Showing papers in "Journal of Biomedical Materials Research in 2001"

Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling

Abstract: A number of different processing techniques have been developed to design and fabricate three-dimensional (3D) scaffolds for tissue-engineering applications. The imperfection of the current techniques has encouraged the use of a rapid prototyping technology known as fused deposition modeling (FDM). Our results show that FDM allows the design and fabrication of highly reproducible bioresorbable 3D scaffolds with a fully interconnected pore network. The mechanical properties and in vitro biocompatibility of polycaprolactone scaffolds with a porosity of 61 +/- 1% and two matrix architectures were studied. The honeycomb-like pores had a size falling within the range of 360 x 430 x 620 microm. The scaffolds with a 0/60/120 degrees lay-down pattern had a compressive stiffness and a 1% offset yield strength in air of 41.9 +/- 3.5 and 3.1 +/- 0.1 MPa, respectively, and a compressive stiffness and a 1% offset yield strength in simulated physiological conditions (a saline solution at 37 degrees C) of 29.4 +/- 4.0 and 2.3 +/- 0.2 MPa, respectively. In comparison, the scaffolds with a 0/72/144/36/108 degrees lay-down pattern had a compressive stiffness and a 1% offset yield strength in air of 20.2 +/- 1.7 and 2.4 +/- 0.1 MPa, respectively, and a compressive stiffness and a 1% offset yield strength in simulated physiological conditions (a saline solution at 37 degrees C) of 21.5 +/- 2.9 and 2.0 +/- 0.2 MPa, respectively. Statistical analysis confirmed that the five-angle scaffolds had significantly lower stiffness and 1% offset yield strengths under compression loading than those with a three-angle pattern under both testing conditions (p < or = 0.05). The obtained stress-strain curves for both scaffold architectures demonstrate the typical behavior of a honeycomb structure undergoing deformation. In vitro studies were conducted with primary human fibroblasts and periosteal cells. Light, environmental scanning electron, and confocal laser microscopy as well as immunohistochemistry showed cell proliferation and extracellular matrix production on the polycaprolactone surface in the 1st culturing week. Over a period of 3-4 weeks in a culture, the fully interconnected scaffold architecture was completely 3D-filled by cellular tissue. Our cell culture study shows that fibroblasts and osteoblast-like cells can proliferate, differentiate, and produce a cellular tissue in an entirely interconnected 3D polycaprolactone matrix.

Gene-expression profiling of human osteoblasts following treatment with the ionic products of Bioglass 45S5 dissolution.

Abstract: The effect of the ionic products of Bioglass 45S5 dissolution on the gene-expression profile of human osteoblasts was investigated by cDNA microarray analysis of 1,176 genes. Treatment with the ionic products of Bioglass 45S5 dissolution increased the levels of 60 transcripts twofold or more and reduced the levels of five transcripts to one-half or less than in control. Markedly up-regulated genes included RCL, a c-myc responsive growth related gene, cell cycle regulators such as G1/S specific cyclin D1, and apoptosis regulators including calpain and defender against cell death (DAD1). Other significantly up-regulated genes included the cell surface receptors CD44 and integrin beta1, and various extracellular matrix regulators including metalloproteinases-2 and -4 and their inhibitors TIMP-1 and TIMP-2. The identification of differentially expressed genes by cDNA microarray analysis has offered new insights into the mode of action of bioactive glasses and has proven to be an effective tool in evaluating their osteoproductive properties.

Biodegradable polymeric scaffolds for musculoskeletal tissue engineering.

Abstract: Biodegradable scaffolds have played an important role in a number of tissue engineering attempts over the past decade. The goal of this review article is to provide a brief overview of some of the important issues related to scaffolds fabricated from synthetic biodegradable polymers. Various types of such materials are available; some are commercialized and others are still in the laboratories. The properties of the most common of these polymers are discussed here. A variety of fabrication techniques were developed to fashion polymeric materials into porous scaffolds, and a selection of these is presented. The very important issue of scaffold architecture, including the topic of porosity and permeability, is discussed. Other areas such as cell growth on scaffolds, surface modification, scaffold mechanics, and the release of growths factors are also reviewed. A summary outlining the common themes in scaffold-related science that are found in the literature is presented.

Material fundamentals and clinical performance of plasma-sprayed hydroxyapatite coatings: A review

Abstract: The clinical use of plasma-sprayed hydroxyapatite (HA) coatings on metal implants has aroused as many controversies as interests over the last decade. Although faster and stronger fixation and more bone growth have been revealed, the performance of HA-coated implants has been doubted. This article will initially address the fundamentals of the material selection, design, and processing of the HA coating and show how the coating microstructure and properties can be a good predictor of the expected behavior in the body. Further discussion will clarify the major concerns with the clinical use of HA coatings and introduce a comprehensive review concerning the outcomes experienced with respect to clinical practice over the past 5 years. A reflection on the results indicates that HA coatings can promote earlier and stronger fixation but exhibit a durability that can be related to the coating quality. Specific relationships between coating quality and clinical performance are being established as characterization methods disclose more information about the coating.

