Joo L. Ong

Diffusion in musculoskeletal tissue engineering scaffolds: Design issues related to porosity, permeability, architecture, and nutrient mixing

The field of tissue engineering continues to advance with the discovery of new biomaterials, growth factors and scaffold fabrication techniques. However, for the ultimate success of a tissue engineered construct the issue of nutrient transport to the scaffold interior needs to be addressed. Often, the requirements for adequate nutrient supply are at odds with other scaffold design parameters such as mechanical properties as well as scaffold fabrication techniques, leading to incongruities in finding optimal solutions. The goal of this review article is to provide an overview of the various engineering design factors that promote movement of nutrients, waste and other biomolecules in scaffolds for musculoskeletal tissue engineering applications. The importance of diffusion in scaffolds and how it is influenced by porosity, permeability, architecture, and nutrient mixing has been emphasized. Methods for measuring porosity and permeability have also been outlined. The different types of biomaterials used, scaffold fabrication techniques implemented and the pore sizes/porosities obtained over the past 5 years have also been addressed.

Contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium.

Chitosan, a derivative of the bio-polysaccharide chitin, has shown promise as a bioactive material for implant, tissue engineering and drug-delivery applications. The aim of this study was to evaluate the contact angle, protein adsorption and osteoblast precursor cell attachment to chitosan coatings bonded to titanium. Rough ground titanium (Ti) coupons were solution cast and bonded to 91.2% de-acetylated chitosan (1 wt% chitosan in 0.2% acetic acid) coatings via silane reactions. Non-coated Ti was used as controls. Samples were sterilized by ethylene oxide gas prior to experiments. Contact angles on all surfaces were measured using water. 5 × 104 cells/ml of ATCC CRL 1486 human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, were used for the cell attachment study. SEM evaluations were performed on cells attached to all surfaces. Contact angles and cell attachment on all surfaces were statistically analyzed using ANOVA. The chitosan-coated surfaces (76.4 ± 5.1°) exhibited a significantly greater contact angle compared to control Ti surfaces (32.2±6.1°). Similarly, chitosan-coated surfaces exhibited significantly greater (P < 0.001) albumin adsorption, fibronectin adsorption and cell attachment, as compared to the control Ti surfaces. Coating chitosan on Ti surfaces decreased the wettability of the Ti, but increased protein adsorption and cell attachment. Increased protein absorption and cell attachment on the chitosan-coated Ti may be of benefit in enhancing osseointegration of implant devices.

Effect of hydrothermally treated anodic oxide films on osteoblast attachment and proliferation

In this study, the effect of anodization following hydrothermal treatments on osteoblast cell attachment and proliferation were evaluated. The anodic oxide films produced in this study was observed to exhibit overlapping microporous structures with microprojections. In addition, the anodic oxide surfaces were significantly rougher in comparison to control untreated titanium (Ti) surfaces. Following hydrothermal treatments for 2 and 4 h, hydroxyapatite (HA) crystals were observed on anodic surfaces. Using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, no significant difference in the biocompatibility of the treated and untreated Ti surfaces was observed. However, scanning electron micrographs indicated rounded osteoblast cells on control and anodized Ti surfaces, with numerous microvilli after 6 h. In contract, cells cultured on hydrothermally treated surfaces after 6 h incubation were observed to exhibit polygonal shape, flattened, and fully spread. In addition, more cells were observed on Ti surfaces that were hydrothermally treated for 4 h as compared to Ti surfaces that were hydrothermally treated for 2 h. After culturing the cells for 24 h and 4 days, no significant difference was observed for cells cultured on all surfaces. It was concluded from this study that hydrothermally treated surfaces exhibited an effect on early osteoblast attachment.

