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PubMed2004

Systematic variation in osteoblast adhesion and phenotype with substratum surface characteristics.

Time-varying interactions of human fetal osteoblastic cells (hFOB 1.19) with materials of diverse chemical composition and surface energy, including biodegradable lactide/glycolide-based polymers, were assessed using a combination of assays sensitive to different phases of cell-substratum compatibility. Short-term (minutes to hours) cell-attachment-rate assays were used to measure the earliest stages of cell-surface interactions leading to adhesion. Proliferation-rate assays quantifying viability of attached cells were applied as a measure of medium-term (hours to days) cytocompatibility. Both attachment- and proliferation-rate assays were found to strongly correlate with material surface energy, with the exception of a reproducible and significant adhesion preference for fully water-wettable quartz over glass. No such adhesion/proliferation preference was observed for hydrophobized counterparts, and attachment to water-wettable glass was significantly less than that to control tissue culture polystyrene. These results suggest that the amorphous SiO(x) surface was inhibitory to hFOB 1.19 growth whereas putatively crystalline quartz stimulated bioadhesion. Alkaline phosphatase activity was evaluated as a marker for long-term (days) differentiation of hFOB 1.19 cells and did not strongly correlate with surface energy or, in the case of biodegradable polymers, chemical composition.

Alkaline Phosphatase↗
PubMed2004

Endothelial cell functions in vitro cultured on poly(L-lactic acid) membranes modified with different methods.

We recently developed several methods to enhance the cell-polymer interactions. Optimal conditions for each method have been revealed separately by in vitro cell culture. As a practical consideration for construction of tissue-engineered organs, it is necessary to consider which is the most suitable and convenient in clinical applications. To compare the efficiency of these methods with respect to cell functions, poly-L-lactic acid (PLLA) was selected as matrix being modified by 1) aminolysis (PLLA-NH(2)), 2) collagen immobilization with GA (PLLA-GA-Col), 3) chondroitin sulfate (CS)/collagen layer-by-layer (LBL) assembly (PLLA-CS/Col), 4) photo-induced grafting copolymerization of hydrophilic methacrylic acid (MAA) (PLLA-g-PMAA), and 5) further immobilization of collagen with 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride (EDAC) (PLLA-g-PMAA-Col). The surface wettability of the modified PLLA was determined by water contact angle measurements. The cell response to the modified PLLA was quantitatively assessed and compared by using human umbilical endothelial cells (HUVECs) culture. Our results indicate that all the modifications can improve the cytocompatibility of PLLA (e.g., cells can attach with spreading morphology, proliferate and secret vWF and 6-keto-PGF(1 alpha)). All the collagen-modified PLLA showed more positive cell response than those purely aminolyzed or PMAA grafted. Among all the methods, collagen immobilization by LBL assembly or GA bridging after aminolysis is more acceptable for the convenience and applicability to scaffolds.

Biocompatible Materials↗
PubMed2004

Development of photocrosslinkable hyaluronic acid-polyethylene glycol-peptide composite hydrogels for soft tissue engineering.

Hyaluronic acid (HA; also called hyaluronan) is a naturally derived, nonimmunogenic, nonadhesive glycosaminoglycan that has important roles in several wound-healing processes. In previous work, we created photocrosslinkable glycidyl methacrylate-HA (GMHA) hydrogel biomaterials that were cytocompatible, biologically active, and had a decreased rate of hyaluronidase degradation compared with native HA. The goal of the studies presented herein was to explore peptide conjugation techniques to further adjust the material and biological properties of the GMHA hydrogels. We conjugated GMHA with acrylated forms of polyethylene glycol (PEG) and PEG-peptides to yield GMHA-PEG-peptide composite hydrogels. By varying the reactant concentrations, we created stable hydrogels with high peptide conjugation efficiencies (up to 80%), controllable peptide concentrations (in the range of 1-6 micromol peptide per milliliter of hydrogel), and defined physicochemical properties (e.g., swelling ratio, enzymatic degradation rate). These composite hydrogels may prove to be a promising scaffolding biomaterial for a variety of soft tissue engineering applications.

