Biomaterials (v.27, #13)

Calendar (I).

Fabrication methods of porous metals for use in orthopaedic applications by Garrett Ryan; Abhay Pandit; Dimitrios Panagiotis Apatsidis (2651-2670).
Implant stability is not only a function of strength but also depends on the fixation established with surrounding tissues [Robertson DM, Pierre L, Chahal R. Preliminary observations of bone ingrowth into porous materials. J Biomed Mater Res 1976;10:335–44]. In the past, such stability was primarily achieved using screws and bone cements. However, more recently, improved fixation can be achieved by bone tissue growing into and through a porous matrix of metal, bonding in this way the implant to the bone host. Another potentially valuable property of porous materials is their low elastic modulus. Depending on the porosity, moduli can even be tailored to match the modulus of bone closer than solid metals can, thus reducing the problems associated with stress shielding. Finally, extensive body fluid transport through the porous scaffold matrix is possible, which can trigger bone ingrowth, if substantial pore interconnectivity is established [Cameron HU, Macnab I, Pilliar RM. A porous metal system for joint replacement surgery. Int J Artif Organs 1978;1:104–9; Head WC, Bauk DJ, Emerson Jr RH. Titanium as the material of choice for cementless femoral components in total hip arthroplasty. Clin Orthop 1995;85–90].Over the years, a variety of fabrication processes have been developed, resulting in porous implant substrates that can address unresolved clinical problems. The advantages of metals exhibiting surface or bulk porosity have led researchers to conduct systematic research aimed at clarifying the fundamental aspects of interactions between porous metals and hard tissue. This review summarises all known methods for fabricating such porous metallic scaffolds.
Keywords: Porosity; Bone ingrowth; Scaffold; Mechanical properties; Rapid prototyping;

Bone formation enhanced by implanted octacalcium phosphate involving conversion into Ca-deficient hydroxyapatite by Osamu Suzuki; Shinji Kamakura; Takenobu Katagiri; Masanori Nakamura; Baohong Zhao; Yoshitomo Honda; Ryutaro Kamijo (2671-2681).
The present study was designed to investigate whether hydrolysis of synthetic octacalcium phosphate (OCP) into hydroxyapatite affects bone formation. Mouse bone marrow stromal ST-2 cells and primary calvarial osteoblastic cells were cultured on the dishes pre-coated with OCP or its hydrolyzed Ca-deficient hydroxyapatite (OCP hydrolyzate; HL). The capacity of proliferation and differentiation was determined up to day 20. Granules of OCP and HL were implanted into critical-size rat calvaria defects for 4 and 12 weeks, and then bone formation was measured by histomorphometry. Structural changes of incubated and implanted OCP were determined by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The proliferation of both ST-2 and primary osteoblasts cultured on OCP or HL was initially inhibited, whereas their differentiation to osteoblasts was promoted at last. Implantation of OCP in bone defect more significantly enhanced bone formation than that of HL until 12 weeks. OCP tended to convert to apatite in vitro and in vivo. The conversion of the implanted OCP was ascertained to advance gradually with implantation periods. Taken together, these results suggest that OCP supports appositional bone formation and OCP-apatite conversion may be involved in this stimulatory capacity of OCP.
Keywords: Bone formation; Octacalcium phosphate (OCP); Hydroxyapatite (HA); Conversion; Biomineralization;