Functionalized silk-based biomaterials for bone formation

Abstract: Silks are being reassessed as biomaterial scaffolds due to their unique mechanical properties, opportunities for genetic tailoring of structure and thus function, and recent studies clarifying biocompatibility. We report on the covalent decoration of silk films with integrin recognition sequences (RGD) as well as parathyroid hormone (PTH, 1-34 amino acids) and a modified PTH 1-34 (mPTH) involved in the induction of bone formation. Osteoblast-like cell (Saos-2) responses to the decorated silk films indicate that the proteins serve as suitable bone-inducing matrices. Osteoblast-like cell adhesion was significantly increased on RGD and PTH compared to plastic, mPTH, and the control peptide RAD. At 2 weeks of culture, message levels of alkaline phosphatase were similar on all substrates, but by 4 weeks, alkaline phosphatase mRNA was greatest on RGD. At 2 weeks of culture, alpha 1(I) procollagen mRNA was elevated on silk, RGD, RAD, and PTH, and hardly detectable on mPTH and plastic. However, by 4 weeks RGD demonstrated the highest level compared to the other substrates. Osteocalcin message levels detected by RT-PCR were greatest on RGD at both time points. Calcification was also significantly elevated on RGD compared to the other substrates with an increase in number and size of the mineralized nodules in culture. Thus, RGD covalently decorated silk appears to stimulate osteoblast-based mineralization in vitro.

Surface modification of neural recording electrodes with conducting polymer/biomolecule blends

Abstract: The interface between micromachined neural microelectrodes and neural tissue plays an important role in chronic in vivo recording Electrochemical polymerization was used to optimize the surface of the metal electrode sites Electrically conductive polymers (polypyrrole) combined with biomolecules having cell adhesion functionality were deposited with great precision onto microelectrode sites of neural probes The biomolecules used were a silk-like polymer having fibronectin fragments (SLPF) and nonapeptide CDPGYIGSR The existence of protein polymers and peptides in the coatings was confirmed by reflective microfocusing Fourier transform infrared spectroscopy (FTIR) The morphology of the coating was rough and fuzzy, providing a high density of bioactive sites for interaction with neural cells This high interfacial area also helped to lower the impedance of the electrode site and, consequently, to improve the signal transport Impedance spectroscopy showed a lowered magnitude and phase of impedance around the biologically relevant frequency of 1 kHz Cyclic voltammetry demonstrated the intrinsic redox reaction of the doped polypyrrole and the increased charge capacity of the coated electrodes Rat glial cells and human neuroblastoma cells were seeded and cultured on neural probes with coated and uncoated electrodes Glial cells appeared to attach better to polypyrrole/SLPF-coated electrodes than to uncoated gold electrodes Neuroblastoma cells grew preferentially on and around the polypyrrole/CDPGYIGSR-coated electrode sites while the polypyrrole/CH(3)COO(-)-coated sites on the same probe did not show a preferential attraction to the cells These results indicate that we can adjust the chemical composition, morphology, electronic transport, and bioactivity of polymer coatings on electrode surfaces on a multichannel micromachined neural probe by controlling electrochemical deposition conditions

Photocrosslinkable polysaccharides for in situ hydrogel formation

Abstract: In situ photopolymerization is an exciting new technique for tissue engineering. Two photocrosslinkable polysaccharides composed of alginate and hyaluronan are described that upon photolysis form soft, flexible, and viscoelastic hydrogels. The degree of methacrylate modification and thus covalent affects mechanical properties such as swelling, compression, and creep compliance. Significant swelling is observed in aqueous solution; these hydrogels can swell up to 14 times their dry weight. Both hydrogels exhibit low phase angles and (G*) values indicative of viscoelastic materials. The hyaluronan based hydrogel is stronger and more resilient than the corresponding alginate gel. SEM and AFM studies on both hydrogels show smooth and uniform surfaces at the macroscopic level with salient features observed only on the nanometer scale. Rapid polymerization by an optical trigger allows for controlled in situ photopolymerization in a minimally invasive manner, indicating that these hydrogels are relevant for biomedical applications such as sealing wounds and reconstructing soft tissues.

