Bone tissue engineering on calcium phosphate-coated titanium plates utilizing cultured rat bone marrow cells: a preliminary study

The use of osteoinductive in vitro tissue-coated implants in orthopaedic and dental surgery (e.g. revision hip arthroplasty), could result in a better fixation of these implants. However, this tissue engineering technology has only proved to be effective in porous materials and not on flat implant surfaces. In this study we have demonstrated that calcium phosphate-coated titanium plates with a layer of cultured osteogenic cells and their extracellular matrix can initiate bone formation in vivo. Both primary and subcultured rat bone marrow cells were grown on to biomimetic calcium phosphate-coated titanium plates. After 7 d of culture, in the presence or absence of dexamethasone, the implants were subcutaneously implanted in nude mice for 4 wk. Control samples, which consisted of calcium phosphate-coated plates without cultured cells and porous calcium phosphate particles with or without cultured cells, were also implanted subcutaneously. At autopsy, no bone formation could be detected on any of the control samples without cells and samples with subcultured cells, which were primary cultured in medium without dexamethasone. In contrast, clear de novo bone formation could be observed on the calcium phosphate-coated plates and in the porous calcium phosphate particles with primary or subcultured cells, which had been continuously cultured in medium with dexamethasone. These results indicate that this hybrid technology offers great potential for the fixation of flat bone replacement implants (e.g. artificial hips) in inferior bone in the future. © 1998 Kluwer Academic Publishers     

Osteoblast cell response to nanoscale SiO2/ZrO2 particulate-reinforced titanium composites and scaffolds by powder metallurgy

The strength of porous pure titanium (Ti) scaffold decreases dramatically with the introduction of porosity and might become lower than that of natural bone when with high porosity. To simultaneously meet the requirements of low-elastic modulus and appropriate strength for implant materials, it is necessary to develop new biocompatible Ti-based composites that are stronger than those currently available while providing low-elastic modulus and adequate strength when they are scaffolded into a porous structure. In this study, new particulate-reinforced Ti-based composites with nanoscale oxide particles of SiO₂ and ZrO₂ were prepared using a powder metallurgical method. The strengths of the new particulate-reinforced titanium composites were found to be significantly higher than that of a pure Ti. Cell culture results revealed that the articulate-reinforced titanium composites showed excellent biocompatibility and cell adhesion. Human osteoblast-like SaOS2 cells grew and spread well on the surfaces of the new titanium composites. The present study illustrated the feasibility of using the particulate-reinforced titanium composites as an orthopaedic implant material.     

Osteoblast proliferation and maturation on bioactive fiber-reinforced composite surface

The objective of this study was to evaluate the proliferation and osteogenic potential of bone-marrow derived osteoblast-like cells on fiber-reinforced composite (FRC) substrates with and without bioactive glass surface modification. Three FRC materials were fabricated for the study: (a) grit-blasted FRC, (b) grit-blasted FRC with bidirectional net reinforcement and (c) FRC with bioactive glass (BAG) coating. Rat bone-marrow derived osteoblast-like cells were harvested and cultured on experimental material plates and on cp. titanium plates (control) for 21 days. The materials' surfaces were characterized by roughness testing and scanning electron microscopy. Cell growth and differentiation kinetics were subsequently investigated by evaluating proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) and bone sialoprotein (BSP) production. On day 14, the cell proliferation was significantly lower (P < 0.05) on FRC-BAG than on titanium and FRC. The proliferation on the other three materials was equal throughout the experiment. The maximal ALP activities on FRC, FRC-Net, and titanium were observed on day 21, whereas FRC-BAG had already reached the maximal level on day 14. Expression of osteoblastic markers (OC, BSP) indicates that the fastest osteogenic differentiation takes place on FRC after 7 days. In contrast, a slower differentiation process was observed on titanium than on any other tested material (P < 0.015) at 21 days, as was confirmed by increased mRNA expression of OC and BSP. It can be concluded that the proliferation and maturation of osteoblast-like cells on FRC appears to be comparable to titanium. Presence of BAG enhances cell maturation.     