Osteoinductive porous titanium implants: Effect of sodium removal by dilute HCl treatment by Mitsuru Takemoto; Shunsuke Fujibayashi; Masashi Neo; Jun Suzuki; Tomiharu Matsushita; Tadashi Kokubo; Takashi Nakamura (2682-2691).
In a previous study, we observed that chemically and thermally treated plasma-sprayed porous titanium possesses intrinsic osteoinductivity and that bone formation occurs after 12 months in the muscles of beagle dogs. The aim of this study was to optimize the surface treatment and to accelerate the osteoinductivity. Previous studies have reported that sodium removal converts the sodium titanate layer on the surface of an alkali-treated titanium plate into a more bioactive titania layer. In this study, we developed a dilute hydrochloric acid (HCl) treatment for porous titanium, which removed sodium from the complexly shaped porous structure more effectively than conventional hot water treatment. Three types of surface treatments were applied: (a) alkali and heat treatment (AH treatment); (b) alkali, hot water, and heat treatment (Water–AH treatment); and (c) alkali, dilute HCl, hot water, and heat treatment (HCl–AH treatment). The osteoinductivity of the materials implanted in the back muscles of adult beagle dogs was examined at 3, 6, and 12 months. The HCl–AH-treated porous bioactive titanium implant had the highest osteoinductivity, with induction of a large amount of bone formation within 3 months. The dilute HCl treatment was considered to give both chemical (titania formation and sodium removal) and topographic (etching) effects on the titanium surface, although we cannot determine which is the predominant factor. Nevertheless, adding the dilute HCl treatment to the conventional chemical and thermal treatments is a promising candidate for advanced surface treatment of porous titanium implants.
Keywords: Osteoinduction; Osteogenesis; Titanium; Porous; Metal surface treatment; Titanium oxide;

The response of osteoblasts to nanocrystalline silicon-substituted hydroxyapatite thin films by Eng San Thian; Jie Huang; Serena M Best; Zoe H Barber; Roger A Brooks; Neil Rushton; William Bonfield (2692-2698).
Magnetron co-sputtering has been employed to fabricate thin nanocrystalline coatings of silicon-substituted hydroxyapatite (SiHA) of different Si compositions: 0.8 wt%, 2.2 wt%, and 4.9 wt%. A human osteoblast-like (HOB) cell model was used to study the long-term interaction between the HOB cells and coatings in vitro. Results showed that the number of cells growing on all coated titanium (Ti) samples were statistically significantly higher than on uncoated Ti. In addition, HOB cells growing on all SiHA surfaces displayed enhanced cell spreading, with extensive extracellular matrix synthesis. DNA staining revealed normal phenotype nuclear morphology for HOB cells, with several dense chromosomes surrounded by a periphery of intact nuclear membrane. Furthermore, immunofluorescent staining indicated that cells showed improved adhesion on the coated surfaces with increasing Si content, developing mature cytoskeletons with numerous distinct and well-defined actin stress fibres in the cell membranes. Results also demonstrated that the bone mineralisation process was greatest in the presence of the highest Si level (4.9 wt%). However, at very early culturing time point, cells did not attach so readily on the surface of this coating due to rapid dissolution. Thus, this work suggests that a Si content of 2.2 wt% may be the optimum loading to improve the bioactive property of HA thin films.
Keywords: Actin; Biomineralisation; Cell Morphology; Hydroxyapatite Coating; Osteoblast; Silicon;

Influences of ammonia plasma treatment on modifying depth and degradation of poly(l-lactide) scaffolds by Yuqing Wan; Chifeng Tu; Jian Yang; Jianzhong Bei; Shenguo Wang (2699-2704).
Hydrophobicity of poly(l-lactide) scaffolds is a main drawback in obtaining a sufficient mass of seeded cells for satisfying the requirements of tissue engineering. Plasma treatment is a useful technique to enhance the hydrophilicity of the scaffolds. However, the effect of this technique on the modifying depth and degradation of the scaffolds should be considered. In this paper, the influence of NH3 plasma treatment on the modifying depth and degradation of scaffolds were investigated. The results showed that the modifying depth of the scaffolds increased with treating time and the plasma power ranging from 20 to 80 W influenced the depth slightly. However, the degradation of the scaffolds increased with increasing treatment time and plasma power. The results also showed that the plasma intruded the scaffolds gradually from top to bottom. For a 4 mm thick scaffold, the optimized treatment condition was 20 W of power in a 30 Pa ammonia atmosphere for 30 min of treating time. Under this condition, the integrity of scaffold could be relatively well kept. NH3 plasma treatment enabled the penetration of cells into scaffolds and facilitated the proliferation of cells in them.
Keywords: Poly(l-lactide); Plasma; Surface modification; Degradation; Cell scaffold; Cell affinity;