Hemocompatibility, biocompatibility, inflammatory and in vivo studies of primary reference materials low-density polyethylene and polydimethylsiloxane: a review

Abstract: In 1984, low-density polyethylene (LDPE) and polymethylsiloxane (PDMS), two primary reference materials (PRM), were made available by the National Heart, Lung, and Blood Institute (NHLBI) as discriminatory tools for the validation of standardized and novel in vitro and in vivo tests in the evaluation of biomaterials. This article reviews the results and conclusions obtained by several studies investigating the hemocompatibility, in vitro biocompatibility, inflammatory response, and in vivo tissue reactions of these two reference materials. Variable results obtained with LDPE and PDMS in ex vivo hemocompatibility studies were attributed to the type of animal model used, the flow velocity of the circulating blood, the time of exposure, and the methodology used to measure blood cell adhesion or activation at the surface of the materials. In contrast, both the LDPE and PDMS appeared to be suitable reference materials when used in in vitro biocompatibility, inflammatory response, and in vivo studies. However, caution must be taken when interpreting the results, because gamma sterilization of these two materials as well as their origin (for example PDMS) are two critically important factors. In conclusion, we see a definite need for standardized hemocompatible parameters and better high-quality hemocompatibility studies on PRM. This review also suggests other materials as potential PRM candidates, namely, Biomer and Intramedic polyethylene.

Engineering new bone tissue in vitro on highly porous poly(α-hydroxyl acids)/hydroxyapatite composite scaffolds

Abstract: Engineering new bone tissue with cells and a synthetic extracellular matrix (scaffolding) represents a new approach for the regeneration of mineralized tissues compared with the transplantation of bone (autografts or allografts). In the present work, highly porous poly(L-lactic acid) (PLLA) and PLLA/hydroxyapatite (HAP) composite scaffolds were prepared with a thermally induced phase separation technique. The scaffolds were seeded with osteoblastic cells and cultured in vitro. In the pure PLLA scaffolds, the osteoblasts attached primarily on the outer surface of the polymer. In contrast, the osteoblasts penetrated deep into the PLLA/HAP scaffolds and were uniformly distributed. The osteoblast survival percentage in the PLLA/HAP scaffolds was superior to that in the PLLA scaffolds. The osteoblasts proliferated in both types of the scaffolds, but the cell number was always higher in the PLLA/HAP composite scaffolds during 6 weeks of in vitro cultivation. Bone-specific markers (mRNAs encoding bone sialoprotein and osteocalcin) were expressed more abundantly in the PLLA/HAP composite scaffolds than in the PLLA scaffolds. The new tissue increased continuously in the PLLA/HAP composite scaffolds, whereas new tissue formed only near the surface of pure PLLA scaffolds. These results demonstrate that HAP imparts osteoconductivity and the highly porous PLLA/HAP composite scaffolds are superior to pure PLLA scaffolds for bone tissue engineering. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res 54: 284–293, 2001

Control of pore structure and size in freeze-dried collagen sponges.

Abstract: Because of many suitable properties, collagen sponges are used as an acellular implant or a biomaterial in the field of tissue engineering. Generally, the inner three-dimensional structure of the sponges influences the behavior of cells. To investigate this influence, it is necessary to develop a process to produce sponges with a defined, adjustable, and homogeneous pore structure. Collagen sponges can be produced by freeze-drying of collagen suspensions. The pore structure of the freeze-dried sponges mirrors the ice-crystal morphology after freezing. In industrial production, the collagen suspensions are solidified under time- and space-dependent freezing conditions, resulting in an inhomogeneous pore structure. In this investigation, unidirectional solidification was applied during the freezing process to produce collagen sponges with a homogeneous pore structure. Using this technique the entire sample can be solidified under thermally constant freezing conditions. The ice-crystal morphology and size can be adjusted by varying the solute concentration in the collagen suspension. Collagen sponges with a very uniform and defined pore structure can be produced. Furthermore, the pore size can be adjusted between 20-40 microm. The thickness of the sponges prepared during this research was 10 mm.

Hydroxylapatite binds more serum proteins, purified integrins, and osteoblast precursor cells than titanium or steel.

Abstract: The implant material hydroxylapatite (HA) has been shown in numerous studies to be highly biocompatible and to osseointegrate well with existing bone; however, the molecular mechanisms at work behind this osseointegration remain largely unexplored. One possibility is that the implant, exposed to the patient's blood during surgery, adsorbs known cell adhesive proteins such as fibronectin and vitronectin from the serum. Osteoblast precursors could then adhere to these proteins through integrin-mediated mechanisms. In the present study, we have used a quantitative ELISA assay to test the hypothesis that hydroxylapatite will adsorb more fibronectin and vitronectin from serum than two commonly used hard-tissue materials, commercially pure titanium, and 316L stainless steel. We further used the ELISA, as well as a standard cell adhesion assay, to test the hypothesis that increased protein adsorption will lead to better binding of purified integrins alpha5beta1 and alpha(v)beta3 and osteoblast precursor cells to the HA than to the metals. Our results show that fibronectin, vitronectin, alpha5beta1, alpha(v)beta3, and osteoblast precursor cells do indeed bind better to HA than to the metals, suggesting that improved integrin-mediated cell binding may be one of the mechanisms leading to better clinical bone integration with HA-coated implants.

Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites.