Osteoblast response and calcium deposition on phospholipid modified surfaces

In this study, the effect of calcium phosphate complexed phospholipid (Ca-PL-PO4) coatings on solid surfaces on the in vitro calcium (Ca) deposition and on the osteoblast responses was evaluated. Commercially available phospholipids were converted to their Ca-PL-PO4, and were coated on glass Petri dishes. The coated dishes were immersed in the simulated body fluid for up to 14 days under sterilized conditions at 37 degrees C, and the amount of calcium (Ca) deposited was quantified. Similarly, by measuring the alkaline phosphatase specific activity, the differentiation of osteoblast precursor cells were evaluated after seeding the cells on Ca-PL-PO4 coated cell culture plastics. It was observed that all Ca-PL-PO4 enhanced Ca deposition on coated surfaces. The, polar head group of phospholipids in coated surfaces was observed to have an influence on the Ca deposition as well as the osteoblast differentiation. Among the phospholipids evaluated, phosphatidylserine (Ca-PS-PO4) exhibited the strongest calcium deposition and more enhanced alkaline phosphatase specific activity. It was therefore concluded from this study that Ca-PS-PO4 surface modification may be an alternative method for enhancing bone-implant interactions.     

Biomimetic bone scaffolds based on chitosan and calcium phosphates

This paper reports a novel biomimetic technique for obtaining chitosan-calcium phosphate composite scaffolds (Cs-CP) by calcium phosphate precipitation from its precursors,CaCl₂ and NaH₂PO₄ in the presence of ammonia and Cs, followed by product freeze-drying. The structural and morphological analysis showed the arrangement of CP onto Cs fibers, while Cs acting as glue to bind together the CP crystals. The presence of different forms of CP was evidenced, including hydroxyapatite, which is known to be involved in the formation of the new bone tissue in living organisms. Great mechanical properties of the scaffolds indicate their potential use in bone tissue engineering.     

Evaluation of the attachment,proliferation, and differentiation of osteoblast on a calcium carbonate coating on titanium surface

Titanium has been reported to have some limitations in dental and orthopaedic clinical application. This study described a coating process using a simple chemical method to prepare calcium carbonate coatings on smooth titanium (STi) and sandblasted and acid-etched titanium (SATi), and evaluated the biological response of the materials in vitro. The surfaces of STi, SATi, calcium carbonate coated STi (CC-STi) and calcium carbonate coated SATi (CC-SATi) were characterized for surface roughness, contact angles, surface morphology and surface chemistry. The morphology of MG63 cells cultured on the surfaces was observed by SEM and Immuno-fluorescence staining. Cell attachment/proliferation was assessed by MTT assay, and cell differentiation was evaluated by alkaline phosphatase (ALP) activity. MG63 was found to attach favorably to calcium carbonate crystals with longer cytoplasmic extensions on CC-STi and CC-SATi, resulting in lower cell proliferation but higher ALP activity when compared to STi and SATi respectively. Moreover, CC-SATi is more favorable than CC-STi in terms of biological response. In conclusion, the calcium carbonate coatings on titanium were supposed to improve the osteointegration process and stimulate osteoblast differentiation, especially in early stage. And this method could possibly be a feasible alternative option for future clinical application.     

Cell growth and function on calcium phosphate reinforced chitosan scaffolds

Macroporous chitosan scaffolds reinforced by calcium phosphate powders such as hydroxyapatite (HA) or calcium phosphate invert glass were fabricated using a thermally induced phase separation technique. Human osteoblast-like MG63 cells were cultured on the composite scaffolds for up to 11 days, and the cell growth and function were analyzed. The cell growth is much faster on the chitosan/HA scaffolds incorporated with the glass (CHG) than on the chitosan/HA scaffold without the glass (CH). The total protein content of cells were quantified and increased over time on both composites (CH, CHG) but was significantly higher on CHG after 7 days of culture. The cells on CHG also expressed significantly higher amount of alkaline phosphatase at days 7 and 11 and osteocalcin at day 7 than those on CH. The results suggested that the addition of glass in chitosan/hydroxyapatite composite scaffolds might enhance the proliferation and osteoblastic phenotype expression of MG63 cells. However, the chitosan-matrix scaffolds did not show higher phenotype expression of MG63 cells, in comparison with the TCPS plate, probably due to the degradation of chitosan and release of acidic byproducts. Larger amount of soluble calcium phosphate invert glasses should be added into the scaffolds to prevent chitosan from fast degradation that may affect the differentiation of osteoblast cells.     