Osteoblast precursor cell activity on HA surfaces of different treatments

The clinical success of dental implants is governed by implant surfaces and bone cell responses that promote rapid osseointegration and long-term stability. The specific objective of this study was to investigate osteoblast precursor cell responses to hydroxyapatite (HA) surfaces of different treatments. Since the nature of bone cell responses in vitro is influenced by the properties of HA ceramics, this study was divided into two components: a chemical and crystallographic characterization of the HA ceramics and an in vitro cell culture study. The sintered HA samples were observed to have the highest crystallite size as compared to the as-received HA and calcined HA samples. No differences in the surface roughness and chemical composition were observed among the sintered, calcined, and as-received HA surfaces. In concurrence with the X-ray diffraction, high resolution XPS resolution of Ca 2p also indicated a higher crystallinity on sintered HA samples as compared to the calcined and as-received HA samples. As indicated by increased alkaline phosphatase-specific activity, increased cell-surface and matrix-associated protein, and 1.25 (OH2) vitamin D3-stimulated osteocalcin production, a more differentiated osteoblast-like phenotype was observed on the sintered HA surfaces compared to the as-received HA and calcined HA surfaces. An increased osteoblast-like cell activity on the sintered HA surfaces suggested that the crystallite size of HA surfaces may play an important role in governing cellular response.

Morphological behavior of osteoblast-like cells on surface-modified titanium in vitro.

In recent papers, we reported the results of a study on the graded porous titanium coatings on titanium by plasma spraying and amino-group ion implantation. The paper is to preliminarily evaluate the biocompatibility of surface-modified titanium through 2, 5 and 7 days cell culture in vitro. Cell morphology was observed by a scanning electron microscope. Cell proliferation and type I collagen synthesis were measured by 3(4.5-dimethyl-thiazole-2-yl)2,5-diphenyl tetrazolium bromide (MTT) and enzyme-linked immunosorbent assay (ELISA), respectively. Our experimental results showed that osteoblast-like cells attached and spread well on surface-modified titanium. Cells were observed to grow into the pores and form extracellular matrix. MTT and ELISA results showed no detrimental effect on the development of cell. These studies support the biocompatibility of surface-modified titanium.

Effects of Dissolved Calcium and Phosphorous on Osteoblast Responses

The dissolution behavior of hydroxyapatite (HA) and its effect on the initial cellular response is of both fundamental and clinical importance. In this study, plasma-sprayed HA coatings were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Calcium (Ca) and inorganic phosphorous (Pi) ions released from plasma-sprayed HA coatings within 3 weeks were measured by flame atomic absorption and colorimetrically molybdenum blue complex, respectively. To investigate the effect of dissolution of HA coatings on osteoblast response, additional Ca and Pi were added into the cell culture media to simulate the dissolution concentrations. Human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the biological responses to enhanced Ca and Pi media over 2 weeks. Osteoblast differentiation and mineralization were measured by alkaline phosphatase-specific assay and 1,25 (OH)2 vitamin D3 stimulated osteocalcin production. The coatings exhibited an HA-type structure. FTIR indicated the possible presence of carbonates on the coatings. A dissolution study indicated a continual increase in Ca and Pi over time. In the cell culture study, enhanced osteoblast differentiation occurred in the presence of additional Ca concentration in the cell culture media. However, additional Pi concentration in the cell culture media was suggested to slow down osteoblast differentiation and mineralization.

Protein adsorption and osteoblast responses to different calcium phosphate surfaces.

