Showing papers in "Journal of Biomedical Materials Research in 1995"

Effect of titanium surface roughness on proliferation, differentiation, and protein synthesis of human osteoblast-like cells (MG63)

Abstract: The effect of surface roughness on osteoblast proliferation, differentiation, and protein synthesis was examined. Human osteoblast-like cells (MG63) were cultured on titanium (Ti) disks that had been prepared by one of five different treatment regimens. All disks were pretreated with hydrofluoric acid-nitric acid and washed (PT). PT disks were also: washed, and then electropolished (EP); fine sandblasted, etched with HCl and H 2 SO 4 , and washed (FA); coarse sandblasted, etched with HCl and H 2 SO 4 , and washed (CA); or Ti plasma-sprayed (TPS). Standard tissue culture plastic was used as a control. Surface topography and profile were evaluated by brightfield and darkfield microscopy, cold field emission scanning electron microscopy, and laser confocal microscopy, while chemical composition was mapped using energy dispersion X-ray analysis and elemental distribution determined using Auger electron spectroscopy. The effect of surface roughness on the cells was evaluated by measuring cell number, [ 3 H]thymidine incorporation into DNA, alkaline phosphatase specific activity, [ 3 H]uridine incorporation into RNA, [ 3 H]proline incorporation into collagenase digestible protein (CDP) and noncollagenase-digestible protein (NCP), and [ 35 S]sulfate incorporation into proteoglycan. Based on surface analysis, the five different Ti surfaces were ranked in order of smoothest to roughest: EP, PT, FA, CA, and TPS. A TiO 2 layer was found on all surfaces that ranged in thickness from 100 A in the smoothest group to 300 A in the roughest. When compared to confluent cultures of cells on plastic, the number of cells was reduced on the TPS surfaces and increased on the EP surfaces, while the number of cells on the other surfaces was equivalent to plastic. [ 3 H]Thymidine incorporation was inversely related to surface roughness. Alkaline phosphatase specific activity in isolated cells was found to decrease with increasing surface roughness, except for those cells cultured on CA. In contrast, enzyme activity in the cell layer was only decreased in cultures grown on FA- and TPS-treated surfaces. A direct correlation between surface roughness and A and CDP production was found. Surface roughness had no apparent effect on NCP production. Proteoglycan synthesis by the cells was inhibited on all the surfaces studied, with the largest inhibition observed in the CA and EP groups. These results demonstrate that surface roughness alters osteoblast proliferation, differentiation, and matrix production in vitro. The results also suggest that implant surface roughness may play a role in determining phenotypic expression of cells in vivo

Neovascularization of synthetic membranes directed by membrane microarchitecture

Abstract: Transplantation of tissues enclosed within a membrane device designed to protect the cells from immune rejection (immunoisolation) provides an opportunity to treat a variety of disease conditions. Successful implementation of immunoisolation has been hampered by the foreign-body reaction to biomaterials. We screened a variety of commercially available membranes for foreign-body reactions following implantation under the skin of rats. Histologic analysis revealed that neovascularization at the membrane-tissue interface occurred in several membranes that had pore sizes large enough to allow complete penetration by host cells (0.8-8 microns pore size). When the vascularization of the membrane-tissue interface of 5-microns-pore-size polytetrafluoroethylene (PTFE) membranes was compared to 0.02-microns-pore-size PTFE membranes, it was found that the larger pore membranes had 80-100-fold more vascular structures. The increased vascularization was observed even though the larger pore membrane was laminated to a smaller pore inner membrane to prevent cell entry into the prototype immunoisolation device. This significantly higher level of vascularization was maintained for 1 year in the subcutaneous site in rats.

The use of xenogeneic small intestinal submucosa as a biomaterial for Achille's tendon repair in a dog model

Abstract: A study was conducted to evaluate the tissue response to a xenogeneic biomaterial when this material was used to repair an experimentally induced Achilles tendon defect in the dog. Twenty dogs had a 1.5 cm segmental defect of the Achilles tendon created surgically which was then repaired with acellular connective tissue derived from porcine small intestinal submucosa (SIS). The animals were sacrificed at 1, 2, 4, 8, 12, 16, 24, and 48 weeks and the neotendons examined for uniaxial longitudinal tensile strength, morphologic appearance, hydroxyproline (collagen) content, and disappearance of the originally implanted SIS material over time. The contralateral normal Achilles tendons served as controls as did four additional dogs that had a 1.5 cm segmental Achilles tendon defect created surgically without subsequent surgical repair with SIS. Results showed the SIS remodeled neotendons to be stronger than the musculotendinous origin or the boney insertion (> 1000 N) by 12 weeks after surgery and to consist of organized collagen-rich connective tissue similar to the contralateral normal tendons. The four dogs in which no SIS was implanted showed inferior strength at the comparable time points of 4, 8, 12, and 16 weeks. Immunohistochemical studies suggest that the SIS biomaterial becomes degraded within the first eight weeks and serves as a temporary scaffold around which the body deposits appropriate and organized connective tissue. SIS is a promising biomaterial worthy of further investigation for orthopedic soft tissue applications.