Biomimetic calcium phosphate crystal mineralization on electrospun cellulose-based scaffolds

Novel cellulose based-scaffolds were studied for their ability to nucleate bioactive calcium phosphate crystals for future bone healing applications. Cellulose-based scaffolds were produced by electrospinning cellulose acetate (CA) dissolved in a mixture of acetone/dimethylacetamide (DMAc). The resulting nonwoven CA mats containing fibrils with diameters in the range of 200 nm to 1.5 μm were saponified by NaOH/ethanol for varying times to produce regenerated cellulose scaffolds. Biomimetic crystal growth nucleated from the fiber surface was studied as a function of surface chemistry. Regenerated cellulose scaffolds of varying treatments were soaked in simulated body fluid (SBF) solution. Scaffolds that were treated with CaCl(2), a mixture of carboxymethyl cellulose (CMC) and CaCl(2), and NaOH and CaCl(2), were analyzed using X-ray photoelectron spectroscopy, X-ray powder diffraction, and scanning electron microscopy to understand the growth of bioactive calcium phosphate (Ca-P) crystals as a function of surface treatment. The crystal structure of the nucleated Ca-P crystals had a diffraction pattern similar to that of hydroxyapatite, the mineralized component of bone. The study shows that the scaffold surface chemistry can be manipulated, providing numerous routes to engineer cellulosic substrates for the requirements of scaffolding.     

Fabrication and characterization of porous calcium polyphosphate scaffolds

Porous calcium polyphosphate (CPP) scaffolds with different polymerization degree and crystalline phases were prepared, and then analyzed by scanning electron microscopy (SEM), Thermmogravimetry (TG) and X-ray diffraction (XRD). Number average polymerization degree was calculated by analyzing the calcining process of raw material Ca(H₂PO₄)₂, as a polycondensation reaction. Amorphous CPP were prepared by the quenching from the melt of Ca(H₂PO₄)₂ after calcining, and CPP with different polymerization degree was prepared by controlling the calcining time. Meanwhile, CPP with the same polymerization degree was prepared to amorphous or different crystalline phases CPP which was made from crystallization of amorphous CPP. In vitro degradation studies using 0.1 M of tris-buffered solution were performed to assess the effect of polymerization degree or crystalline phases on mechanical properties and weight loss of the samples. With the increase of polymerization degree, the weight loss during the degradation decreased, contrarily the strength of CPP increased. The degradation velocity of amorphous CPP, α-CPP, β-CPP and γ-CPP with the same polymerization degree decreased in turn at the same period. The full weight loss period of CPP can be controlled between 17 days and more than 1 year. The results of this study suggest that CPP ceramics have potential applications for bone tissue engineering.     

Reduced fibroblast adhesion and proliferation on plasma-modified titanium surfaces

Soft tissue complications are clinically relevant problems after osteosynthesis of fractures. The goal is to develop a method for reduction of fibroblast adhesion and proliferation on titanium implant surfaces by plasma polymerisation of the organo-silicon monomer hexamethyldisiloxane (HMDSO). HMDSO was deposited under continuous wave conditions in excess oxygen (ppHMDSO surface) and selected samples were further modified with an additional oxygen plasma (ppHMDSO + O₂ surface). Surface characterization was performed by scanning electron microscopy, profilometry, water contact angle measurements, infrared reflection absorption spectroscopy and X-ray photoelectron spectroscopy. In our experimental setup the mechanical properties, roughness and topography of the titanium were preserved, while surface chemistry was drastically changed. Fibroblast proliferation was assessed by alamarBlue assay, cell morphology by confocal microscopy visualization of eGFP-transducted fibroblasts, and cell viability by Annexine V/propidium iodide assay. Both modified surfaces, non-activated hydrophobic ppHMDSO and activated hydrophilic ppHMDSO + O₂ were able to dramatically reduce fibroblast colonization and proliferation compared to standard titanium. However, this effect was more strongly pronounced on the hydrophobic ppHMDSO surface, which caused reduced cell adhesion and prevented proliferation of fibroblasts. The results demonstrate that plasma modifications of titanium using HMDSO are valuable candidates for future developments in anti-adhesive and anti-proliferative coatings for titanium fracture implants.     