Chemical Phenomena↗
PubMed2004

Nanometer surface roughness increases select osteoblast adhesion on carbon nanofiber compacts.

Carbon nanofibers have exceptional theoretical mechanical properties (such as low weight-to-strength ratios) that, along with possessing nanoscale fiber dimensions similar to crystalline hydroxyapatite found in bone, suggest strong possibilities for use as an orthopedic/dental implant material. To determine, for the first time, cytocompatibility properties pertinent for bone prosthetic applications, osteoblast (bone-forming cells), fibroblast (cells contributing to callus formation and fibrous encapsulation events that result in implant loosening), chondrocyte (cartilage-forming cells), and smooth muscle cell (for comparison purposes) adhesion were determined on carbon nanofibers in the present in vitro study. Results provided evidence that, compared to conventional carbon fibers, nanometer dimension carbon fibers promoted select osteoblast adhesion. Moreover, adhesion of other cells was not influenced by carbon fiber dimensions. In fact, smooth muscle cell, fibroblast, and chondrocyte adhesion decreased with an increase in either carbon nanofiber surface energy or simultaneous change in carbon nanofiber chemistry. To determine properties that selectively enhanced osteoblast adhesion, similar cell adhesion assays were performed on polymer (specifically, poly-lactic-co-glycolic; PLGA) casts of carbon fiber compacts previously tested. Compared to PLGA casts of conventional carbon fibers, results provided the first evidence of enhanced select osteoblast adhesion on PLGA casts of nanophase carbon fibers. The summation of these results demonstrate that due to a high degree of nanometer surface roughness, carbon fibers with nanometer dimensions may be optimal materials to selectively increase osteoblast adhesion necessary for successful orthopedic/dental implant applications.

Alloys↗
PubMed2005

Development of fungal mycelia as a skin substitute: characterization of keratinocyte proliferation and matrix metalloproteinase expression during improvement in the wound-healing process.

SACCHACHITIN membranes, prepared from the waste residue of the fruiting body of Ganoderma taugae, were used in our previous study to enhance skin wound healing in animal models. In the present study, the effects of the membrane on the growth of keratinocytes and the activity of matrix metalloproteinases (MMPs), as well as on the healing of skin wounds in humans, were estimated. Fresh human foreskin was employed as the source of the keratinocyte culture, and a modified keratinocyte-SFM medium supplemented with 0.2 ng/mL of recombinant epidermal growth factor and 30 microg/mL bovine pituitary extract was used to enhance the successful growth of keratinocytes under an atmosphere of 5% CO2, at 37 degrees C. The results indicated that 0.01% SACCHACHITIN enhanced the proliferation of keratinocytes in the culture on the fourth and fifth days, and cells showed neither morphological alteration nor disordered proliferation. This evidence clearly indicated that SACCHACHITIN was not cytotoxic to and was safe for the growth of keratinocytes. Thus, SACCHACHITIN might play a positive role in the proliferation and differentiation of keratinocytes around wounds and in accelerated wound healing of epidermal tissue. In addition, microscopic observations during the growth of keratinocytes showed that normal proliferation and differentiation took place along the margin of the SACCHACHITIN membrane. This indicates that SACCHACHITIN is possibly cytocompatible with keratinocytes. Electrophoretic analysis and inhibition tests for the binding effect of SACCHACHITIN on MMPs showed that SACCHACHITIN reduced MMPs in extracellular matrix degradation and facilitated establishment of an extracellular matrix around wounds; these effects resulted in rapid wound healing. SACCHACHITIN was used as a skin dressing for patients who had skin chronicle ulcer, which had not healed for over 7 months. Preliminary clinical observations showed that the wound improved and began to heal. An analysis of MMPs by ELISA in tissue of the wound indicated a significant decrease in MMP levels.

