Manufacture and evaluation of bioactive and biodegradable materials and scaffolds for tissue engineering

For tissue regeneration and tissue engineering applications, a number of bioactive and biodegradable composites, either porous or non-porous, were fabricated. The newly developed materials included tricalcium phosphate reinforced polyhydroxybutyrate and its copolymer, poorly crystallized hydroxyapatite reinforced chitin, and plasma sprayed hydroxyapatite reinforced poly(L-lactic acid). It was shown that these new materials could be successfully produced using the manufacturing techniques adopted. In vitro experiments revealed that the incorporation of bioceramic particles in biodegradable polymers rendered the composites bioactive and significantly improved the ability of composites to induce the formation of bone-like apatite on their surfaces. Degradation of composite scaffolds in simulated body fluid was observed and could be due to the simultaneous degradation of polymer matrix and dissolution of bioceramic particles.     

Electrophoretic deposition of silicon-substituted hydroxyapatite/poly(ε-caprolactone) composite coatings

Silicon-substituted hydroxyapatite/poly(ε-caprolactone) composite coatings were prepared on titanium substrate by electrophoretic deposition in n-butanol and chloroform mixture. The effect of the concentration of poly(ε-caprolactone) in suspension on the morphology and the microstructure of coatings were investigated, furthermore, the thermal behavior and in vitro bioactivity were also investigated. The results show that the coarse and accidented silicon-substituted hydroxyapatite/poly(ε-caprolactone) composite coatings were obtained by electrophoretic deposition when the concentration of poly(ε-caprolactone) in suspension was 6–16 g/l. The adsorption of poly(ε-caprolactone) on the surface of Si–HA particles hinders the electrophoretic deposition of Si–HA. The shear-testing experiments indicated that the addition of poly(ε-caprolactone) in suspension is in favor of improving the bonding strength of the coatings. After immersion in simulated body fluid for 8 days, silicon-substituted hydroxyapatite/poly(ε-caprolactone) composite coatings have the ability to induce the bone-like apatite formation.     

生物活性钛涂层

真空等离子喷涂的钛涂层经 ５．０ｍｏｌ／Ｌ ＮａＯＨ溶液处理后,将其浸泡在含 Ｃａ^２＋、ＨＰＯ_４^２－的模拟生理体液（ＦＣＳ和ＳＢＦ）中,考察涂层诱导羟基磷灰石生长过程,并评价其生物活性．用ＳＥＭ观察碱处理前后和在模拟生理体液中浸泡后钛涂层的形貌,用ＡＥＳ分析了碱处理前后钛涂层的表面成分;用ＸＲＤ、ＦＴ－ＩＲ和ＥＤＳ表征浸泡后涂层表面生长物的结构和成分;并测量了处理后钛涂层在浸泡过程中溶液中离子浓度和ｐＨ值的变化．结果表明,经处理的钛涂层在模拟生理体液中能诱导羟基磷灰石在其表面生长;在ＳＢＦ和ＦＣＳ分别形成碳酸羟基磷灰石层和含氧磷灰石的羟基磷灰石层．钛涂层的活性是由于碱处理后表面形成了网状和纤维状结构的Ｎａ－Ｔｉ－Ｏ化合物．这种化合物在模拟生理溶液中释放Ｎａ^＋,吸收Ｈ^＋;形成水化钛酸盐,诱导羟基磷灰石成核生长．     

Dynamic study of calcium phosphate formation on porous HA/TCP ceramics

Bone-like apatite formation on porous calcium phosphate ceramics was investigated in static simulated body fluid (SBF) and dynamic SBF at different flowing rates. The results of a 14-day immersion in static SBF showed that the formation of bone-like apatite occurred both on the surface and in the pores of the samples. When SBF flowed at the physiological flow rate in muscle (2 ml/100 ml⋅min), bone-like apatite could be detected only in internal surface of the pores of samples. The result that bone-like apatite formation could only be found in the pores when SBF flowed at physiological flow rate was consistent with that of porous calcium phosphate ceramics implanted in vivo: osteoinduction was only detected inside the pores of the porous calcium phosphate ceramics. This result implicates that the bone-like apatite may play an important role in the osteoinduction of Ca-P materials. The dynamic model used in this study may be better than usually used static immersion model in imitating the physiological condition of bone-like apatite formation. Dynamic SBF method is very useful to understand bone-like apatite formation in vivo and the mechanism of ectopic bone formation in calcium phosphate ceramics.     