Electrospinning polyaniline-contained gelatin nanofibers for tissue engineering applications by Mengyan Li; Yi Guo; Yen Wei; Alan G. MacDiarmid; Peter I. Lelkes (2705-2715).
Polyaniline (PANi), a conductive polymer, was blended with a natural protein, gelatin, and co-electrospun into nanofibers to investigate the potential application of such a blend as conductive scaffold for tissue engineering purposes. Electrospun PANi–contained gelatin fibers were characterized using scanning electron microscopy (SEM), electrical conductivity measurement, mechanical tensile testing, and differential scanning calorimetry (DSC). SEM analysis of the blend fibers containing less than 3% PANi in total weight, revealed uniform fibers with no evidence for phase segregation, as also confirmed by DSC. Our data indicate that with increasing the amount of PANi (from 0 to ∼5% w/w), the average fiber size was reduced from 803±121 nm to 61±13 nm ( p < 0.0 1 ) and the tensile modulus increased from 499±207 MPa to 1384±105 MPa ( p < 0.0 5 ). The results of the DSC study further strengthen our notion that the doping of gelatin with a few % PANi leads to an alteration of the physicochemical properties of gelatin. To test the usefulness of PANi-gelatin blends as a fibrous matrix for supporting cell growth, H9c2 rat cardiac myoblast cells were cultured on fiber-coated glass cover slips. Cell cultures were evaluated in terms of cell proliferation and morphology. Our results indicate that all PANi-gelatin blend fibers supported H9c2 cell attachment and proliferation to a similar degree as the control tissue culture-treated plastic (TCP) and smooth glass substrates. Depending on the concentrations of PANi, the cells initially displayed different morphologies on the fibrous substrates, but after 1week all cultures reached confluence of similar densities and morphology. Taken together these results suggest that PANi-gelatin blend nanofibers might provide a novel conductive material well suited as biocompatible scaffolds for tissue engineering.
Keywords: Electrospinning; Polyaniline (PANi); Gelatin; Tissue engineering; H9c2 cardiac myoblasts;

Micro-architecture of calcium phosphate granules and fibrin glue composites for bone tissue engineering by Damien Le Nihouannen; Laurent Le Guehennec; Thierry Rouillon; Paul Pilet; Melitta Bilban; Pierre Layrolle; Guy Daculsi (2716-2722).
Calcium phosphate ceramics are currently used as bone graft substitutes in various types of clinical applications. Fibrin glue is also used in surgery due to its haemostatic, chemotactic and mitogenic properties. By combining these two biomaterials, new composite scaffolds were prepared. In this study, we attempt to analyse whether thrombin concentration in the fibrin glue could influence the properties of the composite. The association between fibrin glue and calcium phosphate ceramic granules was characterized at the ultra structural level. Micro and macroporous biphasic calcium phosphate ceramic granules with a diameter of 1–2 mm composed of hydroxyapatite and beta-tricalcium phosphate (60/40) were associated to fibrin glue. The composites were observed by scanning and transmission electron microscopy and microcomputed tomography. Fibre thickness, porosity and homogeneity of the fibrin clot were modified by increased the thrombin concentration. Mixing fibrin glue with calcium phosphate granules (1:2) did not modify the microstructure of the fibrin clot in the composite. Nevertheless, thrombin interacted with the bioceramic by inducing the nucleation of crystalline precipitate at the ceramic/fibrin glue interface. Combining fibrin sealant and calcium phosphate ceramics could lead to new scaffolds for bone tissue engineering with the synergy of the properties of the two biomaterials.
Keywords: Biphasic calcium phosphate; Fibrin; Composite; Scaffold; Bone tissue engineering;

Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34+ cells by Xue-Song Jiang; Chou Chai; Yue Zhang; Ren-Xi Zhuo; Hai-Quan Mao; Kam W. Leong (2723-2732).
The interaction between integrins and extracellular matrix proteins play an important role in the regulation of hematopoiesis. Human hematopoietic progenitor cells express very late antigen-4 (VLA-4) and VLA-5, which mediate their interaction with fibronectin by recognizing the connecting segment-1 (CS-1 and RGD motifs, respectively. In this study, we investigated the ex vivo expansion of human umbilical cord blood (UCB) CD34+ cells on synthetic substrates surface-immobilized with peptides containing the CS-1 binding motif (EILDVPST) and the RGD motif (GRGDSPC). These peptides were covalently conjugated to poly(ethylene terephthalate) (PET) film at a surface density of 2.0–2.3 nmol/cm2. UCB CD34+ cells were cultured for 10 days in serum-free medium supplemented with recombinant human thrombopoietin, stem cell factor, flt3-ligand and interleukin 3. The highest cell expansion fold was observed on the CS-1 peptide-modified surface, where total nucleated cells, total colony forming unit, and long-term culture initiating cells were expanded by 589.6±58.6 (mean±s.d.), 76.5±8.8, and 3.2±0.9-fold, respectively, compared to unexpanded cells. All substrates surface-immobilized with peptides, including the control peptides, were more efficient in supporting the expansion of CD34+, CFU-GEMM and LTC-ICs than tissue culture polystyrene surface. Nevertheless, after 10-days of ex vivo expansion from 600 CD34+ cells, only cells cultured on CS-1-immobilized surface yielded positive engraftment, even though the frequency was low. PET surface immobilized with RGD peptide was less efficient than that with CS-1 peptide. Our results suggest that covalently immobilized adhesion peptides can significantly influence the proliferation characteristics of cultured UCB CD34+ cells.
Keywords: Stem cell; Scaffold; Fibronectin; Extracellular matrix; HSC expansion;

Interaction of human valve interstitial cells with collagen matrices manufactured using rapid prototyping by Patricia M. Taylor; Eleftherios Sachlos; Sally A. Dreger; Adrian H. Chester; Jan T. Czernuszka; Magdi H. Yacoub (2733-2737).
Rapid prototyping is a novel process for the production of scaffolds of predetermined size and three-dimensional shape. The aim of the study was to determine the feasibility of this technology for producing scaffolds for tissue engineering an aortic valve and the optimal concentration of collagen processed in this manner that would maintain viability and promote proliferation of human valve interstitial cells. Scaffolds of 1%, 2% and 5% w/v bovine type-I collagen were manufactured using rapid prototyping. Valve interstitial cells isolated from three human aortic valves were seeded on the scaffolds and cultured for up to 4 weeks. Cell viability was assessed using the CellTiter 96® Aqueous One Solution Cell Proliferation Assay and cell death by lactate dehydrogenase (LDH) measurement. Valve interstitial cells remained viable and proliferated within the collagen scaffolds. Cells consistently proliferated to a greater extent on 1% collagen scaffolds rather than either 2% or 5% collagen and after 4 weeks reached 212±33.1%, 139±25.9% and 129±38.3% (mean±SD) of their initial seeding density on 1%, 2% and 5% collagen scaffolds, respectively. LDH analysis demonstrated that there was minimal cell death indicating that the collagen scaffold was not toxic to human valve interstitial cells. Rapid prototyping provides a route to optimize biological scaffold designs for tissue engineering cardiac valves. This technology has the versatility to create scaffolds that are compatible with the specific needs of the valve interstitial cells and should enhance cell viability, proliferation and function.
Keywords: Cell viability; Collagen matrices; ECM; Heart valves; Rapid prototyping; Tissue engineering;