Abstract: Chitosan/hydroxyapatite (HAp) composites with a homogeneous nanostructure have been prepared by a co-precipitation method. According to TEM observations, HAp crystallites in the composites formed elliptic aggregations 230 nm in length and 50 nm in width. The typical length of the aggregations corresponded approximately to that of a chitosan molecule. The size of the constituent HAp crystallites was found to be predominantly 30 nm in length and 10 nm in width, and the c-axes were well aligned in parallel with the chitosan molecules in the respective aggregations. The growth of the HAp crystallites is considered to occur at nucleation sites, most probably forming the complexes with amino groups on chitosan with calcium ions. The compact composites obtained have been found to be mechanically flexible, and this flexibility has been improved further by heating at 120 degrees C for 20 min in an autoclave with saturated steam pressure.

Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering

Abstract: Chitosan scaffolds reinforced by beta-tricalcium phosphate (beta-TCP) and calcium phosphate invert glass were fabricated with a low-cost, bioclean freeze-drying technique via thermally induced phase separation. The microstructure, mechanical performance, biodegradation, and bioactivity of the scaffolds were studied. The composite scaffolds were macroporous, and the pore structures of the scaffolds with beta-TCP and the glass appeared very different. Both the compressive modulus and yield strength of the scaffolds were greatly improved, and reinforced microstructures were achieved. The bioactivity tests showed a continuous decrease in both Ca and P concentrations of a simulated body fluid (SBF) after the scaffolds with beta-TCP were immersed in the SBF for more than 20 h, which suggests that an apatite layer might be formed on the scaffolds. However, the same was not observed for the pure chitosan scaffolds or the scaffolds incorporated with the glass. This was further confirmed by micrographs from scanning electron microscopy. This study suggests that the desirable pore structure, biodegradation rate, and bioactivity of the composite scaffolds might be achieved through controlling the ratio of chitosan and calcium phosphates or beta-TCP and the glass.

Characterization of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses.

Abstract: The ability of bioactive glasses to form a bond to living bone and also to stimulate bone-cell proliferation depends on the chemical composition and on the surface texture of the glasses. In this work, the differences in physical properties between the melt-derived 45S5 and sol-gel–derived 58S Bioglass® powders of various particle-size ranges were studied. The powders were characterized for particle-size distribution by laser spectrometry, for specific surface area and porosity by nitrogen sorption analysis, and for morphological features by scanning electron microscopy. Melt-derived 45S5 powders exhibited a low-porosity texture with surface area in the range 0.15–2.7 m2/g. In contrast, the sol-gel–derived powders exhibited a highly mesoporous texture, with surface area in the range of 126.5–164.7 m2/g and a large fraction of 6–9 nm pore sizes. These differences in texture, as well as variations in chemical composition, account for significant changes in the resorption and in vivo responses. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 58: 734–740, 2001

Microtubular architecture of biodegradable polymer scaffolds

Abstract: It is a relatively new approach to generate tissues with mammalian cells and scaffolds (temporary synthetic extracellular matrices). Many tissues, such as nerve, muscle, tendon, ligament, blood vessel, bone, and teeth, have tubu- lar or fibrous bundle architectures and anisotropic proper- ties. In this work, we have designed and fabricated highly porous scaffolds from biodegradable polymers with a novel phase-separation technique to generate controllable parallel array of microtubular architecture. Porosity as high as 97% has been achieved. The porosity, diameter of the microtu- bules, the tubular morphology, and their orientation are con- trolled by the polymer concentration, solvent system, and temperature gradient. The mechanical properties of these scaffolds are anisotropic. Osteoprogenitor cells are seeded in these three-dimensional scaffolds and cultured in vitro. The cell distribution and the neo-tissue organization are guided by the microtubular architecture. The fabrication technique can be applied to a variety of polymers, therefore the deg- radation rate and cell-matrix interactions can be controlled by the chemical composition of the polymers and the incor- poration of bioactive moieties. These microtubular scaffolds may be used to engineer a variety of tissues with anisotropic architecture and properties. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 469-477, 2001

Pore diameter of more than 100 microm is not requisite for bone ingrowth in rabbits.

Abstract: The optimal pore size for bone ingrowth is claimed to be 100-400 microm. With the use of a highly standardized experimental model, the present study reevaluated whether a pore size of 100 microm is the threshold value for bone ingrowth into porous structures under non-load-bearing conditions. Titanium triangle-shaped plates 250 or 500 microm thick were perforated with the use of a laser in order to create standard-sized holes ( 50, 75, 100, and 125 microm) in multiple rows. The amount of bone ingrowth through the implant holes was studied in the cancellous bone of the distal rabbit femur. Twelve weeks after implantation, detailed analysis of bone ingrowth was performed with computerized image analysis of backscattered electron imaging techniques of scanning electron microscopy. The results showed that the amount of ingrown new bone was independent of the pore size and implant thickness. The median value for bone ingrowth varied between 64 and 78%. A striking feature was the formation of secondary osteonal structures even in the smallest holes. Based on these results, there is no threshold value for new bone ingrowth in pore sizes ranging from 50 to 125 microm under non-load-bearing conditions.