Properties of biocomposites based on titanium scaffolds with a different porosity

Open-porous titanium scaffolds have been widely investigated for orthopaedic and dental applications because of their ability to form composites via bone ingrowth into pores and promote implant fixation with mother bone. In this work, porous titanium scaffolds coated with a diamond-like carbon were produced, and their ability to form biocomposites was evaluated through in vivo experiments. Three types of the open-porous scaffolds made of spongy titanium granules (porosity 0.3, 0.4 and 0.5, Young’s modulus 4.4, 3.5 and 0.6 GPa) were implanted into a bone defect of sheep. Time dependences of the Young’s modulus of titanium scaffold–bone tissue biocomposites were determined through the measurement of Young’s modulus of the extracted scaffolds after 4, 8, 24 and 52 weeks of surgery. The Young’s modulus of biocomposite is dependent not only on the time of composite formation but also on the porosity of scaffold.     

Vaterite deposition on biodegradable polymer foam scaffolds for inducing bone-like hydroxycarbonate apatite coatings

Hydroxycarbonate apatite (HCA) coatings on the surface of bioresorbable materials for bone tissue engineering scaffolds were produced using macroporous poly(DL-lactide) (PDLLA) foams impregnated by calcium carbonate in vaterite crystalline form. Stable and homogeneous vaterite deposition on PDLLA foams was achieved using a slurry dipping technique. In vitro studies in simulated body fluid (SBF) were performed to induce formation of (HCA) on the surface of vaterite/PDLLA composite foams. HCA was detected after immersion of foams in SBF for 7 days. Hence, depositing vaterite on materials followed by immersion in SBF is confirmed to induce HCA coatings on the surface of the material. The HCA coated, bioactive and resorbable PDLLA foams are intended for use as bone tissue engineering scaffolds.     

Preliminary study on the adhesion and proliferation of human osteoblasts on starch-based scaffolds

Up to today, several techniques have been used to produce biodegradable porous scaffolds for tissue engineering. In this work, a new technique based on extrusion by using blowing agents in combination with a 50:50 (wt.%) blend of starch/cellulose acetate (SCA) was studied. The results show that by using this technique it was possible to obtain scaffolds with 70% of porosity and a fully interconnected network of pores, with sizes ranging from 200 to 500 μm. After their production, the mechanical properties of these scaffolds were tested, presenting a compressive modulus of 124.6±27.2 MPa and a compressive strength of 8.0±0.9 MPa. These values are within the best found in the literature and show that by using this technique, it is possible to produce scaffolds that, from a mechanical standpoint, may be suitable for bone tissue engineering. Cell culturing experiments showed that cells were viable and that there were no signs of cellular death after 3 weeks of culture. Finally, biochemical assays demonstrate that cells maintained the osteogenic phenotype throughout the experiment and deposition of mineralized extracellular matrix could be detected.     

Tenocyte proliferation on collagen scaffolds protects against degradation and improves scaffold properties

Tissue engineering scaffolds encourage cell proliferation whilst degrading to facilitate tissue regeneration. Their mechanical properties therefore change, decreasing due to scaffold degradation and increasing due to extracellular matrix deposition. This work compares the changing properties of collagen scaffolds incubated in culture medium, with and without human tenocytes, in order to investigate the relationship between degradation and tenocyte proliferation. The material properties of scaffolds are compared over 26 days using mechanical testing, differential scanning calorimetry, infra-red spectroscopy, and histology and biochemical assays. For medium-only scaffolds, the mechanical properties decrease rapidly, while culture medium sulfhydryl content increases significantly, with no significant changes in the denaturation temperature of scaffold collagen content. Conversely, the mechanical properties and collagen content of tenocyte-seeded scaffolds increase significantly while culture medium sulfhydryl content decreases and denaturation temperature remains the same. These results indicate that tenocytes proliferation both reduces the degradation of collagen scaffolds incubated in culture medium and produces scaffolds with improved properties.     