Morphological and chemical characterisation of biomimetic bone like apatite formation on alkali treated Ti6Al4V titanium alloy

The present study is an attempt to enhance the apatite-forming ability of titanium metal induced by the alkaline (NaOH) treatment. A cell free culture medium, acellular DMEM solution was utilised to develop bone-like apatite on alkali-treated titanium alloy surface. The main advantage of this process is the development of bone like apatite with essential trace elements on the metallic substrate by using the DMEM culture medium as a soaking medium. The formed apatite deposits were investigated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDXS). The obtained results suggest that the method utilized in this work can be successfully applied to obtain deposition of uniform coatings of crystalline hydroxyapatite on alkali treated titanium substrates.     

Dissolution behaviour of plasma sprayed apatite coatings

Understanding the interaction of bioactive coatings with aqueous media is essential for development of systems possessing rapid osteointegration and durability. An in vitro study of a commercial, plasma sprayed hydroxyapatite coating has been undertaken. The coating behaviour when test coupons were immersed in water, simulated body fluid and foetal calf serum has been examined. The principal aim was to characterise, in detail, any structural changes to the coatings and, in particular, examine features of any new layers formed. The amorphous phase of the coating showed preferential dissolution in all media. The rate of dissolution was greatest in water and the process was initially retarded in the foetal calf serum. A nanocrystallite apatite layer was shown to precipitate on the coatings in all media although this was significantly enhanced in simulated body fluid. The features of this layer (e.g., lattice parameters, crystallite size etc.) were quantified by adopting a novel approach to the X-ray diffraction data analysis. The results are discussed in the context of similar studies and implications for in vivo behaviour.     

Formation of carbonate apatite on calcium phosphate coatings containing silver ions

The prerequisite for bioactive materials to bond to living bone is the formation of biologically equivalent carbonate apatite on their surfaces in the body. Calcium phosphate ceramic surfaces can be transformed to a biological apatite through a series of surface reactions including dissolution–precipitation and ion exchange. In the present work, apatite coatings with different crystallinity, compositions and crystal sizes, including a well-crystallized hydroxyapatite coating, were synthesized electrochemically and doped with silver ions in silver nitrate solution at room temperature. The formation of a new carbonate apatite on the surface of these coatings was investigated in an acellular simulated body fluid with ion concentrations comparable with those of human blood plasma, using scanning electron microscopy and Fourier transform-infrared spectroscopy. The results show that small quantities of silver ions incorporated into apatite coatings may have a strong stimulatory effect on the formation of carbonate apatite without adversely affecting the chemical stability of these coatings.     

Production and characterization of HA and SiHA coatings

Plasma sprayed hydroxyapatite (HA) coatings on metallic prostheses have been used clinically in dentistry and orthopedics since the mid 1980s. The coating properties are dependent on the spraying parameters. Since silicon-substituted hydroxyapatite (SiHA) has been shown to offer improved bioactivity over phase pure HA, SiHA coatings have the potential for enhanced performance in clinical application. In this study, phase pure HA and 0.8 wt% SiHA powders were synthesized with similar particle size distribution and morphology. The powders were plasma sprayed onto Ti–6Al–4V substrates at 37 kW and 40 kW plasma gun input power respectively. Four kinds of samples were prepared, HAC 37, HAC 40, SiHAC 37 and SiHAC 40. Materials characterization showed that the coatings were of relatively high phase purity. In vitro cell culture demonstrated that human osteoblast cells grew well on all samples, with the highest cell growth observed on SiHA coatings produced under the lower plasma gun input power.     