Structure, metallurgy, and mechanical properties of a porous tantalum foam.

Abstract: This study evaluated a porous tantalum biomaterial (Hedrocel) designed to function as a scaffold for osseous ingrowth. Samples were characterized for structure, Vickers microhardness, compressive cantilever bending, and tensile properties, as well as compressive and cantilever bending fatigue. The structure consisted of regularly arranged cells having struts with a vitreous carbon core with layers of CVI deposited crystalline tantalum. Microhardness values ranged from 240-393, compressive strength was 60 +/- 18 MPa, tensile strength was 63 +/- 6 MPa, and bending strength was 110 +/- 14 MPa. The compressive fatigue endurance limit was 23 MPa at 5 x 10(6) cycles with samples exhibiting significant plastic deformation. SEM examination showed cracking at strut junctions 45 degrees to the axis of the applied load. The cantilever bending fatigue endurance limit was 35 MPa at 5 x 10(6) cycles, and SEM examination showed failure due to cracking of the struts on the tension side of the sample. While properties were variable due to morphology, results indicate that the material provides structural support while bone ingrowth is occurring. These findings, coupled with the superior biocompatibility of tantalum, makes the material a candidate for a number of clinical applications and warrants further and continued laboratory and clinical investigation.

Mechanical properties of the dentinoenamel junction: AFM studies of nanohardness, elastic modulus, and fracture.

Abstract: The dentinoenamel junction (DEJ) is a complex and poorly defined structure that unites the brittle overlying enamel with the dentin that forms the bulk of the tooth. In addition, this structure appears to confer excellent toughness and crack deflecting properties to the tooth, and has drawn considerable interest as a biomimetic model of a structure uniting dissimilar materials. This work sought to characterize the nanomechanical properties in the region of the DEJ using modified AFM based nanoindentation to determine nanohardness and elastic modulus. Lines of indentations traversing the DEJ were made at 1-2 microm intervals from the dentin to enamel along three directions on polished sagittal sections from three third molars. Nanohardness and elastic modulus rose steadily across the DEJ from bulk dentin to enamel. DEJ width was estimated by local polynomial regression fits for each sample and location of the mechanical property curves for the data gradient from enamel to dentin, and gave a mean value of 11.8 microm, which did not vary significantly with intratooth location or among teeth. Nanoindentation was also used to initiate cracks in the DEJ region. In agreement with prior work, it was difficult to initiate cracks that traversed the DEJ, or to produce cracks in the dentin. The fracture toughness values for enamel of 0.6-0.9 MPa . m(1/2) were in good agreement with recent microindentation fracture results. Our results suggest that the DEJ displays a gradient in structure and that nanoindenation methods show promise for further understanding its structure and function.

Attachment of fibronectin to poly(vinyl alcohol) hydrogels promotes NIH3T3 cell adhesion, proliferation, and migration.

Abstract: Hydrogels have been used in biology and medicine for many years, and they possess many properties that make them advantageous for tissue engineering applications. Their high water content and tissue-like elasticity are similar to the native extracellular matrix of many tissues. In this work, we investigated the potential of a modified poly(vinyl alcohol) (PVA) hydrogel as a biomaterial for tissue engineering applications. First, the ability of NIH3T3 fibroblast cells to attach to PVA hydrogels was evaluated. Because of PVA's extremely hydrophilic nature, important cell adhesion proteins do not adsorb to PVA hydrogels, and consequently, cells are unable to adhere to the hydrogel. By covalently attaching the important cell adhesion protein fibronectin onto the PVA hydrogel surface, the rate of fibroblast attachment and proliferation was dramatically improved, and promoted two-dimensional cell migration. These studies illustrate that a fibronectin-modified PVA hydrogel is a potential biomaterial for tissue engineering applications.

Injection molding of chondrocyte/alginate constructs in the shape of facial implants.

Abstract: Over one million patients per year undergo some type of procedure involving cartilage reconstruction. Polymer hydrogels, such as alginate, have been shown to be effective carriers for chondrocytes in subcutaneous cartilage formation. The goal of our current study was to develop a method to create complex structures (nose bridge, chin, etc.) with good dimensional tolerance to form cartilage in specific shapes. Molds of facial implants were prepared using Silastic ERTV. Suspensions of chondrocytes in 2% alginate were gelled by mixing with CaSO(4) (0.2 g/mL) and injected into the molds. Constructs of various cell concentrations (10, 25, and 50 million/mL) were implanted in the dorsal aspect of nude mice and harvested at times up to 30 weeks. Analysis of implanted constructs indicated progressive cartilage formation with time. Proteoglycan and collagen constructs increased with time to approximately 60% that of native tissue. Equilibrium modulus likewise increased with time to 15% that of normal tissue, whereas hydraulic permeability decreased to 20 times that of native tissue. Implants seeded with greater concentrations of cells increased proteoglycan content and collagen content and equilibrium and decreased permeability. Production of shaped cartilage implants by this technique presents several advantages, including good dimensional tolerance, high sample-to-sample reproducibility, and high cell viability. This system may be useful in the large-scale production of precisely shaped cartilage implants.