Enhancement of tissue engineered bone formation by a low pressure system improving cell seeding and medium perfusion into a porous scaffold by Juyong Wang; Yoshinori Asou; Ichiro Sekiya; Shinichi Sotome; Hisaya Orii; Kenichi Shinomiya (2738-2746).
To obtain more extensive bone formation in composites of porous ceramics and bone marrow stromal cells (BMSCs), we hypothesized that a low-pressure system would serve to facilitate the perfusion of larger number of BMSCs into the porous scaffold, enhancing bone formation within the composites. After culturing BMSCs in osteogenic medium, porous blocks of β-tricalcium phosphate (β-TCP) were soaked in the cell suspension. Composites of the block and BMSCs were put immediately into a vacuum desiccator. Low pressure was applied to the low pressure group, while controls were left at atmospheric pressure. Composites were incubated in vitro or subcutaneously implanted into syngeneic rats, then analyzed biologically and histologically. In the in vitro group, cell suspension volume, cell seeding efficiency, alkaline phosphatase (ALP) activity, and DNA content in the β-TCP blocks were significantly higher in low pressure group than in the controls. Scanning electron microscopy (SEM) demonstrated that a greater number of cells covered the central parts of the composites in the low pressure group. ALP activity in the composites was increased at 3 and 6 weeks after implantation into rats. Histomorphometric analysis revealed more uniform and extensive bone formation in the low pressure group than in the controls. The application of low pressure during the seeding of BMSCs in perfusing medium into a porous scaffold is useful for tissue-engineered bone formation.
Keywords: Tricalcium phosphate; Low pressure system; Marrow stromal cells; Bone formation;

Evaluation of the anterior cruciate ligament, medial collateral ligament, achilles tendon and patellar tendon as cell sources for tissue-engineered ligament by James A. Cooper; LeeAnn O. Bailey; Janell N. Carter; Cynthia E. Castiglioni; Michelle D. Kofron; Frank K. Ko; Cato T. Laurencin (2747-2754).
This study investigated four different connective tissue cell types to determine which cell type should be the source for seeding a tissue-engineered anterior cruciate ligament (ACL) replacement. Cells derived from the ACL, medial collateral ligament (MCL), achilles tendon (AT), and patellar tendon (PT) of New Zealand White rabbits were isolated and cultured. Each cell type was cultured in vitro after seeding on three-dimensional (3-D) braided polymer scaffolds and on tissue culture polystyrene that served as a control. Samples were evaluated and compared for their morphology, proliferation, and gene expression of fibronectin, type I and type III collagen. Scanning electron microscopy (SEM) photomicrographs verified cell attachment of all four types of connective tissue fibroblasts to the scaffolds. Preliminary results comparing proliferation indicate that cells obtained from the PT and AT have the fastest proliferation. Whereas gene expression of the phenotypic markers measured using real-time reverse transcription polymerase chain reaction (RT-PCR) indicates ACL cells have the highest gene expression for the matrix markers. This leads to the question of which cell type should be the cell source for tissue-engineering of ligament, the highly proliferating cells or the differentiated matrix producing cells. This study would suggest that ACL differentiated matrix producing cells are the most suitable cells for further study and development of a tissue-engineered ligament.
Keywords: Tissue engineering; Ligament; Tendon; Scaffold; Biomaterials; Real-time reverse transcription polymerase chain reaction (RT-PCR);

Planktonic and attached cells of strains of Candida albicans, C. glabrata and C. krusei with varied susceptibilities to fluconazole (FCZ) were compared for their relative susceptibilities to Ag+ via cell recovery and flow cytometric analyses. All strains lost membrane permeability and were non-recoverable upon culture after 1 h exposure in morpholino-ethanesulfonic acid (MES) buffer fortified with ⩽2.0 μg/ml Ag+. Cells attached to silicone over a 2-h period demonstrated enhanced tolerance to FCZ and to a lesser degree to Ag+. Minimal inhibitory concentrations of Ag+ in defined media increased in the order C. glabrata, C. krusei, C. albicans. Susceptibilities to Ag+ did not correlate with tolerance or resistance to FCZ.
Keywords: Candida albicans; C. glabrata; C. krusei; Fluconazole tolerance; Silver;