Effect of surface topology on the osseointegration of implant materials in trabecular bone.

Abstract: The importance of surface topology and implant material composition on osseointegration in trabecular bone was investigated using three commercially used implant materials and surface-texturing procedures which included blasting, high temperature acid etching, and hydroxyapatite (HA) coating. Surface roughness and spacing parameters were measured for each implant group with a laser interferometric profilometer. Cylindrical implants were press-fit into trabecular bone sites in the knee of mature miniature pigs. After 12 weeks in situ, osseointegration was evaluated by (1) mechanical pushout tests to measure bone-implant interface strength and (2) quantitative morphometric measurements of the percent implant surface covered by bone. We found that HA-coated implants showed superior osseointegration in terms of both pushout failure load and surface coverage by bone measurements. An excellent correlation (r2 = .90) was found between the average roughness of the implant surface and pushout failure load. New methods for altering the local topologic and/or chemical state of the implant surface (i.e., by acid etching) may provide an important new avenue of research for improving the osseointegrative properties of orthopedic materials. © 1995 John Wiley & Sons, Inc.

Attachment and growth of cultured fibroblast cells on silk protein matrices

Abstract: The attachment and growth of L-929 cells on films made of Bombyx mori silk proteins--fibroin and sericin and their mixtures--was studied by a cell culture method. Both cell attachment and growth were dependent on a minimum of around 90% sericin in the mixture. The results from electron micrography as well as from the DSC measurements supported the notion that the mixture of the two proteins fibroin and sericin has a phase-separated structure in the solid state. The observed minimum of sericin in the cell attachment and growth is thought to be a result of this phase-separated structure. Films of pure component proteins (i.e., 100% fibroin or sericin) exhibited as high a cell attachment and growth as collagen, a widely used mammalian cell culture substrate. However, a morphological study of the attached cells revealed that the cells attached to silk fibroin were extended and had a spindle shape, just like the cells attached to collagen, while the cells attached to the silk sericin had a different shape. It is concluded, therefore, that the attachment condition on silk fibroin is ideal for the viability, growth and function of the cells.

Physical crosslinking of collagen fibers: Comparison of ultraviolet irradiation and dehydrothermal treatment

Abstract: The strength, resorption rate, and biocompatibility of collagenous biomaterials are profoundly influenced by the method and extent of crosslinking. We compared the effects of two physical crosslinking methods, ultraviolet irradiation (UV) (254 nm) and dehydrothermal (DHT) treatment, on the mechanical properties and molecular integrity of collagen fibers extruded from an acidic dispersion of type I bovine dermal collagen. Collagen fibers exposed to UV irradiation for 15 min had ultimate tensile strength (54 MPa) and modulus (184 MPa) values greater than or equivalent to values for fibers crosslinked with DHT treatment for 3 or 5 days. UV irradiation is a rapid and easily controlled means of increasing the mechanical strength of collagen fibers. Characterization of collagen extracted from the crosslinked samples by dilute acetic acid and limited pepsin digestion indicate that both UV and DHT treatments cause fragmentation of at least a portion of the collagen molecules. Partial loss of the native collagen structure may influence attachment, migration, and proliferation of cells on collagen fiberbased ligament analogs. These issues are currently being addressed in our laboratory. © 1995 John Wiley & Sons, Inc.

Isolation of predominantly submicron-sized UHMWPE wear particles from periprosthetic tissues

Abstract: A method of tissue digestion using sodium hydroxide was applied to the isolation and recovery of ultra-high-molecular-weight polyethylene (UHMWPE) particles from tissues around failed total hip replacements. Density gradient ultracentrifugation of the digested tissues was performed to separate the UHMWPE from cell debris and other particulates. Fourier transform infrared spectroscopy and differential scanning calorimetry (DSC) verified that the recovered particles were UHMWPE. When viewed by scaning electron microscopy, individual particles were clearly observed and were either rounded or elongated. The majority were submicron in size. The application of this method to the study or particles from periprosthetic tissues may elucidate aspects of biomaterial particles size and shape that are important to the biologic response to, and clinical outcome of, total joint replacement. © 1995 John wiley & Sons, Inc.

Formation and characterization of anodic titanium oxide films containing Ca and P.

Abstract: Commercially pure titanium was anodized in an electrolytic solution that was dissolved calcium and phosphorus compounds in water, and an AOFCP (anodic titanium oxide film containing Ca and P) was formed. It was found that sodium beta-glycerophosphate (beta-GP) and calcium acetate (CA) were suitable for the electrolytes to form the AOFCP having an equivalent Ca/P ratio to hydroxyapatite (HA). The AOFCP was characterized by scanning electron microscopy (SEM), an energy-dispersive X-ray microanalysis (EDX), and X-ray diffraction (XRD). Numerous micropores and microprojections were observed on the AOFCP by SEM. The composition of the AOFCP, which was measured by EDX, changed according to beta-GP and CA concentration, and the electrolytic voltage. Ca and P in the AOFCP seem to be incorporated into the TiO2 matrix from CA and beta-GP in the electrolyte during the anodic oxidation. Despite the existence of Ca and P in the AOFCP, no calcium phosphate peak was detected by XRD, and the AOFCP consisted of anatase and only a little rutile. The AOFCP, whose contents of Ca and P were low, had a high adhesive strength after soaking in a simulated body fluid for 300 days. When the AOFCP having an equivalent Ca/P ratio to HA was hydrothermally heated at 300 degrees C, HA crystals were precipitated on the AOFCP and completely covered the surface.