Cell Differentiation↗
PubMed2005

Development of a resorbable macroporous cellulosic material used as hemostatic in an osseous environment.

The control of bleeding is a frequently encountered therapeutic problem, particularly during dental surgery. The most efficient substances used to resolve this problem are not risk-free because of their animal or human origins, so cellulosic materials are potentially of interest. The aim of this study was to develop a resorbable macroporous cellulosic material for use as a resorbable hemostatic agent in bone sites. The degradation and the cytocompatibility of the cellulosic material versus controls were evaluated and its behaviour in vivo was studied. An original process using calcium carbonate powder as inverse matrix was used to develop a macroporous material. In order to predegrade the cellulosic material for hemostatic use, oxidation was performed with periodate. A dialdehyde component unstable at physiological pH was thus obtained. The material was found to have cytotoxicity, biocompatibility, and resorption properties similar to control but its hemostatic power was higher.

Absorbable Implants↗
PubMed2006

Calcium carbonate-calcium phosphate mixed cement compositions for bone reconstruction.

The feasibility of making calcium carbonate-calcium phosphate (CaCO(3)-CaP) mixed cements, comprising at least 40% (w/w) CaCO(3) in the dry powder ingredients, has been demonstrated. Several original cement compositions were obtained by mixing metastable crystalline CaCO(3) phases with metastable amorphous or crystalline CaP powders in aqueous medium. The cements set within at most 1 h at 37 degrees C in atmosphere saturated with water. The hardened cement is microporous and exhibits weak compressive strength. The setting reaction appeared to be essentially related to the formation of a highly carbonated nanocrystalline apatite phase by reaction of the metastable CaP phase with part or almost all of the metastable CaCO(3) phase. The recrystallization of metastable CaP varieties led to a final cement consisting of a highly carbonated poorly crystalline apatite analogous to bone mineral associated with various amounts of vaterite and/or aragonite. The presence of controlled amounts of CaCO(3) with a higher solubility than that of the apatite formed in the well-developed CaP cements might be of interest to increase resorption rates in biomedical cement and favors its replacement by bone tissue. Cytotoxicity testing revealed excellent cytocompatibility of CaCO(3)-CaP mixed cement compositions.

Biocompatible Materials↗
PubMed2006

Co-electrospun poly(lactide-co-glycolide), gelatin, and elastin blends for tissue engineering scaffolds.

In this study, we describe composite scaffolds composed of synthetic and natural materials with physicochemical properties suitable for tissue engineering applications. Fibrous scaffolds were co-electrospun from a blend of a synthetic biodegradable polymer (poly(lactic-co-glycolic acid), PLGA, 10% solution) and two natural proteins, gelatin (denatured collagen, 8% solution) and alpha-elastin (20% solution) at ratios of 3:1:2 and 2:2:2 (v/v/v). The resulting PLGA-gelatin-elastin (PGE) fibers were homogeneous in appearance with an average diameter of 380 +/- 80 nm, which was considerably smaller than fibers made under identical conditions from the starting materials (PLGA, 780 +/- 200 nm; gelatin, 447 +/- 123 nm; elastin, 1060 +/- 170 nm). Upon hydration, PGE fibers swelled to an average fiber diameter of 963 +/- 132 nm, but did not disintegrate. Importantly, PGE scaffolds were stable in an aqueous environment without crosslinking and were more elastic than those made of pure elastin fibers. To investigate the cytocompatibility of PGE, we cultured H9c2 rat cardiac myoblasts and rat bone marrow stromal cells (BMSCs) on fibrous PGE scaffolds. We found that myoblasts grew equally as well or slightly better on the scaffolds than on tissue-culture plastic. Microscopic evaluation confirmed that myoblasts reached confluence on the scaffold surfaces while simultaneously growing into the scaffolds. Histological characterization of the PGE constructs indicated that BMSCs penetrated into the center of scaffolds and began proliferating shortly after seeding. Our results suggest that fibrous scaffolds made of PGE and similar biomimetic blends of natural and synthetic polymers may be useful for engineering soft tissues, such as heart, lung, and blood vessels.