TEM-EDX study of mechanism of bonelike apatite formation on bioactive titanium metal in simulated body fluid.

Abstract: Bioactive titanium metal, which forms a bonelike apatite layer on its surface in the body and bonds to the bone through the apatite layer, can be prepared by NaOH and heat treatments to form an amorphous sodium titanate layer on the metal. In the present study, the mechanism of apatite formation on the bioactive titanium metal has been investigated in vitro. The metal surface was examined using transmission electron microscopy and energy dispersive X-ray spectrometry as a function of the soaking time in a simulated body fluid (SBF) and complemented with atomic emission spectroscopy analysis of the fluid. It was found that, immediately after immersion in the SBF, the metal exchanged Na(+) ions from the surface sodium titanate with H(3)O(+) ions in the fluid to form Ti-OH groups on its surface. The Ti-OH groups, immediately after they were formed, incorporated the calcium ions in the fluid to form an amorphous calcium titanate. After a long soaking time, the amorphous calcium titanate incorporated the phosphate ions in the fluid to form an amorphous calcium phosphate with a low Ca/P atomic ratio of 1.40. The amorphous calcium phosphate thereafter converted into bonelike crystalline apatite with a Ca/P ratio of 1.65, which is equal to the value of bone mineral. The initial formation of the amorphous calcium titanate is proposed to be a consequence of the electrostatic interaction of negatively charged units of titania, which are dissociated from the Ti-OH groups, with the positively charged calcium ions in the fluid. The amorphous calcium titanate is speculated to gain a positive charge and to interact with the negatively charged phosphate ions in the fluid to form the amorphous calcium phosphate, which eventually stabilizes into bonelike crystalline apatite.

Biodegradable polymer/hydroxyapatite composites: Surface analysis and initial attachment of human osteoblasts

Abstract: Biodegradable polymer/hydroxyapatite (HA) composites have potential application as bone graft substitutes. Thin films of polymer/HA composites were produced, and the initial attachment of primary human osteoblasts (HOBs) was assessed to investigate the biocompatibility of the materials. Poly(epsilon-caprolactone) (PCL) and poly(L-lactic acid) (PLA) were used as matrix materials for two types of HA particles, 50-microm sintered and submicron nonsintered. Using ESEM, cell morphology on the surfaces of samples was investigated after 90 min, 4 h, and 24 h of cell culture. Cell activity and viability were assessed after 24 h of cell culture using Alamar blue and DNA assays. Surface morphology of the polymer/HA composites and HA exposure were investigated using ESEM and EDXA, respectively. ESEM enabled investigation of both cell and material surface morphology in the hydrated condition. Combined with EDXA it permitted chemical and visual examination of the composite. Differences in HA exposure were observed on the different composite surfaces that affected the morphology of attached cells. In the first 4 h of cell culture, the cells were spread to a higher degree on exposed HA regions of the composites and on PLA than they were on PCL. After 24 h the cells were spread equally on all the samples. The cell activity after 24 h was significantly higher on the polymer/HA composites than on the polymer films. There was no significant difference in the activity of the cells on the various composite materials. However, cells on PCL showed higher activity compared to those on PLA. A polymer surface exhibiting "point exposure" of HA appeared to provide a novel and favorable substrate for primary cell attachment. The cell morphology and activity results indicate a favorable cell/material interaction and suggest that PLA and PCL and their composites with HA may be candidate materials for the reconstruction of bony tissue. Further investigations regarding long-term biomaterial/cell interactions and the effects of acidic degradation products from the biodegradable polymers are required to confirm their utility.

The effects of surface chemistry and adsorbed proteins on monocyte/macrophage adhesion to chemically modified polystyrene surfaces.

Abstract: Monocytes and macrophages play critical roles in inflammatory responses to implanted biomaterials. Monocyte adhesion may lead to macrophage activation and the foreign body response. We report that surface chemistry, preadsorbed proteins, and adhesion time all play important roles during monocyte adhesion in vitro. The surface chemistry of tissue culture polystyrene (TCPS), polystyrene, Primaria, and ultra low attachment (ULA) used for adhesion studies was characterized by electron spectroscopy for chemical analysis. Fibrinogen adsorption measured by (125)I-labeled fibrinogen was the lowest on ULA, higher on TCPS, and the highest on polystyrene or Primaria. Monocyte adhesion on protein preadsorbed surfaces for 2 h or 1 day was measured with a lactate-dehydrogenase method. Monocyte adhesion decreased over time. The ability of preadsorbed proteins to modulate monocyte adhesion was surface dependent. Adhesion was the lowest on ULA, higher and similar on TCPS or polystyrene, and the highest on Primaria. Monocyte adhesion on plasma or fibrinogen adsorbed surfaces correlated positively and linearly to the amount of adsorbed fibrinogen. Preadsorbed fibronectin, immunoglobulin G, plasma, or serum also promoted adhesion compared with albumin preadsorbed or uncoated surfaces. Overall, biomaterial surface chemistry, the type and amount of adsorbed proteins, and adhesion time all affected monocyte adhesion in vitro.