Anchorage of TiO2‐blasted, HA‐coated, and machined implants: An experimental study with rabbits

Abstract: The purpose of this study was to evaluate the histometrical and biomechanical anchorage of TiO2-blasted implants and TiO2-blasted implants coated with hydroxyapatite. The control implants were machined. Twenty-six rabbits had a total of 156 implants placed in the proximal part of the tibia. Each rabbit had a machined, a TiO2-blasted, and a TiO2-blasted, HA-coated implant placed in each tibia. After a healing period of 3 and 12 weeks, respectively, the implants placed in the right tibia were used for removal torque test, and the implants placed in the left tibia were used for histomorphometrical measurements. Preoperatively, implants from the same batches were examined topographically with a TopScan 3D system. The TiO2-blasted implants demonstrated significantly higher removal torque values than the machined implants, and they also had a significantly more irregular surface. Furthermore, significantly higher bone-to-implant contact length fractions were measured adjacent to the TiO2-blasted implants in contrast to the machined implants. The advantages of a TiO2-blasted surface were more pronounced after 3 weeks than after 12 weeks. The results demonstrated that it was possible to influence the anchorage of implants by altering the surface structure morphology. The new method with TiO2 blasting on the titanium surface improves the anchorage of implants but is not yet practicable for HA coating.

Articular cartilage repair using allogeneic perichondrocyteseeded biodegradable porous polylactic acid (PLA): A tissue‐engineering study

Abstract: Efforts to expand treatment options for articular cartilage repair have increasingly focused on the implantation of cell-polymer constructs. The purpose of this study is to determine the suitability of porous D,D-L,L-polylactic acid as a carrier for delivering repair cells obtained from rib perichondrium into full-thickness articular cartilage defects. In vitro characterization of perichondrocyte-polylactic acid composite grafts was combined with in vivo assessment of the early articular cartilage repair in a clinically relevant model. Using a fluorescent double-stain protocol to visualize live and dead cells in situ, primary cells cultured from perichondrium were found to be capable of attaching to and surviving within a porous D,D-L,L-polylactic acid matrix. These perichondrocyte-polylactic acid composite grafts were then implanted within osteochondral defects drilled into the left medial femoral condyles of 16 adult New Zealand white rabbits. Experimental animals were sacrificed 6 weeks after implantation and the repair tissue was evaluated grossly, histologically, and biochemically. Grossly, 96% (15/16) of the experimental animals demonstrated repairs consisting of a smooth, firm neocartilage which appeared similar in color and texture to the surrounding articular surface. Matrix staining for cartilaginous protein was seen surrounding chondrocyte-like cells in the cartilage regions of the repair. Cellular alignment was found to be related to scaffold architecture. These results suggest that scaffolds composed of porous D,D-L,L-polylactic acid support the growth of cartilaginous repair tissue and are compatible with both in vitro and in vivo survival of chondrogenic cells.

Field emission SEM comparison of four postfixation drying techniques for human dentin

Abstract: Critical-point drying (CPD) is generally considered essential for the preparation of biologic specimens for electron microscopy. Several attempts have been made to introduce alternative techniques. More recently, this problem has arisen in dentistry, because of the new developments in dentin bonding. The present study focuses on three alternative techniques to CPD: hexamethyldisilzane (HMDS) drying, Peldri II drying, and air drying. Twenty-four dentin disks were obtained from noncarious extracted human molars by microtome sectioning parallel to the occlusal surface. The dentin surfaces were etched with polymer-thickened, silica-free, 10% phosphoric acid semigel, fixed, dehydrated, and dried with one of the four techniques. The specimens were observed in two perpendicular planes, showing dentinal tubules in longitudinal view and crosssection, using a field emission scanning electron microscope. The intertubular demineralized dentin zone was composed of three different successive layers, which did not substantially differ between CPD and Peldri II drying, but were more evident in HMDS-dried specimens: first, an upper layer of denatured collagen and residual smear layer particles, with sectioned collagen fibrils and few open intertubular pores; second, an intermediate layer of closely packed cross-sectioned collagen fibers; and third, a deeper layer with unfilled spaces, scattered hydroxyapatite crystals, and few collagen fibers. HMDS drying seemed to preserve better the collagen network and the microporosity of the demineralized dentin surface. Moreover, HMDS drying is easy to perform. The air-drying method caused some artefacts, such as surface collapsing and thickening of the denatured collagen layer. © 1995 John Wiley & Sons, Inc.

Protein-rejecting ability of surface-bound dextran in end-on and side-on configurations: comparison to PEG.