Plasma spraying of biologically derived hydroxyapatite on implantable materials

Plasma spraying of hydroxyapatite (HA) coatings on human implants is considered to provide a promising means of enhancing their biocompatibility and improving tissue growth. This paper briefly describes a method of extracting HA powder from a biological source, namely human teeth. The physical and chemical characteristics of the derived powder are studied and the suitability of this powder for plasma spraying applications is ascertained. The deposited coatings are found to retain the chemistry characteristic of the apatite. Typical results of metallographic and scanning electron microscopy (SEM) studies and hardness measurements on the sprayed HA coatings are presented.     

Formation of highly oriented hydroxyapatite in hydroxyapatite/titanium composite coating by radio-frequency thermal plasma spraying

Highly oriented hydroxyapatite (HA) coatings with excellent adhesion were successfully obtained on titanium (Ti) and titanium alloy through a radio-frequency thermal plasma spraying method. The ratio of HA and Ti powders supplied into the plasma was precisely controlled by two microfeeders so as to change the composition from Ti-rich to HA-rich toward the upper layer of the formed coatings. The bond (tensile) strength of the HA/Ti composite coatings was ca. 40–50 MPa. XRD patterns showed that the topmost HA layer of the coatings had an apatite structure with (00l) preferred orientation. The degree of this orientation showed a tendency to increase with an increase in the substrate temperature during spraying.     

Immersion behavior of hydroxyapatite (HA) powders before and after sintering

The immersion behavior of two different hydroxyapatite (HA) powders before and after sintering was investigated by soaking them in simulated body fluid (SBF) for various periods. The results showed that the mechanism of formation of bone-like apatite on the two HA powders was different due to their different phase composition. Moreover, after being sintered at a proper elevated temperature, the bioactivity of HA powders was increased.     

Microstructure and chemistry affects apatite nucleation on calcium phosphate bone graft substitutes

The bioactivity of calcium phosphate bone grafts of varying chemistry and strut-porosity was compared by determining the rate of formation of hydroxycarbonate apatite crystals on the material surface after being soaked in simulated body fluid for up to 30 days. Three groups of silicate-substituted hydroxyapatite material were tested, with each group comprising a different quantity of strut-porosity (23, 32, and 46 % volume). A commercially available porous β-tricalcium phosphate bone graft substitute was tested for comparison. Results indicate that strut-porosity of a material affects the potential for formation of a precursor to bone-like apatite and further confirms previous findings that β-tricalcium phosphate is less bioactive than hydroxyapatite.     

Dynamic study of calcium phosphate formation on porous HA/TCP ceramics

Bone-like apatite formation on porous calcium phosphate ceramics was investigated in static simulated body fluid (SBF) and dynamic SBF at different flowing rates. The results of a 14-day immersion in static SBF showed that the formation of bone-like apatite occurred both on the surface and in the pores of the samples. When SBF flow at the physiological flow rate in muscle (2 ml/100 ml min1), bone-like apatite could be detected only in internal surface of the pores of samples. The result that bone-like apatite formation could only be found in the pores when SBF flown at physiological flow rate was consistent with that of porous calcium phosphate ceramics implanted in vivo: osteoinduction was only detected inside the pores of the porous calcium phosphate ceramics. This result implicates that the bone-like apatite may play an important role in the osteoinduction of Ca-P materials. The dynamic model used in this study may be better than usually used static immersion model in imitating the physiological condition of bone-like apatite formation. Dynamic SBF method is very useful to understand bone-like apatite formation in vivo and the mechanism of ectopic bone formation in calcium phosphate ceramics.     