Biomimetic calcium phosphate coatings on nitric-acid-treated titanium surfaces

This study describes biomimetic calcium phosphate (Ca-P) coatings formation under simulated physiological conditions on Ti surfaces that go through nitric acid treatment (NT). In the present study, nitric acid treatment was used to treat Ti specimens so that Ti specimens could have the ability to induce Ca-P formation. After careful selection of the NT parameters, Ca-P coatings success fully formed on the nitric-acid-treated Ti surfaces in a supersaturated calcium phosphate solution (SCPS) and in the simulated body fluid (SBF). Before NT, the Ti specimen should go through mixed acid etching to increase its surface roughness because rough surfaces lead to good adherence between coatings and substrates. Amorphous Ca-P coatings were formed on the Ti surfaces by immersing the NT Ti specimens in SBF, while octacalcium phosphate (OCP) coatings were formed in the SCPS after 3 days of immersion. The study firstly proved that nitric acid treatment is not only just for surface passivation but also is another bioactive treatment as an alternative to the alkaline treatment and two-step method. The experimental results also confirmed that the conventional nitric acid treatment of a titanium surface does not increase the titanium oxide on the Ti surfaces. However, extending the nitric acid treatment time and enhancing the nitric acid treatment temperature help to increase Ti surface ability of Ca-P induction in simulated physiological environments. Ti specimens that had 600 min of NT at 60 °C had the best Ca-P induction ability under biomimetic conditions.     

Human-osteoblast proliferation and differentiation on grit-blasted and bioactive titanium for dental applications

Physico-chemical and topographical surface quality of commercially pure titanium (c.p. Ti) dental implants is one of the most influencing factors in the improvement of their osseointegration. In this sense, previously, a two-step method (2S) for obtaining bioactive blasted-rough titanium surfaces was developed for improving short-term (due to its bioactivity) and long-term (due to its roughness) osseointegration. This 2S-method consists of: (1) Grit blasting on titanium surface in order to roughen it, and (2) thermo-chemical (TCh) treatment in order to obtain a bioactive surface with bone-bonding ability. The aim of the present work is to evaluate the in vitro human-osteoblast response (proliferation, differentiation – ALP activity- and cell morphology-studied by environmental scanning electron microscopy) of rough c.p. Ti (grit blasted), bioactive c.p. Ti (thermo-chemically treated) and rough-bioactive c.p. Ti (2S-treated). Different grit materials (Al₂O₃ and SiC) have been used in order to investigate their influence. The results showed that cell adhesion was statistically higher for the rough and bioactive surfaces, whatever the grit used. Cells proliferated very well on all the c.p. Ti surfaces. If comparing groups with and without TCh (all other treatments being equal) the ALP was always higher in the groups with TCh, indicating stimulation of osteoblast differentiation because of TCh, more significantlly in the groups that were first blasted. Those ALP results were accompanied by a decrease in the value of proliferation, which shows the good behavior of the cells. This results suggest that a rough and bioactive-titanium surface obtained by 2S-treatment enhances adhesion and differentiation activity of human osteoblasts cells.     

Physicochemical properties and cytotoxicities of Sr-containing biphasic calcium phosphate bone scaffolds