Abstract: There is much interest in attaching polyethylene glycol (PEG) and other hydrophilic, neutral polymers to surfaces to reduce the extent of protein and cell adsorption. Interestingly, these same surface-bound polymers are effective in masking surface charge and reducing electrokinetic effects such as particle electrophoretic mobility, streaming potential, and electroosmosis. It is apparent that similar molecular properties are responsible for both protein and cell rejection and reduction of electrokinetic effects. In this work we compared the fibrinogen-rejecting ability and the effect on electrophoretic mobility of three polymer coatings bound to polystyrene. The three polymers were side-bound dextran, end-bound dextran, and end-bound PEG. The results of these measurements were used to elucidate the importance of polymer packing density and polymer layer thickness on protein adsorption and reduction of electrokinetic effects. Protein adsorption appears not to be sensitive to polymer layer thickness or the presence of dilute polymer tails in a surface coating, while electrokinetic effects are. Protein adsorption is, however, very sensitive to the availability of exposed surface. Finally, the unique effectiveness of PEG is apparent in this research as in previous studies. © 1995 John Wiley & Sons, Inc.

Characterization of thin hydroxyapatite layers formed on anodic titanium oxide films containing Ca and P by hydrothermal treatment.

Abstract: An anodic titanium oxide film containing Ca and P (AOFCP) was formed on commercially pure titanium which was anodized in an electrolytic solution of dissolved β-glycerophosphate (β-GP) and calcium acetate (CA). Hydroxyapatite (HA) crystals were precipitated by hydrothermally heating the AOFCP at 300°C. After hydrothermal treatment, the film was characterized by scanning electron microscopy (SEM), energy-dispersive X-ray microanalysis (EDX), and tensile tests. The morphology, composition, and amount of HA crystals precipitated were significantly affected by the composition of the electrolytes. Nearstoichiometric HA crystals with high crystallinity were precipitated completely covering the AOFCP surface at specific electrolyte concentrations. The HA layers were thin at 1–2 μm in thickness. The adhesive strength of the film increased with decreasing electrolyte concentration and the maximum value was about 40 MPa. In vitro tests for 300 days suggested that the stability of the film was high. The high adhesive strength may result from the AOFCP existing as an intermediate layer between the HA layer and a titanium substrate. The intervention of the AOFCP may have prevented abrupt changes in Ca and P content at an HA coating-titanium interface as seen in a plasma-sprayed one. The porous TiO2 matrix of the AOFCP may be suitable for nucleation sites of HA crystals, as well as SiO2 matrix of silicate bioactive glasses or glass ceramics. © 1995 John Wiley & Sons, Inc.

Effect of parallel surface microgrooves and surface energy on cell growth.

Abstract: To evaluate the effect of surface treatment and surface microtexture on cellular behavior, smooth and microtextured silicone substrata were produced. The microtextured substrata possessed parallel surface grooves with a width and spacing of 2.0 (SilD02), 5.0 (SilD05), and 10 microns (SilD10). The groove depth was approximately 0.5 microns. Subsequently, these substrata were either left untreated (NT) or treated by ultraviolet irradiation (UV), radiofrequency glow discharge treatment (RFGD), or both (UVRFGD). After characterization of the substrata, rat dermal fibroblasts (RDF) were cultured on the UV, RFGD, and UVRFGD treated surfaces for 1, 3, 5, and 7 days. Comparison between the NT and UV substrata revealed that UV treatment did not influence the contact angles and surface energies of surfaces with a similar surface topography. However, the contact angles of the RFGD and UVRFGD substrata were significantly smaller than those of the UV and NT substrata. The dimension of the surface microevents did not influence the wettability characteristics. Cell culture experiments revealed that RDF cell growth on UV-treated surfaces was lower than on the RFGD and UVRFGD substrata. SEM examination demonstrated that the parallel surface grooves on the SilD02 and SilD05 substrata were able to induce stronger cell orientation and alignment than the events on SilD10 surfaces. By combining all of our findings, the most important conclusion was that physicochemical parameters such as wettability and surface free energy influence cell growth but play no measurable role in the shape and orientation of cells on microtextured surfaces.

Hydrogel-based three-dimensional matrix for neural cells

Abstract: The ability to organize cells in three dimensions (3D) is an important component of tissue engineering. This study sought to develop an extracellular matrix (ECM) equivalent with a physicochemical structure capable of supporting neurite extension from primary neural cells in 3D. Rat embryonic day 14 striatal cells and chick embryonic day 9 dorsal root ganglia extended neurites in 3D in agarose hydrogels in a gel concentration-dependent manner. Primary neural cells did not extend neurites above a threshold agarose gel concentration of 1.25% wt/vol. Gel characterization by hydraulic permeability studies revealed that the average pore radius of a 1.25% agarose gel was 150 mm. Hydraulic permeability studies for calculating average gel pore radius and gel morphology studies by environmental and scanning electron micrography showed that the average agarose gel por size decreased exponentially as the gel concentration increased. It is hypothesized that the average gel porosity plays an important role in determining the ability of agarose gels to support neurite extension. Lamination of alternating nonpermissive, permissive, and nonpermissive gel layers facilitated the creation of 3D neural tracts in vitro. This ability of agarose hydrogels to organize, support, and direct neurite extension from neural cells may be useful for applications such as 3D neural cell culture and nerve regeneration. Agarose hydrogel substrates also offer the possibility of manipulating cells in 3D, and may be used as 3D templates for tissue engineering efforts in vitro and in vivo.

