Furthermore the presence of BMP-2 resulted in an upregulation of FAK, while collagen had an impact on the gene expression of Smad5. Therefore these different strategies can be combined in order to enhance the osteoblast differentiation of hMSC on PLLA based nanofiber scaffold. By doing this, different signal transduction pathways seem to be up regulated.     

Physico-chemical and in vitro biological evaluation of strontium/calcium silicophosphate glass

Strontium is known to reduce bone resorption and stimulate bone formation. Incorporation of strontium into calcium phosphate bioceramics has been widely reported. In this work, calcium and calcium/strontium silicophosphate glasses were synthesized from the sol–gel process and their rheological, thermal, and in vitro biological properties were studied and compared to each other. The results showed that the gel viscosity and thus the rate of gel formation increased by using strontium in glass composition and by increasing aging temperature. In strontium-containing glass, the crystallization temperature increased and the type of the crystallized phase was different to that of strontium-free glass. Both glasses favored precipitation of calcium phosphate layer when they were soaked in simulated body fluid; however strontium seemed to retard the rate of precipitation slightly. The in vitro biodegradation rate of the strontium/calcium silicophosphate glass was higher than that of strontium-free one. The cell culture experiments carried out using rat calvaria osteoblasts showed that the incorporation of strontium into the glass composition stimulated proliferation of the cells and enhanced their alkaline phosphatase activity, depending on cell culture period.     

Mineralization of Collagen‐Coated Electrospun Poly(lactide‐co‐glycolide) Nanofibrous Mesh to Enhance Growth and Differentiation of Osteoblasts and Bone Marrow Mesenchymal Stem Cells

To obtain the biomimetic scaffolding materials for bone tissue engineering, poly(lactide‐co‐glycolide) (PLGA) nanofibrous mesh (NFM) was mineralized in a 5× simulated body fluid (SBF) for different time after it was treated by air plasma for 15 min and subsequent collagen coating. The apatite particles were nucleated on the surface of individual nanofibers, gradually grew up, and finally covered the whole NFM surface. The mineral aggregates were mainly composed of tiny hydroxyapatite (HA) nanoparticles, whose content reached a constant value of 54 µg · cm^?2 after 9 days. The collagen coating and apatite deposition enhanced the NFM strength pronouncedly too. In vitro cell culture demonstrated that the non‐ or less mineralized NFMs were more beneficial of cell spreading and proliferation than those highly mineralized NFMs, but the latter ones could strongly promote secretion of alkaline phosphatase (ALP) by osteoblasts after cultured for 14 days. Moreover, the highly mineralized NFMs also could significantly up‐regulated ALP activity and calcium synthesis of bone marrow mesenchymal stem cells (BMSCs), demonstrating that these NFMs are more favorable of the osteoblast phenotype expression and osteogenic induction. Therefore, the biomimetic apatite deposited PLGA/collagen NFM could be a promising candidate scaffold for bone tissue engineering.     

Novel bone scaffolds of electrospun polycaprolactone fibers filled with nanoparticles

Novel bone scaffolding materials were successfully fabricated by electrospinning from polycaprolactone (PCL) solutions containing nanoparticles of calcium carbonate (CaCO3) or hydroxyapatite (HA). The potential use of the electrospun fibrous scaffolds for bone regeneration was evaluated in vitro with human osteoblasts (SaOS2) in terms of attachment, proliferation, and alkaline phosphatase (ALP) activity of the cells that were cultured directly on the scaffolds. The results were compared with those on corresponding solution-cast film scaffolds and tissue-culture polystyrene plate (TCPS). It was found that all of the fibrous scaffolds promoted much better adhesion and proliferation of cells than the corresponding film scaffolds and TCPS. Interestingly, the cells that were seeded on all of the fibrous scaffolds appeared to be well-expanded and attach on the fiber surface very well even only about 1 hr in culture, while those seeded on all of the film scaffolds and the glass substrate were still in round shape. Among the various fibrous scaffolds investigated, the one that was filled with 1.0% HA showed the highest ALP activity. Finally, all of the fibrous scaffolds exhibited much greater tensile strength at yield than all of the corresponding film scaffolds.     

泡沫复制法制备多孔钛及表面水热碱处理改性

采用聚氨酯泡沫复制法成功的制备出孔径可控、孔隙率高且三维贯通的多孔钛.采用水热碱处理法对多孔钛进行表面改性,以提高其生物活性.改性后多孔钛的表面形貌发生了改变,呈均匀分布的三维网状微纳米结构.将改性后的多孔钛浸泡在SCPS中检测其矿化能力.研究结果表明,改性后的多孔钛能加速磷灰石的矿化,其中三维网状微纳米结构是加速矿化的主要因素.同时考察了改性后的多孔钛表面大鼠颅盖骨成骨细胞(MC3T3-E1)的黏附铺展情况.细胞培养表明细胞在改性后的多孔钛表面能较好的黏附铺展.上述研究结果表明水热碱处理改性后的多孔钛具有较好的生物活性及生物相容性.     