Animals↗
PubMed2007

Fabrication and in vitro characterization of porous biodegradable composites based on phosphate glasses and oligolactide-containing polymer networks.

Degradable porous composite materials for use as temporary bone replacement or tissue engineering scaffolds were produced using a methacrylate-modified oligolactide polymer network and phosphate invert glasses in the system P2O5-CaO-MgO-Na2O-(TiO2). Porous glasses with an open interconnective porosity were produced by a salt sintering process. Compressive strengths were significantly enhanced by polymer coating of the inner surface of the porous glasses or by fabrication of glass powder-reinforced porous polymer specimens. In vitro degradation in simulated body fluid showed a degradation pattern of the composites which could be modulated by the composition and resulting solubility of the incorporated glass phase. Cytocompatibility of the composites was investigated in a FDA/EtBr viability assay using an MC3T3-E1 osteoblast-like cell line and showed good biocompatibility of the materials in vitro.

3T3 Cells↗
PubMed2007

Increased osteoblast adhesion on nanograined hydroxyapatite and tricalcium phosphate containing calcium titanate.

Depending on the coating method utilized and subsequent heat treatments (such as through the use of plasma-spray deposition), inter-diffusion of atomic species across titanium (Ti) and hydroxyapatite (HA) coatings may result. These events may lead to structural and compositional changes that consequently cause unanticipated HA phase transformations which may clearly influence the performance of an orthopedic implant. Thus, the objective of the present in vitro study was to compare the cytocompatibility properties of chemistries that may form at the Ti:HA interface, specifically HA, tricalcium phosphate (TCP), HA doped with Ti, and those containing calcium titanate (CaTiO(3)). In doing so, results of this study showed that osteoblast (bone-forming cells) adhesion increased with greater CaTiO(3) substitutions in either HA or TCP. Specifically, osteoblast adhesion on HA and TCP composites with CaTiO(3) was almost 4.5 times higher than that over pure HA. Material characterization studies revealed that enhanced osteoblast adhesion on these compacts may be due to increasing shrinkage in the unit lattice parameters and decreasing grain size. Although all CaTiO(3) composites exhibited excellent osteoblast adhesion results, Ca(9)HPO(4)(PO(4))(5)OH phase transformation into TCP/CaTiO(3) increased osteoblast adhesion the most; because of these reasons, these materials should be further studied for orthopedic applications.

Bone Substitutes↗
PubMed2007

Preparation and assessment of glutaraldehyde-crosslinked collagen-chitosan hydrogels for adipose tissue engineering.

A portfolio of crosslinked chitosan:collagen blends was prepared, and their microarchitecture and water binding capacity were studied to investigate their application for adipose tissue engineering. Glutaraldehyde (GA) concentration had little effect on scaffold morphology or water binding capacity. However, the processing freezing temperature prior to lyophilization affected both. In vitro cytocompatibility of pre-adipocytes (PAs) was assessed for a candidate collagen:chitosan blend using two assays. Results confirm the viability of PAs on GA-crosslinked collagen:chitosan scaffolds. A rat subcutaneous pocket assay was employed to assess PA-seeded scaffolds in vivo. Animal tests proved that PA-seeded scaffolds were biocompatible, could induce vascularization, and form adipose tissue.

Absorbable Implants↗
PubMed2005

Preparation and evaluation of ammonia-treated collagen/chitosan matrices for liver tissue engineering.