In vitro evaluation of cytotoxicity of diepoxy compounds used for biomaterial modification

Abstract: The toxicity of various diepoxy compounds used for biomaterials crosslinking was investigated with a cell culture method and compared with an in vivo method. The neutral red uptake by cells was used to count the number of cells still alive after contact with the diepoxy compounds, because this method was more sensitive in cell counting than the other four methods studied in this work. The amount of neutral red taken up by cells depended strongly on the activity of cells in comparison with other methods; only small amounts of neutral red were taken up when cells were in a low activity state even if they were still alive. The in vitro toxicity of diepoxy compounds evaluated by the neutral red method revealed a good correlation with that found by the in vivo Draize test. The in vitro cytotoxicity to a cell line of L929 was closely related to that of primary culture cells of the normal rabbit cornea epidermal cell. The toxicity of diepoxy compounds was lower as their chain was longer, probably because of the lower chemical reactivity. All the diepoxy compounds investigated in this study exhibited lower cytotoxicity than formaldehyde, glutaraldehyde, and a water-soluble carbodiimide.

Development of fibroblast-seeded ligament analogs for ACL reconstruction

Abstract: We fabricated “ligament analogs” in vitro, by seeding highstrength resorbable collagen fiber scaffolds with intraarticular (anterior cruciate ligament, ACL) or extraarticular (patellar tendon, PT) rabbit fibroblasts. Fibroblasts attached, proliferated, and secreted new collagen on the ligament analogs in vitro. Fibroblast function depended on the tissue culture substrate (ligament analog vs. tissue culture plate) and the origin of the fibroblasts (ACL vs. PT). PT fibroblasts proliferated more rapidly than ACL fibroblasts when cultured on ligament analogs. Collagen synthesis by ACL and PT fibroblasts was approximately tenfold greater on ligament analogs than on tissue culture plates. The composition, structure, and geometry of the collagen fiber scaffolds may promote collagen synthesis within ligament analogs in vitro., Ligament analogs roughly approximate the structure and strength of native ligament tissue. Ongoing in vivo studies suggest that autogenous fibroblast-seeded ligament analogs remain viable after implantation into the knee joint. With further development, ligament analogs may be useful as implants for ACL reconstruction surgery. © 1995 John Wiley & Sons, Inc.

Biodegradable sponges for hepatocyte transplantation

Abstract: Liver cell transplantation may provide a means to replace lost or deficient liver tissue, but devices capable of delivering hepatocytes to a desirable anatomic location and guiding the development of a new tissue from these cells and the host tissue are needed. We have investigated whether sponges fabricated from poly-L-lactic acid (PLA) infiltrated with polyvinyl alcohol (PVA) would meet these requirements. Highly porous sponges (porosity = 90-95%) were fabricated from PLA using a particulate leaching technique. To enable even and efficient cell seeding, the devices were infiltrated with the hydrophilic polymer polyvinyl alcohol (PVA). This reduced their contact angle with water from 79 to 23 degrees, but did not inhibit the ability of hepatocytes to adhere to the polymer. Porous sponges of PLA infiltrated with PVA readily absorbed aqueous solutions into 98% of their pore volume, and could be evenly seeded with high densities (5 x 10(7) cells/mL) of hepatocytes. Hepatocyte-seeded devices were implanted into the mesentery of laboratory rats, and 6 +/- 2 x 10(5) of the hepatocytes engrafted per sponge. Fibrovascular tissue invaded through the devices' pores, leading to a composite tissue consisting of hepatocytes, blood vessels and fibrous tissue, and the polymer sponge.

Interspecies variation of compressive biomechanical properties of the meniscus

Abstract: Various animal models have been used to investigate the normal and reparative properties of the knee meniscus. Yet, only limited data on meniscal biomechanical properties of various animals are available. It was therefore the objective of this study to compare measurements of meniscal biomechanical properties between six species: human, bovine, monkey, canine, sheep, and porcine. Uniaxial confined compression tests were conducted on 1-mm-thick, 4-mm-diameter meniscal discs, and the viscoelastic creep deformation was obtained. Two biomechanical parameters, the aggregate modulus (HA) and permeability (K), were found by implementing the linear biphasic theory and a newly developed nonlinear regression scheme. A one-way analysis of variance was conducted along with Student-Newman-Keuls comparison tests to assess the differences in these parameters among the species. Sheep menisci exhibited HA and K values most similar to human menisci. The water content of each specimen was also measured and correlated significantly with K. The interspecies variations found in material properties of the knee meniscus indicate the need for caution in extrapolating data on the biomechanical behavior of the human meniscus from animal models.