Bone tissue engineering in a rotating bioreactor using a microcarrier matrix system.

Abstract: A novel approach was utilized to grow in vitro mineralized bone tissue using lighter-than-water, polymeric scaffolds in a high aspect ratio rotating bioreactor. We have adapted polymer microencapsulation methods for the formation of hollow, lighter-than-water microcarriers of degradable poly(lactic-co-glycolic acid). Scaffolds were fabricated by sintering together lighter-than-water microcarriers from 500 to 860 μm in diameter to create a fully interconnected, three-dimensional network with an average pore size of 187 μm and aggregate density of 0.65 g/mL. Motion in the rotating bioreactor was characterized by numerical simulation and by direct measurement using an in situ particle tracking system. Scaffold constructs established a near circular trajectory in the fluid medium with a terminal velocity of 98 mm/s while avoiding collision with the bioreactor wall. Preliminary cell culture studies on these scaffolds show that osteoblast-like cells readily attached to microcarrier scaffolds using controlled seeding conditions with an average cell density of 6.5 × 104 cells/cm2. The maximum shear stress imparted to attached cells was estimated to be 3.9 dynes/cm2. In addition, cells cultured in vitro on these lighter-than-water scaffolds retained their osteoblastic phenotype and showed significant increases in alkaline phosphatase expression and alizarin red staining by day 7 as compared with statically cultured controls. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 242–253, 2001

Degradation behaviors of biodegradable macroporous scaffolds prepared by gas foaming of effervescent salts.

Abstract: Biodegradable polymeric scaffolds for tissue engineering were fabricated by a gas-foaming/salt-leaching method using a combination of two effervescent salts, ammonium bicarbonate and citric acid. Poly(D,L-lactic-co-glycolic acid) (PLGA) in a state of gel-like paste was first produced by precipitation of PLGA dissolved in chloroform into ethanol. The polymer slurry was mixed with sieved particles of ammonium bicarbonate, molded, and then immersed in an aqueous solution of citric acid to generate macroporous scaffolds. The scaffolds had relatively homogeneous pore structures throughout the matrix and showed an average pore size of 200 μm and over 90% porosity. By adjusting the concentration of citric acid in the aqueous medium, it was possible to control porosity as well as mechanical strength of the scaffolds. In vitro degradation studies of three different scaffolds having lactic/glycolic acid molar ratios of 75/25, 65/35, and 50/50 exhibited marked swelling behaviors at different critical time points. The swollen matrices had a hydrogel-like internal structure. It was found that massive water uptake into the degrading scaffolds induced matrix swelling, which facilitated the hydrolytic scission of PLGA chains with concomitant disintegration of the matrices. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 55: 401–408, 2001

Calcium alginate gel: A biocompatible and mechanically stable polymer for endovascular embolization

Abstract: The development and optimization of calcium alginate for potential use in endovascular occlusion was investigated by testing its in vitro and in vivo mechanical stability and biocompatibility. The compressive resistance, rheology, and polymer yield of reacted alginate, and the polymer viscosity of unreacted alginate, were assessed. Biocompatibility was tested by injecting calcium alginate into the kidney capsule of rats. The reactivity of alginates with various structures and levels of purity were compared visually and histologically. Results suggest that calcium alginate is a biocompatible and mechanically stable gel for endovascular applications. Purified alginates exhibited compressive strength of 22 kPa and above at 40% compression, with no significant loss in elasticity. Purified alginate strength was significantly higher than that of crude alginates (p < 0.08). Purified alginates also exhibited significantly lower tissue reaction than crude alginates (p < 0.05). Of the alginates tested, purified high guluronic acid alginates (PHG) exhibited optimal strength and polymer yield, increased biocompatibility, and decreased viscosity. Clinical embolization treatments may be improved with the development of stable and biocompatible polymers such as calcium alginate. Possible uses of improved endovascular polymers include treating arteriovenous malformations (AVMs), aneurysms, blood flow to tumors, and vascular hemorrhaging.