Apatite-forming ability of polyglutamic acid hydrogels in a body-simulating environment

Artificial joints can replace damaged joints provided the surrounding bone is sufficiently dense. However, elderly patients generally have reduced osteoporosis-associated bone density. Therefore, restitution of bone density is essential to ensure implantation. Injectable and resorbable bioactive fillers with bone-bonding ability (osteoconductivity) are promising, as osteoporosis can be reversed with minimal invasion. Osteoconduction occurs through the surface formation of biologically active hydroxyapatite via reactions with body fluids. Heterogeneous nucleation of the hydroxyapatite is catalysed by specific surface functional groups. In addition, release of Ca²⁺ ions into the surrounding fluids enhances apatite nucleation by increasing its degree of supersaturation. We tested injectable bioactive filler made from cross-linked polyglutamic acid (PGA). This has many carboxyl groups that facilitate apatite nucleation. An insoluble hydrogel can be formed by cross-linkage. We exposed PGA gels to a simulated body fluid for 7 days. Trace amounts of calcium phosphate were formed, but were not identified as bone-like apatite by X-ray diffraction. However, formation of a bone-like apatite layer was detected using pre-treatment with CaCl₂ solutions (>0.01 mol dm⁻³) dose dependently. Thus, this chemically cross-linked PGA gel could induce the heterogeneous nucleation of hydroxyapatite in a body environment, and this was enhanced by pre-treatment with CaCl₂.     

Plasma Spraying of Al2O3 and ZrSiO4 to Form ZrO2- Mullite Composites

Zirconia is effective in improving fracture toughness of a number of ceramics when introduced as a reinforcement either in the form of participates, dispersed phase or whiskers because of its unique tetragonal-monoclinic (t → m) transformation. In this paper, the authors attempt to prepare ZrO₂, reinforced mullite by plasma spraying mixtures of zircon and alumina. Pre-mixed powders of zircon and alumina are injected onto a D.C. plasma jet. The plasma sprayed particles are collected in distilled water and analyzed. The results indicate that the plasma sprayed powders consist of zirconia, zircon, and alumina. It was found that fine grained, even amorphous and chemically homogeneous composite powders could be obtained by ball milling and plasma spraying. Recrystallization of amorphous phases and formation of mullite occurred at about 1OOO^°C in plasma sprayed powders. This value is more than 500^°C lower than the formation of mullite in asmilled powders. Uniform coatings with good structural integrity were obtained by plasma spraying. The relative quantity of mullite in coatings after heat treatment is about 4 times as much as that obtained in the spheroidized powders. Preheat treatment of the spheroidized powder promoted dissociation of zircon. Zirconia remained as tetragonal under 1000^°C in the sprayed coatings.     

In vitro dissolution and corrosion study of calcium phosphate coatings elaborated by pulsed electrodeposition current on Ti6Al4V substrate

Calcium-deficient hydroxyapatite (Ca-def-HAP) coatings on titanium alloy (Ti6Al4V) substrates are elaborated by pulsed electrodeposition. In vitro dissolution/precipitation process is investigated by immersion of the coated substrate into Dulbecco’s Modified Eagle Medium (DMEM) from 1 h to 28 days. Calcium and phosphorus concentrations evolution in the biological liquid are determined by Induced Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) for each immersion time. Physical and chemical characterizations of the coating are performed by scanning electron microscopy (SEM) associated to Energy Dispersive X-ray Spectroscopy (EDXS) for X-ray microanalysis. Surface modifications are investigated by an original method based on the three-dimensional reconstruction of SEM images (3D-SEM). Moreover, corrosion measurements are carried out by potentiodynamic polarization experiments. The results show that the precipitation rate of the Ca-def HAP coating is more pronounced in comparison with that of stoichiometric hydroxyapatite (HAP) used as reference. The precipitated bone-like apatite coating is thick, homogenous and exhibits an improved link to the substrate. Consequently, the corrosion behaviour of the elaborated prosthetic material is improved.     