Mechanical properties of the binary titanium-zirconium alloys and their potential for biomedical materials

Abstract: Mechanical properties of titanium-zirconium binary alloys were investigated in order to reveal their possible use for new biomedical materials and to collect useful data for alloy design through a hardness test, a tensile test, and optical microscopy. The hardness of the alloy containing 50% zirconium was approximately 2.5 times as large as the hardness of pure titanium and pure zirconium. Tensile tests showed a similar tendency. No changes between hardness of as cast specimens and as homogenized specimens were observed, nor were changes in microstructures noted. Comparisons between the Ti-6Al-4V alloy and the Ti-Zr-6Al-4V alloy indicated that a titanium-zirconium alloy could provide a base material for a new biomedical alloy. From these results, it was concluded that new alloys for biomedical materials should be designed as titanium-zirconium base alloys. © 1995 John Wiley & Sons, Inc.

Polyvinylidene fluoride (PVDF) as a biomaterial: From polymeric raw material to monofilament vascular suture

Abstract: This study identified the effects of various manufacturing processes on the crystalline microstructure, mechanical properties, and biocompatibility of a polyvinylidene fluoride (PVDF) suture To achieve this, changes in the crystalline microstructure and the tensile behavior of PVDF monofilaments were monitored in vitro after different thermal processing, coloration, and sterilization treatments In addition, the in vivo biocompatibility of the manufactured and sterilized PVDF suture was assessed by using it to anastomose a preclotted polyester vascular prosthesis as a thoracoabdominal bypass in a series of dogs The tissue response was followed by histologic and scanning electron microscopy over implantation periods ranging from 4 h to 6 months Differential scanning calorimetry and infrared spectroscopy (FTIR-ATR) showed that thermal processing and the addition of a coloring agent had a direct effect on modifying the crystalline microstructure and hence changing the mechanical properties For example, thermal processing converted some of the alpha phase into the beta and gamma polymorphs, whereas coloration led only to a major increase in the beta-to-alpha ratio The tensile properties were found to be optimized when the relative proportion of the beta and gamma phases combined compared to the alpha form gave rise to an FTIR A509/A532 absorption ratio between 40 and 45 Sterilization was found to cause some modifications to the crystalline microstructure near the surface of the monofilaments, but it did not change their mechanical properties Pathologic examination of the anastomotic regions after different periods of implantation revealed a minimal cellular response, with no mineralization, intimal hyperplasia, or excessive fibrous tissue reaction This good biocompatibility, together with other desirable characteristics such as ease of manipulation and satisfactory mechanical strength, makes PVDF an attractive alternative monofilament suture material for cardiovascular surgery

Formation and properties of a synthetic bone composite: hydroxyapatite-collagen.

Abstract: Composites composed of microcrystalline calcium-deficient hydroxyapatite (HAp) and collagen were formed at 38 degrees C via an acid-base reaction between calcium phosphate precursors in the presence of a collagen matrix. Formation of composites having HAp:collagen weight ratios of 4.5:1, 11:1, and 22:1, along with that of pure mineral were investigated. Isothermal calorimetry and X-ray diffraction indicated complete reaction within 5 h resulting in hardened monoliths. The rate of HAp formation increased with an increase in the proportion of collagen present. Electron microscopy indicated that the acceleratory effect of collagen was associated with the provision of nucleation sites for HAp crystallization. Analysis of the solution chemistry also showed that collagen affected the calcium and phosphate concentrations and the pH. While collagen was shown to effect the kinetics of HAp formation, the rate limiting step, as shown by X-ray diffraction and solution chemistry, was the dissolution of the acidic calcium phosphate precursor, CaHPO4. Preliminary mechanical data indicated that the Young's modulus, yield strength, and work to fracture were at the lower end of the range of those values reported for bone. The porosities observed in these composites suggest that they might be osteoinductive while their compositions should allow their eventual resorption. Thus, microstructure, kinetics, and mechanical data suggest that these composites might be suitable as bone substitutes which form in vivo.

Estimation of ideal mechanical strength and critical porosity of calcium phosphate cement.

Abstract: The ideal mechanical strength and critical porosity of calcium phosphate cement (CPC) were estimated to help determine ways to improve its properties. CPC at various porosities was made by packing CPC paste, at various powder-to-liquid (P/L) ratios (2.0-6.0), into a mold under various pressures (0-173 MPa). The mechanical strength of CPC, in terms of diametral tensile strength (DTS), increased with decreases in porosity. Intercrystalline fracture was observed in specimens made without the application of pressure, while fracture within the crystals increased with the packing pressure. These observations support the application of the relationship between DTS and porosity in fractographic equations. The ideal wet DTS and critical porosity of CPC were estimated to be 102 MPa and 63%, respectively. The minimum porosity of the currently used CPC was approximately 26-28%, even when it was packed under 173 MPa, and the maximum DTS value was thus approximately 13-14 MPa. Because reducing the porosity of currently used CPC would be difficult, we conclude that in CPC-related research, we should focus on ways in which to accelerate bone-replacing behavior, in addition to improving the mechanical strength of CPC.

Release of hexavalent chromium from corrosion of stainless steel and cobalt-chromium alloys.