This study demonstrates a new biomaterial system composed of Sr-containing hydroxyapatite (Sr-HA) and Sr-containing tricalcium phosphate (Sr-TCP), termed herein Sr-containing biphasic calcium phosphate (Sr-BCP). Furthermore, a series of new Sr-BCP porous scaffolds with tunable structure and properties has also been developed. These Sr-BCP scaffolds were obtained by in situ sintering of a series of composites formed by casting various Sr-containing calcium phosphate cement (Sr-CPC) into different rapid prototyping (RP) porous phenol formaldehyde resins, which acted as the negative moulds for controlling pore structures of the final scaffolds. Results show that the porous Sr-BCP scaffolds are composed of Sr-HA and Sr-TCP. The phase composition and the macro-structure of the Sr-BCP scaffold could be adjusted by controlling the processing parameters of the Sr-CPC pastes and the structure parameters of the RP negative mould, respectively. It is also found that both the compressive strength (CS) and the dissolving rate of the Sr-BCP scaffold significantly vary with their phase composition and macropore percentage. In particular, the compressive strength achieves a maximum CS level of 9.20 ± 1.30 MPa for the Sr-BCP scaffold with a Sr-HA/Sr-TCP weight ratio of 78:22, a macropore percentage of 30% (400–550 μm in size) and a total-porosity of 63.70%, significantly higher than that of the Sr-free BCP scaffold with similar porosity. All the extracts of the Sr-BCP scaffold exhibit no cytotoxicity. The current study shows that the incorporation of Sr plays an important role in positively improving the physicochemical properties of the BCP scaffold without introducing obvious cytotoxicity. It also reveals a potential clinical application for this material system as bone tissue engineering (BTE) scaffold.     

An electrodeposition method of calcium phosphate coatings on titanium alloy

Calcium phosphates coatings were deposited onto titanium alloy discs via en electrodeposition method. Titanium alloy discs were blasted with calcium phosphate particles, then etched in a mixture of nitric and fluoric acids and rinsed in demineralized water. The titanium alloy disc (cathode) and platinum mesh (anode) were immersed in a supersaturated calcium phosphate electrolyte buffered at pH 7.4 and connected to a current generator. The microstructure, chemical composition and crystallinity of the electrodeposited coatings were studied as function of time 10–120 min, temperature 25–80°C, current density 8–120 mA/cm², magnesium and hydrogen carbonate amounts (0.1–1 mM). Uniform calcium phosphate coatings were obtained in 30 min but coating thickness increased with deposition time. Raising the temperature of electrolyte resulted in more uniform coatings as ionic mobility increased. Low current density was preferable due to hydrogen gas evolving at the cathode, which disturbed the deposition of calcium phosphate crystals on titanium. The amounts of magnesium and hydrogen carbonate ions affected both the homogeneity and morphology of the coatings. This study showed that the electrodeposition method is efficient for coating titanium with osteoconductive calcium phosphate layers.     

焙烧时间对钛酸钙光催化性能的影响

以CaCO3和TiO2为原料,焙烧温度为1500℃,焙烧时间分别为2、4、6、8、10h的条件下焙烧得到CaTiO3。应用X射线衍射(XRD)、扫描电子显微镜(SEM)、傅里叶红外变换光谱仪(FT-IR)、紫外-可见光漫反射光谱仪(UV-Vis)对焙烧后的物料进行了表征。实验结果表明,焙烧时间对合成CaTiO3的纯度影响很小;焙烧时间的延长使得CaTiO3微观形貌从不规则多边形向光滑曲面形变化,颗粒团聚现象越来越明显,堆积密实,尺寸增大;焙烧时间对钛酸钙光吸收能力的影响很小。在光催化性能评价实验中,随着焙烧时间的延长,亚甲基蓝的降解率和降解速率降低,反应速率常数最多可降低接近50%;钛酸钙降解亚甲基蓝溶液比较符合一级反应动力学特征。     

Photocatalytic activity of neat and silicon-doped titanium(IV) oxide nanofibers prepared by combined sol-gel and electrospinning techniques

In the present contribution, the photocatalytic activity of neat and silicon-doped titanium(IV) oxide or titania fibers obtained by combined sol-gel and electrospinning techniques was reported for the first time. Both the calcination temperature and the presence of the silicon dopant affected a great deal the physical and chemical properties of the as-synthesized titania fibers. Increasing the calcination temperature resulted in the observed decrease in the fraction of the anatase phase and the observed increase in the size of the crystalline domains, while the presence of the silicon dopant caused the crystallite size to decrease and the fraction of the anatase phase to increase. The photocatalytic activity of both the neat and the silicon-doped titania fibers was investigated by following the photooxidative decomposition of methylene blue against that of the reference anatase titania. Both the neat and the silicon-doped titania fibers exhibited much better activity than the reference titania powder. In addition, the presence of silicon dopant enhanced the photocatalytic activity of the obtained titania fibers considerably.