To further enhance the properties of existing collagen/chitosan scaffolds for liver tissue engineering, a very simple method was developed to form noncovalently linked mimic of the liver extracellular matrices. Collagen/chitosan mixtures in various proportions (i.e., 1:0, 3:2, 1:1, 2:3, and 0:1 v/v) were lyophilized or evaporated to form sponges or flat films before they were gelled using an aqueous 25% ammonia solution. The porosities of the obtained sponges were above 90% with various pore sizes. The highest mechanical strength (1.9+/-0.7 MPa) and the lowest degradation time (65+/-1.7 days) were achieved by the collagen/chitosan (1:1) matrices. Hepatocytes cultured on the collagen/chitosan (1:1) matrices exhibited relatively high glutamate-oxaloacetate transaminase and glucose secretion functions 25 days post-seeding. Nuclues of the hepatocytes were more elongated and arranged in certain directions on the 1:1 matrices. The cytocompatibility and enhanced biostability of our new ammonia-treated collagen/chitosan matrices suggest that they could be used as scaffolds for liver tissue engineering.

Amines↗
PubMed2006

Poly(D,L-lactic acid)-block-(ligand-tethered poly(ethylene glycol)) copolymers as surface additives for promoting chondrocyte attachment and growth.

The poly(D,L-lactic acid)-block-(ligand-tethered poly(ethylene glycol)) copolymer was explored to engineer poly(D,L-lactic acid) (PLA) material to promote chondrocyte attachment and growth. The poly(D,L-lactic acid)-block-poly(ethylene glycol) copolymer (PLE) was synthesized by a coupling reaction between PLA and poly(ethylene glycol) (PEG) (M(n) 1000, 2000, and 4000 respectively), with the use of 4,4'-methylenediphenyl diisocyanate (MDI). Then the PLE was activated by methyl sulfonyl chloride and the amino acids or arginine-glycine-aspartic acid tripeptide (RGD) was attached, which was verified by the ninhydrin-UV method. The modified PLA films were simply prepared by blending PLA with PLE derivatives. ATR-FTIR, XPS, contact angle, and AFM results clearly showed that the PEG chain stably enriched on the surface of PLE-modified PLA films. The chondrocyte cytocompatibility test showed the modified PLA films could significantly improve chondrocyte attachment and proliferation.

Cell Adhesion↗
PubMed2006

In vitro studies on the influence of precultural conditioning method on osteoblast reactions of a new type of injectable calcium cement material.

A new injectable dicalcium phosphate dehydrate (DCPD)-based cement material "PD" VitalOs Cement was studied to elucidate the process of equilibrium occurring in the early stage of implantation. The present study investigated the pH variations of the cement sample-immersing culture medium at determined intervals, time-dependent calcium/phosphate release, cell proliferation, and vitality in the cells-cement coculture milieu, after different preculture conditionings of the samples. Measurement of pH variation showed that without renewing the medium, pH value of sample lixiviate medium first dropped and, after 70 h, gradually balanced. When medium was renewed each day, pH value of lixiviate medium first descended and, after 24 h, gradually returned to pH 7.2. The cell viability revealed an excellent cytocompatibility of the cement. Both cell proliferation and vitality test showed that the preculture conditioning treatment is important at least for good performance of osteoblasts growing on the surface of calcium phosphate hydraulic cement (CPHC) samples in vitro. The results of calcium and phosphate assays clearly showed that this cement material can continuously dissolve to release calcium and phosphate in the liquid cell culture environment. The decrease of proliferation in some experimental groups with short conditioning is due to an excess of acid, which still can have some influence on cell growth after 24 h, since the biological milieu is not continuously renewed as in in vivo conditions.

3T3 Cells↗
PubMed2007

Chitosan scaffolds for in vitro buffalo embryonic stem-like cell culture: an approach to tissue engineering.

Three-dimensional (3D) porous chitosan scaffolds are attractive candidates for tissue engineering applications. Chitosan scaffolds of 70, 88, and 95% degree of deacetylation (% DD) with the same molecular weight were developed and their properties with buffalo embryonic stem-like (ES-like) cells were investigated in vitro. Scaffolds were fabricated by freezing and lyophilization. They showed open pore structure with interconnecting pores under scanning electron microscopy (SEM). Higher % DD chitosan scaffolds had greater mechanical strength, slower degradation rate, lower water uptake ability, but similar water retention ability, when compared to lower % DD chitosan. As a strategy to tissue engineering, buffalo ES-like cells were cultured on scaffolds for 28 days. It appeared that chitosan was cytocompatible and cells proliferated well on 88 and 95% DD scaffolds. In addition, the buffalo ES-like cells maintained their pluripotency during the culture period. Furthermore, the SEM and histological study showed that the polygonal buffalo ES-like cells proliferated well and attached to the pores. This study proved that 3D biodegradable highly deacetylated chitosan scaffolds are promising candidates for ES-like cell based tissue engineering and this chitosan scaffold and ES cell based system can be used as in vitro model for subsequent clinical applications.