Abstract: Experiments were undertaken to determine whether hexavalent chromium was released during corrosion of orthopedic implants. Uptake of chromium (Cr) by cells and separation using amberlite resin were the methods used to determine that hexavalent Cr was present. We used salts of chromium as trivalent chromium (chromic chloride) and hexavalent chromium (potassium dichromate) to verify that the amberlite separation technique separates hexavalent Cr into the upper phase and trivalent Cr into the lower phase. The use of the salts also verified that only the hexavalent Cr became red blood cell-associated and that most of this was intracellular rather than membrane bound. The use of the amberlite separation technique demonstrated that the hexavalent Cr in the red blood cells was rapidly reduced to trivalent Cr. Cellular uptake of chromium was documented in red blood cells following corrosion of stainless-steel and cobalt-chromium implants in vivo, in the red blood cells of patients undergoing total joint revisions, and in fibroblasts subjected to products of fretting corrosion of stainless-steel and cobalt-chromium implants. Thus, corrosion of implants can lead to the release of the biologically active hexavalent chromium into the body. This chromium is rapidly reduced to trivalent chromium in cells.

Role for interleukin‐4 in foreign‐body giant cell formation on a poly(etherurethane urea) in vivo

Abstract: Interleukin-4 (IL-4) was previously shown to induce extensive macrophage fusion to form foreign-body giant cells (FBGCs) in vitro. In the present study, our goal was to extend these findings to an in vivo test environment on biomaterials. The subcutaneous cage-implant system was modified for mice to elucidate IL-4 participation in mediating FBGC formation in vivo. Exudate leukocyte concentrations from cages containing poly(etherurethane urea) (PEUU A') and empty cage controls indicated a similar inflammatory response that turned toward resolution by 14 days postimplantation, thus confirming the applicability of the cage-implant system in mice. FBGC kinetic analysis showed that the formation of mouse FBGCs occurs through the fusion of adherent macrophages at a constant rate up to 14 days of implantation. Purified goat anti-mouse IL-4 neutralizing antibody (IL4Ab) or normal goat nonspecific control IgG (gtIgG) at various concentrations, or recombinant murine IL-4 (muIL4) was injected into the implanted cages containing PEUU A' every 2 days for 7 days. The injection of IL4Ab significantly decreased the FBGC density on PEUU A' cage-implanted in mice, when compared with the nonspecific IgG or PBS injection controls. Conversely, the FBGC density was significantly increased by the injection of muIL4 when compared with nonspecific IgG and PBS injection controls. Adherent macrophage density, FBGC morphology, FBGC average size, and size distribution were not significantly different among IL4Ab, nonspecific control gtIgG, muIL4, and PBS control groups. Our data suggest that IL-4 participates in FBGC formation on biomaterials in vivo.

Apatite coated on organic polymers by biomimetic process: improvement in its adhesion to substrate by glow-discharge treatment.

Abstract: A dense, uniform, and highly biologically active bone-like apatite layer can be formed in arbitrary thickness on any kind and shape of solid substrate surface by the following biomimetic method at ordinary temperature and pressure: First, a substrate is set in contact with particles of bioactive CaOSiO2-based glass soaked in a simulated body fluid (SBF) with inorganic ion concentrations nearly equal to those of human blood plasma so that a number of apatite nuclei are formed on the substrate. Second, the substrate is soaked in another solution with ion concentrations 1.5 times those of SBF (1.5SBF) so that the apatite nuclei grow in situ. In the present study, organic polymer substrates were treated with glow-discharge in O2 gas atmosphere, then subjected to the above-mentioned biomimetic process. The induction periods for the apatite nucleation on all the examined organic polymers were reduced from 24 to 6 h, with glow-discharge treatment. The adhesive strengths of the formed apatite layer to the substrates increased from about 4 to 10 MPa for poly(ethylene terephthalate) and poly-ether sulfone, and from 1 ∼ 2 to 6 ∼ 7 MPa even for poly(methyl methacrylate), polyamide 6 and polyethylene. It is supposed that highly polar groups such as carbonyl, ester, hydroxyl, and carboxyl ones formed by glow-discharge treatment increased the affinity of a silicate ion with the substrates to decrease the induction period, and also increased the affinity of the apatite with the substrate to increase the adhesive strength. The apatite-organic polymer composites thus obtained are expected to be useful as bone-repairing materials as well as soft tissue-repairing materials. © 1995 John Wiley & Sons, Inc.

Bioactive material template for in vitro synthesis of bone.

