Key Engineering Materials Vols. 493-494

Abstract: The research of obtained bioglasses in the form of silver-containing powders were presented in this paper. The research conducted in order to determine antimicrobial activity under in vitro conditions of bioglasses. The results confirmed that these bioglasses Z-5, Z-8 and B-I inhibit the growth of most micro-organisms during the tested period and could be used in surgical treatment of the most advanced parodontium illnesses.

Abstract: Bio-absorbable and biomimetic composites (HA-C) of hydroxyapatite (HAp) and collagen were designed utilizing salmon bone and skin at 298K and pH 7.5-7.9 by a dissolution-precipitation method. The HA-C powders were consistuted by Ca²⁺-deficient HAp containing small amounts of Na⁺ and Mg²⁺ ions and I type-collagen. Microstructure and surface characteristics of the HA-C powders gradually changed depending on the composition ratio of HAp to collagen (H/C). The HA-C powder of H/C=3.5 had frock like-agglomerated particles consisted of nano-crystals, micro-pore, and meso-pore so that it exhibited high specific surface area (75m²¥g^-1) and large total pore volume (0.543 cm³¥g^-1). From adsorption isotherms of water vapor at 298K for the HA-C powders, hysteresis - curves of the amounts of water vapor adsorbed were obtained in the adsorption-desorption processes. The amounts of water vapor adsorbed for the H/C=3.5 powder were the highest values under the relative partial pressures of 0.73-0.93. At 2 weeks after implantation of the HA-C powders into the subcutaneous tissue of the back region in rats, collagen was completely bio-absorbed and body fluid permeated into large agglomerated particles, although bio-absorption by multi-giant cell- infiltration was recognized around the surface layers of HAp particles.

Abstract: The aim of this work is to produce and characterise carbonate substituted hydroxyapatite (CHA) reinforced polycaprolactone (PCL) nanocomposites with a controlled degradation rate in order to match the rate of bone in-growth. The ideal degradation time for this purpose is estimated to be around 5-6 months however, in vivo, PCL degrades over a period of 2 to 3 years. It has been reported that NaOH surface treatment can accelerate the degradation of PCL [1-3]. In order to further modify the degradation rate of PCL, the effects of the incorporation of different volume fractions of CHA in samples surface treated with NaOH was investigated. CHA was produced by wet chemical synthesis. Samples comprising 8, 19, 25 wt% uncalcined CHA-PCL composites were produced by twin screw extrusion which were then injection moulded into cylinders. In order to accelerate the degradation rate of PCL, it was surface treated with 5 M NaOH for 3 days prior to PBS studies. The degradation profile was examined by % weight loss and % water uptake measurements. NaOH treatment was observed to erode the polymer surface and the polymer-filler interface. On subsequently degrading the pre-treated samples in PBS, it was observed that with increasing fraction of CHA, the degradation rate in PBS of the sample increased. Up to 8 wt % CHA filler there appeared to be little change in the degradation properties of the NaOH treated samples with the onset occurring after 60 days. However there was a marked acceleration of degradation for samples containing 19 wt% when degradation appeared to occur immediately. In conclusion, the addition of CHA significantly affects the behaviour of PCL.

Abstract: Hydroxyapatite/collagen (HAp/Col) nanocomposites with bone-like self-organized nanostructure show excellent bioactivity in vivo. However, they show quite high absorbability for cationic ions and lower culture medium ionic concentrations which adversely affects bone cell proliferation and osteogenic differentiation in in vitro cell culture condition. To address this limitation, in this study we have supplemented Ca²⁺ and Mg²⁺ ions to the HAp/Col nanocomposite membrane sample prior to cell culture to improve it’s in vitro biological properties. The HAp/Col nanocomposite membrane samples were fabricated by the simultaneous titration method using Ca(OH)₂, type-I atelocollagen and H₃PO₄ as starting precursor materials. Prior to in vitro cell culture experiments, the HAp/Col samples were pretreated with Ca²⁺ and/or Mg²⁺ ions by immersing in 10 ml of 20 mM CaCl₂ solution, 20 mM MgCl₂ solution, or a solution containing 20 mM CaCl₂ and 20 mM MgCl₂ for 7 days. In vitro bone cell-material interactions on the pretreated and untreated HAp/Col samples were studied by culturing MC3T3-E1 cells up to 7 days. Enhanced bone cell proliferation was found on all the pretreated HAp/col samples as confirmed by the CCK-8 assay. Interestingly, the HAp/Col samples pretreated with both Ca²⁺ and Mg²⁺ ions showed the maximum viable bone cell density.