Evaluation of the osteogenesis and biodegradation of porous biphasic ceramic in the human spine by Youzhuan Xie; Daniel Chopin; Christian Morin; Pierre Hardouin; Zhenan Zhu; Jian Tang; Jianxi Lu (2761-2767).
The histological reports on porous biphasic calcium phosphate ceramic (PBC) in human spine are limited. The osteogenesis and biodegradation of PBC are insufficiently known in human. In present study, the undecalcified histological study was carried out on 20 samples retrieved from posterior spinal fusion in order to reveal the osteogenesis and biodegradation of the PBC in human spine. The quantitative study was performed in 14 samples with sufficient size. Newly formed bone was found in all the samples. More new bone was formed in those samples closely in contact with autogenous bone. The PBC degradation particles were present both in the macrophages and around the tissue. However, those phenomena were highly variable among the samples. New bone formation increased with time and decreased with age. The PBC degradation decreased with age, but it did not differ greatly with time. New bone formation was higher and the residual material was lower in the fusion group than that in non-fusion group. The PBC is a kind of osteoconductive material and do not transform into new bone after a relatively long time. The PBC should be well mixed with the autogenous bone in order to achieve high new bone colonization. The PBC degradation particles and related active phagocytotic activity have been noted.
Keywords: Spinal surgery; Calcium phosphate; Biodegradation; Osteogenesis;

Catalytic efficiency of a thrombomodulin-functionalized membrane-mimetic film in a flow model by Po-Yuan Tseng; Sumanas W. Jordan; Xue-Long Sun; Elliot L. Chaikof (2768-2775).
The protein C anticoagulant pathway generates an “on demand” physiologic anticoagulant response, which is initiated when thrombin binds to thrombomodulin (TM), a transmembrane protein constitutively expressed by endothelial cells. A stable, protein C activating membrane-mimetic film was produced on a polyelectrolyte multilayer (PEM) by in situ photopolymerization of a phospholipid assembly containing TM. The monoacrylated phospholipid monomer was initially synthesized and prepared as unilamellar vesicles with varying molar concentrations of TM. Membrane-mimetic films were constructed on planar substrates with defined surface concentrations of catalytically active TM. 125I-labeled radiolabeling demonstrated little change in TM surface concentration over periods of up to 4 weeks. We utilized a parallel plate flow system to investigate the effects of simulated arterial (500 s−1) and venous (50 s−1) shear rates and TM surface concentration (0–1400 fmol cm−2) on the rate and extent of activation of protein C. The rate of production of activated protein C increased with shear rate and TM surface content. However, in agreement with an analysis of reaction kinetics and mass transfer, experimental results demonstrate that reaction rates become saturated at TM surface densities greater than or equal to 800 fmol cm−2. We believe that the design of membrane-mimetic films that have the capacity to activate the protein C pathway will provide a useful strategy for generating “actively” antithrombogenic surfaces.
Keywords: Anticoagulant; Biomimetics; Thrombomodulin; Anti-thrombogenic; Membrane-mimetic;

Structure and properties of clinical coralline implants measured via 3D imaging and analysis by Mark Alexander Knackstedt; Christoph H Arns; Tim J Senden; Karlis Gross (2776-2786).
The development and design of advanced porous materials for biomedical applications requires a thorough understanding of how material structure impacts on mechanical and transport properties. This paper illustrates a 3D imaging and analysis study of two clinically proven coral bone graft samples (Porites and Goniopora). Images are obtained from X-ray micro-computed tomography (micro-CT) at a resolution of 16.8 μm. A visual comparison of the two images shows very different structure; Porites has a homogeneous structure and consistent pore size while Goniopora has a bimodal pore size and a strongly disordered structure. A number of 3D structural characteristics are measured directly on the images including pore volume-to-surface-area, pore and solid size distributions, chord length measurements and tortuosity. Computational results made directly on the digitized tomographic images are presented for the permeability, diffusivity and elastic modulus of the coral samples. The results allow one to quantify differences between the two samples. 3D digital analysis can provide a more thorough assessment of biomaterial structure including the pore wall thickness, local flow, mechanical properties and diffusion pathways. We discuss the implications of these results to the development of optimal scaffold design for tissue ingrowth.
Keywords: Microtomography; Scaffolds; Pore morphology; Diffusivity; Fluid dynamics; Elasticity;