Abstract: We describe the synthesis of a new, porous, modified bioactive glass for use as a template for bone formation in vitro. The porosity of the glass was 36.4%; the pore size ranged from 10-160 mm, and there was no incipient devitrification. Prior to seeding the glass with cells, it was necessary to condition the disks. Optimum conditioning was achieved by immersing the templates in a tris buffer at pH 6.8 for 48 h and then treating the glass with tissue culture medium for 1 h at 37 degrees C. The conditioned glass disks were seeded with 10(6) neonatal rat calvaria osteoblast-like cells; cells on the substrate were maintained in culture for 3-7 days. To prevent pH shifts due to corrosion of the conditioned glass, the medium:glass ratio was maintained at 90 ml/g. We found that the templates were rapidly invaded by cells which maintained the osteoblast phenotype; thus, they exhibited high alkaline phosphatase activity and synthesized type I collagen and osteocalcin. SEM-EDAX showed that the cells elaborated substantial amounts of extracellular matrix and a bonelike tissue was present throughout the entire template thickness. FTIR analysis of material formed in the glass indicated that the mineral phase was a biologic hydroxyapatite. Controls (cells without substrate and substrate without cells) exhibited none of these features. Results of the study suggest that this porous glass can function as a template for generating bone in vitro.

Initial bone matrix formation at the hydroxyapatite interface in vivo

Abstract: Dense, sintered, slip-cast hydroxyapatite rods were implanted transfemorally in young adult rats. The femora were excised after 2 and 4 weeks and, following fixation, either embedded in methyl methacrylate for light microscopy, decalcified and prepared for transmission electron microscopy, or freeze fractured in liquid nitrogen for scanning electron microscopic analysis. The latter was performed on the two tissue fragments that remained after freeze fracturing, from which the first contained the implants and the second comprised tissue that had been immediately adjacent to the hydroxyapatite rods. Undecalcified light microscopic sections revealed extensive bone tissue formation around and in contact with the hydroxyapatite rods. The initial bone matrix apposed to the implant surface, as demonstrated with scanning electron microscopy, was either composed of globular deposits or an organized network of collagen fibers. The deposits, which ranged in size from 0.1–1.1 μm, fused to form a cement-like matrix to which collagen fibers were attached. Degradation of the hydroxyapatite surface resulted in the presence of unidirectionally aligned crystallites, with which the newly formed bone matrix was closely associated. Ultrastructural analysis of the bone-hydroxyapatite interface with transmission electron microscopy revealed a 50–600-nm-wide collagen-free granular zone, comprising one or more 40–100-nm-thick electron-dense layer(s). These structural arrangements most probably partially represent the globular deposits and proteinaceous material adsorbed onto and partially in the degrading hydroxyapatite surface. Although the latter change in surface topography may have enhanced bonding of the cement-like matrix to the hydroxyapatite, the cause for this change in topography and the type of bond formed are, at present, unknown. © 1995 John Wiley & Sons, Inc.

Mechanical properties of xenogeneic small-intestinal submucosa when used as an aortic graft in the dog.

Abstract: Small-intestinal submucosa (SIS) has been shown to induce tissue remodeling in vivo when used as a vascular graft. The present study investigated the physical and mechanical properties of remodeled aortic grafts derived from xenogeneic SIS material. Eight infrarenal aortic grafts were implanted in mongrel dogs. The grafts were explanted at 1 or 2 months and tested for compliance and hoop mechanical properties. The morphologic changes within the grafts were also characterized. The remodeling process produced graft structures which were significantly stronger than both the normal artery (P = .012) and the original SIS graft (P = .0001), and the compliance of these structures was one third that of normal artery and similar to the original SIS grafts. The remodeled grafts were > 10 times the thickness of the implanted SIS. Immunohistochemical analysis of remodeled tissues suggest that the SIS material was degraded and resorbed over time. The remodeling process transformed a material which was physically and mechanically quite different from normal aorta into a blood conduit which had the physical and mechanical properties needed to function in this mammalian arterial system. © 1995 John Wiley & Sons, Inc.

Neuronal cell attachment to fluorinated ethylene propylene films with covalently immobilized laminin oligopeptides YIGSR and IKVAV. II

Abstract: Material surfaces that can mediate cellular interactions by the coupling of specific cell membrane receptors may allow for the design of a biomaterial that can control cell attachment, differentiation, and tissue organization. Cell adhesion proteins have been shown to contain minimum oligopeptide sequences that are recognized by cell surface receptors and can be covalently immobilized on material surfaces. In this study, cell attachment to fluorinated ethylene propylene (FEP) films functionalized with the laminin-derived oligopeptides, YIGSR and a 19-mer IKVAV-containing sequence, was assessed using NG108-15 neuroblastoma and PC12 cells. A radiofrequency glow discharge (RFGD) process that replaces the FEP surface fluorine atoms with reactive hydroxyl functionalities was used to activate the film surfaces. The oligopeptides were then covalently coupled to the surface by their C-terminus using a standard nucleophilic substitution reaction. The covalent attachment of the oligopeptides to the FEP surface was verified using electron spectroscopy for chemical analysis (ESCA). Receptor-mediated NG108-15 cell attachment on the YIGSR-modified films was determined using competitive binding assays. Average cell attachment on the oligopeptide immobilized films in medium containing soluble CDPGYIGSR was reduced by approximately a factor of 2, compared to cell attachment in serum-free medium alone. No significant decrease in cell attachment was noted in medium containing the mock oligopeptide sequence CDPGYIGSK. FEP films immobilized with the 19-mer IKVAV sequence demonstrated a higher percentage of receptor mediated cell attachment on the film surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)