Abstract: In the present study, we have newly developed an artificial bone substitute, which is unidirectional porous β-tricalcium phosphate (UDPTCP). The objective of this study was to examine the effects of high and low porosity substitutes on the balance between new bone formation and β-TCP absorption. Materials and MethodsSix male Japanese white rabbits (weight 3.1–3.5 kg, approximately 18– 21 weeks old) were used for this study. Intra-venous injection of pent barbiturate was administered and the both medial and lateral femoral condyle were exposed. A hole of 5 mm diameter was drilled to a depth of 12 mm in the metaphysis, perpendicular to the long axis of the femur. (Figure 1) Figure 1. Operation procedureIn the next step, a cylindrical UDPTCP test piece measuring 4.8 × 11 mm was implanted in the holes. Within the bone substitute, unidirectional pores ranging from 100 to 300 μm in diameter were made. This unique architecture fostered transmission of fluids and cells into the piece. In this case, the test piece was implanted into the bone perpendicular to the long axis of the femur, and the orientation of uni-directional pore was parallel to the long axis of femur. We prepared two different test pieces having low (69%) and high (74%) porosities. Half of the animals were sacrificed at 3 weeks after the operation and the remaining half at 6 weeks. After removal of the femoral condyle, the specimen was fixed in formalin and demineralized. Specimens were obtained from the central axis of the cylindrical piece as well as from the lateral or medial surfaces at a distance of 4 mm from midline. The histological samples were prepared for H&E and TRAP staining. Results and Discussion 　At 3 weeks interval, woven bone, which was formed along the wall of the substitute, could be observed by H&E staining in both low and high porosity substitutes (Figure 2a, 2b). In addition, there were osteoblast-like cells lining the newly formed bone surface with extensive capillary formation (Figure 3). At 6 weeks, the β-TCP walls had thinned and bone had matured in both the groups (Figure 4a, 4b). However, in the high-porosity group, β-TCP absorption tended to be more prominent (Figure 4). In addition, it was observed that at the center of the piece, β-TCP absorption was more prominent than that in the 4 mm-area obtained from the lateral or medial surfaces. At 3 and 6 weeks interval, activities of osteoclast-like multinuclear cells were seen on the surface of the pore wall as observed by TRAP staining. Figure 2a. Low porosity (69%) Figure 2b. High porosity (74%) Fig.2a and Fig.2b H&E staining (×12.5) after 3 weeks (center of the specimen)Figure 3. Formation of woven bone with osteoblast-like cells lining the low porosity specimen at 3 weeks. (H&E staining ×400) Figure 4a. Low porosity Figure 4b. High porosityFig.4a and Fig. 4b H&E staining at 6 weeks after implantation. In high porosity, dense-pink staining areas are located at peripheral in the field.Figure 5. TRAP-positive multinuclear cells (black arrow) were seen on the wall and in the capillaries.Conclusions　The UDPTCP implanted in the medullar canal of the femur was absorbed by multinuclear cells and quickly replaced by the newly formed bone. Our results are consistent with those of other studies using porous β-TCP [1]. In our preparation, porosity had certain effects on the balance between bone formation and　β-TCP absorption. Because of the unique architecture of unidirectional pores within the β-TCP specimen as well as easy formation of capillary network and access to osteoclasts may have accelerated absorption of the substitute. UDPTCP is very promising scaffolding material for bone regeneration. However, optimization of the porosity of UDPTCP in accordance with its application site is necessary before its clinical use. Reference[1] Naoki Kondo, Akira Ogose, Kunihiko Tokunaga, Tomoyuki Ito, Katsumitsu Arai, Naoko Kudo, Hikaru Inoue, Hiroyuki Irie, Naoto Endo: Bone formation and resorption of highly purified β-tricalcium phosphate in the rat femoral condyle. Biomaterials 26: 5600-5608, October 2005.

Abstract: Calcium phosphate foam could be an ideal bone filler and scaffold for tissue engineering. This paper describes fabrication method of β-tricalcium phosphate (βTCP) foam with fully-interconnected porous structure by employing magnesium oxide (MgO) as βTCP stabilizer. The foam was prepared using the so-called ceramics foam method. MgO was added to calcium carbonate and dicalcium phosphate dihydrate so that 0, 1, 2, 3, 4, 6 and 8 mol% calcium would be substituted by magnesium (Mg) in βTCP structure. After sintering at 1500°C, crystal phase of the obtained foam included α-tricalcium phosphate (αTCP) when no Mg or less than 3 mol% Mg was added. In contrast, crystal phase was single phase βTCP when 3 mol% or higher Mg was added. The compressive strength was approximately 15 kPa and the porosity was above 95% for all specimens. No significant difference was observed between αTCP and βTCP foams in compressive strength and porosity when the sintering temperature was the same.