HA and double-layer HA-P2O5/CaO glass coatings: influence of chemical composition on human bone marrow cells osteoblastic behavior

Human osteoblastic bone marrow derived cells were cultured for 28 days onto the surface of a glass reinforced hydroxyapatite (HA) composite and a commercial type HA plasma sprayed coatings, both in the as-received condition and after an immersion treatment with culture medium during 21 days. Cell proliferation and differentiation were analyzed as a function of the chemical composition of the coatings and the immersion treatment.Cell attachment, growth and differentiation of osteoblastic bone marrow cells seeded onto as-received plasma sprayed coatings were strongly affected by the time-dependent variation of the surface structure occurring during the first hours of culture. Initial interactions leading to higher amounts of adsorbed protein and zeta potential shifts towards negative charges appeared to result in surface structures with better biological performance. Cultures grown onto the pretreated coatings showed higher rate of cell proliferation and increased functional activity, as compared to those grown onto the corresponding as-received materials. However, the cell behavior was similar in the glass composite and HA coatings.The results showed that the glass composites present better characteristics for bone cell growth and function than HA. In addition, this work also provide evidence that the biological performance of the glass composites can be modulated and improved by manipulations in the chemical composition, namely in the content of glass added to HA. © 2001 Kluwer Academic Publishers     

Plasma sprayed hydroxy apatite coatings

This paper describes the method of producing plasma sprayed coatings of hydroxy apatite (HA) on metallic substrates of Ti-6Al-4V. Hydroxy apatite is a material which has similar composition to that of the mineral phase of the human bone. Poor mechanical properties however inhibits its use in the load bearing applications. The powders prepared in our laboratory are sprayed using a plasma spray torch operating in ambient. The deposited coatings are charaterized by X-ray diffraction and found to retain HA in the coatings as per requirements. Paper presented at the poster session of MRSI AGM VI, Kharagpur, 1995.     

Improvement in mechanical properties of plasma sprayed hydroxyapatite coatings by Al2O3 reinforcement

Thermal sprayed hydroxyapatite coatings suffer from poor mechanical properties like tensile strength, wear resistance, hardness, toughness and fatigue. The mechanical properties of hydroxyapatite coatings can be enhanced via incorporation of secondary bioinert reinforcement material. In this study an attempt has been made to improve the mechanical properties of plasma sprayed hydroxyapatite by reinforcing it with 10, 20 and 30% Al₂O₃. The plasma sprayed coatings have been characterized using FE-SEM/EDAX, XRD, AFM and FTIR spectroscopy. Corrosion studies have been done in simulated body fluid and abrasive wear studies have been performed on flat specimens on a disk wear tester. Microhardness, tensile strength and wear resistance are found to be increased with increasing Al₂O₃ content. All types of coatings show superior resistance against corrosion in simulated body fluid.     

Improved stability of plasma-sprayed dicalcium silicate/zirconia composite coating

Dicalcium silicate/zirconia composite coatings were produced on Ti-6Al-4V substrates using atmospheric plasma spraying. Different weight ratios of zirconia (50 wt.%, 70 wt.%, 90 wt.%) were mechanically blended with dicalcium silicate (C₂S) powders as feedstocks. The composite coatings were immersed in a simulated body fluid (SBF) and a Tris-HCl solution for the in vitro appraisement of stability and long-term performance in a biological environment. The ion concentration changes of Ca, Si, and P in SBF and Tris-HCl solution were monitored using inductively-coupled plasma atomic emission spectroscopy (ICP-AES). Compared to the pure C₂S coating, our results show that the dissolution rate of the composite coatings is effectively reduced and the stability is improved by the addition of zirconia. The high content of zirconia in the coatings ensures the long-term performance in biological environment, while dissolution of C₂S in the coatings results in a higher Ca ion concentration in SBF and rapid precipitation of bone-like apatite on the composite coating surfaces indicating good bioconductivity of the coatings.