Titanium oxide nanotubes for bone regeneration

Titanium oxide nanotubes with Ca ions on their surfaces were prepared as 2 mm cylindrical inserts and placed into surgically created bone defects in the femurs of Wistar rats. On day 3, fibroblast-like cells were present on the surface of the nanotube inserts and fibers were observed by scanning electron microscopy (SEM). On day 7, cells with alkaline phosphatase activity were present and identified as osteoblasts by SEM and transmission electron microscopy. New bone matrices were observed in and around the porous nanotube inserts by light microscopy. Compared with clinically used hydroxyapatite and tricalcium phosphate, beta-titanium oxide nanotubes promote faster acquisition and development of osteoblasts and bone tissues and have better bone regenerating ability after one week.     

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     

Poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) based tissue engineering matrices

In this study, the aim was to produce tissue-engineered bone using osteoblasts and a novel matrix material, poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV). In order to prepare a porous PHBV matrix with uniform pore size, sucrose crystals were loaded in the foam and then leached leaving pores behind. The surface of the PHBV matrix was treated with rf-oxygen plasma to increase the surface hydrophilicity. SEM examination of the PHBV matrices was carried out. Stability of PHBV foams in aqueous media was studied. The pH decrease is an indication of the degradation extent. The weight and density were unchanged for a period of 120 days but then a significant decrease was observed for the rest of the study. Osteoblast cells were then isolated from rat bone marrow and seeded onto PHBV matrices. The metabolization and proliferation on the foams was determined with MTS assay which showed that osteoblasts proliferated on PHBV. It was also found that cells proliferated better on large pore size foams (300–500 m) than on the small pore size foams (75–300 m). Production of ALP was measured spectrophotometrically. The present study demonstrated that PHBV matrices are suitable substrates for osteoblast proliferation and differentiation.     

Porous silicon confers bioactivity to polycaprolactone composites in vitro

Silicon is an essential element for healthy bone development and supplementation with its bioavailable form (silicic acid) leads to enhancement of osteogenesis both in vivo and in vitro. Porous silicon (pSi) is a novel material with emerging applications in opto-electronics and drug delivery which dissolves to yield silicic acid as the sole degradation product, allowing the specific importance of soluble silicates for biomaterials to be investigated in isolation without the elution of other ionic species. Using polycaprolactone as a bioresorbable carrier for porous silicon microparticles, we found that composites containing pSi yielded more than twice the amount of bioavailable silicic acid than composites containing the same mass of 45S5 Bioglass. When incubated in a simulated body fluid, the addition of pSi to polycaprolactone significantly increased the deposition of calcium phosphate. Interestingly, the apatites formed had a Ca:P ratio directly proportional to the silicic acid concentration, indicating that silicon-substituted hydroxyapatites were being spontaneously formed as a first order reaction. Primary human osteoblasts cultured on the surface of the composite exhibited peak alkaline phosphatase activity at day 14, with a proportional relationship between pSi content and both osteoblast proliferation and collagen production over 4 weeks. Culturing the composite with J744A.1 murine macrophages demonstrated that porous silicon does not elicit an immune response and may even inhibit it. Porous silicon may therefore be an important next generation biomaterial with unique properties for applications in orthopaedic tissue engineering.     

Osteocompatibility and osteoinductive potential of supermacroporous polyvinyl alcohol-TEOS-Agarose-CaCl2 (PTAgC) biocomposite cryogels

Bone tissue engineering majorly focuses on the development of biomaterials which have the capability to mimic bone as well as the ability to induce bone formation. To this direction, we have prepared supermacroporous polyvinyl alcohol-TEOS-Agarose-CaCl₂ (PTAgC) biocomposite cryogels having a uniform porous structure with an interconnected porosity of 77 ± 0.16 % and pore size of 190 ± 0.78 μm, as determined by scanning electron microscopic and micro-computed tomographic analyses. These biocomposite cryogels show an osteocompatible response towards Saos-2 human osteoblasts as analyzed via MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, alkaline phosphatase (ALP) assay and cell adhesion behaviour showing a flattened morphology of the cells on the cryogel surface. The property of bioactivity was also observed on the surface of these biomaterials. Further, we also explored the osteoinductive potential of these biocomposite cryogels by the analysis of osteogenic differentiation of C2C12 myoblasts after seeding onto these biocomposite cryogels. The results indicate that these biocomposite cryogels indeed show an osteoinductive potential as we could observe the presence of respective markers for different stages during osteoblast maturation. During early timepoints, higher alkaline phosphatase production via ALP assay and BCIP/NBT staining was observed in the case of biocomposite cryogel seeded cells suggesting the osteoblastic differentiation of C2C12 cells. Whereas, during later timepoints, formation of calcium-phosphate like crystals was confirmed by von-kossa staining, further indicating towards the onset of mineralization phase during osteoblast maturation. Therefore, these results suggest that PTAgC biocomposite cryogels can form an important part of bone tissue engineered biomaterials due to their osteocompatible behaviour and osteoinductive potential.