Animals↗
PubMed2007

Biocompatibility of a new radiopaque iodine-containing acrylic bone cement.

Radiopacity in the vast majority of the commercially available acrylic bone cements that are used clinically is provided by particles of either BaSO(4) or ZrO(2). Literature reports have shown these agents to have a detrimental effect on some mechanical properties of the cements as well as on its biological response. We, therefore, have developed a new type of bone cement, for which radiopacity results from the presence of an iodine-containing methacrylic copolymer. The focus of the present work was the comparison of the biocompatibility of this new cement and a commercially available cement that contains barium sulfate. In vitro experiments show that both cements are cytocompatible materials, for which no toxic leachables are found. Implantation of the cements in a rabbit for three months resulted in the occasional presence of a thin fibrous tissue at the cement-bone interface, which is common for acrylic bone cements. Consideration of all the results led to the conclusion that the new cement is as biocompatible as the BaSO(4)-containing one.

Animals↗
PubMed2004

Novel photopolymerizable biodegradable triblock polymers for tissue engineering scaffolds: synthesis and characterization.

For use in micro-patterned scaffolds in tissue engineering, novel diacrylated triblock macromers (PLA-b-PCL-b-PLA, PGA-b-PCL-b-PGA and PCL-b-PEO-b-PCL) were synthesized and characterized by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR) and gel permeation chromatography (GPC). All diacrylated polymers were designed as triblock copolymers and involved biodegradable blocks of relatively non-polar epsilon-caprolactone (CL) and polar monomers such as glycolide (GA), lactide (LA) or ethylene oxide (EO). All triblock polymers were prepared in molecular weights of a few kilo daltons via the anionic ring-opening polymerization (ROP) of the corresponding lactide, glycolide or caprolactone using stannous octoate [Sn(Oct)(2)] as catalyst. The polymers had low polydispersity indices, ranging from 1.23 to 1.56. Biodegradable polymeric networks were prepared with conversions of 72-84% via photopolymerization of the triblock diacrylated polymers with 2,2-dimethoxy-2-phenylacetophenone (DMPA) as photoinitiator. PLA-b-PCL-b-PLA copolymers crumbled easily and were not suitable for micro-patterning. PGA-b-PCL-b-PGA copolymers had higher water contact angles than PCL-b-PEO-b-PCL and were also cytocompatible with Fibroblasts 3T3.

3T3 Cells↗
PubMed1993

[Effect of synthetic hydroxyapatite ceramics on long-term cultures of isolated chondrocytes].

This study deals with the influence of hydroxyapatite-ceramic on long-term cultures of rabbit articular chondrocytes. By additioni of granules of this material into the culture wells improval of cell proliferation by factor 12 over a period of twelve weeks is found. More important is the maintaining of the status of differentiation of the chondrocytes, documented by immunoidentification of the expressed collagen I production over collagen II as marker for chondrocytes. In controls without addition of hydroxyapatite-ceramic there is a dynamic increase of collagen I production over the culture period and morphological alteration of the cell shape leading to fibroblast-like cells. As this dedifferentiation is the main problem concerning chondrocytes long-term cultures, the addition of HAC into the culture medium is a simple and effective method enhancing the productivity of the culture system. Moreover this experimental design with chondrocytes as cells familiar with mineralized matrix gives an important proof of the excellent cytocompatibility of the hydroxyapatite-ceramic used in this study.

Animals↗