Abstract: Carbonate apatite (CO₃Ap) is expected to be an ideal bone substitute since it can harmonize with the bone remodeling cycle. The aim of this study is to fabricate a CO₃Ap bone substitute from gypsum (calcium sulfate, CaSO₄·2H₂O) hardening bodies based on dissolution-precipitation reaction. Calcium sulfate hemihydrate mixed with water at a water-to-powder ratio of 0.5 was packed in a split stainless mold and kept at room temperature for 24 hours to obtain set CaSO₄·2H₂O. The set CaSO₄·2H₂O was hydrothermally treated in the presence of disodium hydrogen phosphate (Na₂HPO₄) and sodium hydrogen carbonate (NaHCO₃). The results of powder X-ray diffraction and Fourier transform infrared spectroscopy indicated that CO₃Ap block could be fabricated from the set CaSO₄·2H₂O block by hydrothermal treatment with Na₂HPO₄ and NaHCO₃. When the treatment temperature was increased, the conversion rate to CO₃Ap increased. However, the carbonate content decreased with increasing treatment temperature.

Abstract: To improve the efficiency of osteogenic repair, we compared 3 types of round granular bone substitutes composed of hydroxyapatite (HA) in a single opened large pore covered by one or more thin shell structure, biphasic HA and tricalcium phosphate (TCP) in a compact granules with small uniform interconnected internal pores, and bioglass(BG) in a compact granules with hierarchical interconnected pores its bone repair efficiency by evaluation of cellular toxicity, cellular attachment and proliferation rate, and osteogenic supportive effect. They were nontoxic and revealed no noxious effect on cellular proliferation and osteoblastic differentiation. The cultured cells were most effectively proliferated on HA granular bone substitute surface. However, the bony repair of calvarial defects was most effective by BCP granular bone substitutes. The implanted BCP and HA granular bone substitutes showed excellent osteoconductive bone growth and favorable bone regeneration within 3 weeks compared to BG granular bone substitutes. All type granular bone substitutes were well incorporated into newly formed bone without foreign body reaction. Except for HA granular bone substitute, some implanted BG and BCP granular bone substitutes were partially resobed by TRAP positive multinucletated cells. These findings suggest that round granular biphasic calcium phosphate bone substitute structured with fully interconnected uniform sized internal pore might be a more promising bone substitute for small-sized none load-bearing bone defects.

Abstract: The comparative investigation of a highly nanoporous bone grafting material (NanoBone S, NBS) and a sintered hydroxyapatite ceramic (Cerabone, CB) aimed to show the influence of the structure of the material on osteoinductivity.NBS consists of synthetic nanocrystalline hydroxyapatite embedded in a porous silica gel matrix. Its specific surface amounts 206 m²/g in contrast to CB with a specific surface of 0.4 m²/g.The biomaterials were implanted in the neck region of 18 sheep and left there for the periods of 6, 12 and 26 weeks. In each case granulate was implanted superficially into the trapezius muscle and into the subcutaneous adipose tissue respectively. The samples were analysed by micro-CT, histochemistry, immunohistochemistry and histomorphometry. In the case of NBS ossicles had developed. An intensive remodelling process was verifiable. The bone formation in CB was marginal.As a basic phenomenon in NBS, the substitution of the original SiO₂ gel matrix by organic molecules forming an organic matrix around the embedded hydroxyapatite seams to be the key event causing these results.

Abstract: The hydration of α−tricalcium phosphate (α−TCP) is known to lead to the formation of an interlinked calcium−deficient hydroxyapatite (CDHA) framework. This apatite resembles the composition and structure of physiologic bone mineral, making it a good candidate for bone reconstruction. The additional presence of a calcium sulfate phase, which has a faster resorption rate than apatite, gives a gradual creation of porosity in the cement. This may in turn enhance bone ingrowth. The aim of this study was to gain deeper understanding of the main characteristics and properties of a bi−phasic α−TCP/α−CSH cement, prepared with an X−ray contrast medium.Isothermal calorimetry has been used to follow the course of the hydration reaction over time as it involves traceable exothermic events with possible contributions from both the calcium phosphate and calcium sulfate components. This was done in an attempt to identify and differentiate the role of each reactive phase. Additionally, the total produced heat from the cement has been used to estimate the degree of conversion and this result has been verified by X−ray diffraction analysis.Furthermore, compressive strength of the α−TCP/α−CSH cement has been measured over time to investigate the possible connection to its thermal evolution signature. Also, the impact of the surrounding environment on the α−TCP and α−CSH conversion was investigated by comparing XRD results between samples that had been kept dry or wet.