Biomimetic coprecipitation of calcium phosphate and bovine serum albumin on titanium alloy

Abstract: Titanium alloy implants were precoated biomimetically with a thin and dense layer of calcium phosphate and then incubated either in a supersaturated solution of calcium phosphate or in phosphate-buffered saline, each containing bovine serum albumin (BSA) at various concentrations, under physiological conditions for 48 h. Coated implants then underwent scanning electron microscopy, immunohistochemical evaluation, Fourier transform infrared spectroscopy, and X-ray diffraction. The quantity of BSA taken up by coatings and the kinetics of protein release were monitored colorimetrically. In coatings prepared by the coprecipitation of calcium phosphate and BSA, protein had become incorporated into the mineral crystal latticework. With increasing BSA concentration, matrices decreased in thickness, became more dense, showed lower crystallinity, and underwent a change in crystal geometry. The octacalcium phosphate structure manifested in the absence of protein was gradually transformed into a carbonated apatite form. Preformed mineral coatings became only superficially mantled with a layer of BSA, and the morphology of the mineral matrices themselves remained unchanged. At equivalent protein concentrations, coatings prepared by the coprecipitation of calcium phosphate released only a minute fraction of its protein component under physiological conditions, whereas preformed mineral matrices showed a burst release of their associated protein within a single 2-h period. The biomimetic coating can be a carrier for osteoinductive agents.

Bone induction by porous glass ceramic made from Bioglass (45S5).

Abstract: Porous glass ceramic, which was prepared from Bioglass® powder (45S5, U.S. Biomaterials) by foaming with diluted H2O2 solution and sintering at 1000 °C for 2 h, was implanted as cylinders (5 mm in diameter and 6 mm in length) in thigh muscles of dogs for 3 months. Histological observation was made on thin un-decalcified sections. Bone formation was histologically found in pores of all implants (X16) retrieved from 16 dogs. The bone tissue was also identified with backscattered scanning electron microscopy observation (BSE) and energy dispersive X-ray microanalysis (EDX). This is the first report of bone induction in soft tissues of animals by glass ceramic that has long been recognized as a bioactive (osteoconductive) biomaterial. The present results justify the impetus to investigate the osteoinductivity of calcium phosphate-based biomaterials, to study the mechanism of bone induction (osteoinduction) by calcium phosphate-based biomaterials, to develop osteoinductive calcium phosphate-based biomaterials, and to examine the relation between osteoinduction and osteoconduction. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 58: 270–276, 2001

Stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin).

Abstract: The study was undertaken to investigate the stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin) at distinct elapsed storage durations. The glutaraldehyde-fixed counterpart was used as a control. Porcine pericardia procured from a slaughterhouse were used as raw materials. After fixation, the fixed tissues were sterilized in a graded series of ethanol solutions and thoroughly rinsed in phosphate buffered saline for 1 day, and then stored in a jar containing sterilized water. The samples were taken out and tested for their stability during the durations of 1day through 6 months after storage. The stability of each study group was tested by measuring its tensile strength, free-amino-group content, and denaturation temperature. Additionally, the cytotoxicity of each test sample and its corresponding storage solution were investigated in vitro using 3T3 fibroblasts. The results were examined using a microscope and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the stability of the genipin-fixed tissue during storage was superior to its glutaraldehyde-fixed counterpart. The differences in stability between the genipin- and glutaraldehyde-fixed tissues during storage may be caused by their differences in crosslinking structure. There was no apparent cytotoxicity for both the genipin-fixed tissue and its corresponding storage solution throughout the entire course of the study, whereas significant cytotoxicity was observed for both the glutaraldehyde-fixed tissue and its storage solution. However, the cytotoxicity of the glutaraldehyde-fixed tissue decreased with increasing elapsed storage duration, whereas that of its corresponding storage solution increased. This suggested that the toxic residues remaining in the glutaraldehyde-fixed tissue leached out slowly into its corresponding storage solution during the course of storage.

Immobilized RGD peptides on surface-grafted dextran promote biospecific cell attachment.

Abstract: Dextran has recently been investigated as an alternative to poly(ethylene glycol) (PEG) for low protein-binding, cell-resistant coatings on biomaterial surfaces. Although antifouling properties of surface-grafted dextran and PEG are quite similar, surface-bound dextran has multiple reactive sites for high-density surface immobilization of biologically active molecules. We recently reported nontoxic aqueous methods to covalently immobilize dextran on material surfaces. These dextran coatings effectively limited cell adhesion and spreading in the presence of serum-borne cell adhesion proteins. In this study we utilized the same nontoxic aqueous methods to graft cell adhesion peptides on low protein-binding dextran monolayer surfaces. Chemical composition of all modified surfaces was verified by X-ray photoelectron spectroscopy (XPS). Surface-grafted cell adhesion peptides stimulated endothelial cell, fibroblast, and smooth muscle cell attachment and spreading in vitro. In contrast, surface-grafted inactive peptide sequences did not promote high levels of cell interaction. Surface-grafted high affinity cyclic RGD peptides promoted cell type-dependent interactions. With dextran-based surface coatings, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in long-term biomaterial implants.