The purpose of this study was to investigate the effect of sintering different calcium phosphate (CaP) surfaces on protein adsorption and osteoblast cell response. As-received and sintered hydroxyapatite (HA) and brushite were used in this study. X-ray diffraction revealed a poorly crystallized HA structure for the unsintered HA and a highly crystallized HA for the sintered HA surfaces. A brushite-type structure was indicated on the unsintered brushite surfaces, whereas sintered brushite surfaces contained mixtures of different CaP phases. Using 1 mg/mL albumin solution, protein was suggested to selectively adsorb on the CaP surfaces. A statistically higher albumin adsorption was observed on unsintered HA (9.5 micrograms/mL) and unsintered brushite (50.1 micrograms/mL) surfaces compared to sintered HA (3.2 micrograms/mL) and sintered brushite (3.4 micrograms/mL) surfaces. In the in vitro study using osteoblast cells, no statistical responses were observed between cells cultured on sintered HA and sintered brushite after 8 days of incubation. However, statistical differences in osteocalcin and protein production were observed between the unsintered HA and unsintered brushite. In addition, statistical differences in protein production, alkaline phosphatase activity, and osteocalcin production were observed between sintered CaP and unsintered CaP surfaces. From the protein adsorption and cell responses observed in this study, it was concluded that CaP surfaces need to be fully characterized prior to implantation.

Deposition of highly adhesive ZrO2 coating on Ti and CoCrMo implant materials using plasma spraying

ZrO(2) (4% CeO(2)) and ZrO(2) (3% Y(2)O(3)) coatings were deposited on titanium (Ti) and CoCrMo implants using plasma spraying and the adhesive, morphological and structural properties of the plasma-sprayed coatings were evaluated. Characterization of these coatings was performed using X-ray diffraction (XRD), scanning electron microscopy (SEM), surface roughness, hardness, and adhesive strength. XRD patterns showed that both the coatings appeared to be primitive tetragonal phase. SEM observations showed that both the ZrO(2) coatings appeared to be rough, porous and melted. The cross-section surface morphology of the coatings, coating-substrate interfaces and substrates without acid etching was very dense and smooth. After acid etching, as compared to the dense ZrO(2) coating-CoCrMo substrate interfaces, the thin gaps appeared within the ZrO(2) coating-Ti substrate interfaces. It is suggested that plasma spraying probably formed an amorphous Ti layer in the coating-Ti substrate interface that can be removed by acid etching. The average surface roughness of ZrO(2) (3% Y(2)O(3)) and ZrO(2) (4% CeO(2)) coatings was correlated to the starting powder size and substrates. No significant difference between the hardness of all coatings and substrates was observed. The adhesive strengths of ZrO(2) (4% CeO(2)) coating to Ti and CoCrMo substrates were higher than 68MPa and significantly greater than that of ZrO(2) (3% Y(2)O(3)) coatings.

Fibronectin adsorption on titanium surfaces and its effect on osteoblast precursor cell attachment

Abstract The objective of this study was to investigate the adsorption of fibronectin on titanium (Ti) surfaces and the effect of pre-adsorbed fibronectin on osteoblast precursor cell attachment in vitro. Two different concentrations of bovine fibronectin were used in this study. Protein adsorption on Ti surfaces was analyzed using the micro bicinchoninic acid (BCA) protein assay. Cell concentration on Ti and fibronectin pre-adsorbed Ti surfaces after 3 h incubation was analyzed using the Vybrant™ cell adhesion assay. Cell morphology on Ti and fibronectin pre-adsorbed Ti surfaces was observed using scanning electron microscopy (SEM). After 180 min incubation, maximum adsorption of bovine fibronectin on Ti surfaces was observed. Fibronectin adsorption on Ti surfaces was observed to be significantly dependent on the initial concentration and the amount of incubation time. In the presence of 1 mg/ml fibronectin pre-adsorbed on Ti surfaces after 15 min, osteoblast precursor cell attachment on Ti surfaces was observed to be enhanced compared with control Ti surfaces, Ti surfaces pre-adsorbed with 1 mg/ml fibronectin for 180 min, and Ti surfaces pre-adsorbed with 0.1 mg/ml fibronectin for 15 and 180 min. No significant difference in cell attachment was observed between control Ti surfaces, Ti surfaces pre-adsorbed with fibronectin for 180 min, and Ti surfaces pre-adsorbed with 0.1 mg/ml fibronectin for 15 and 180 min. In addition, no differences in cell morphology of the attached osteoblast precursor cells on control Ti surfaces and Ti surfaces pre-adsorbed with fibronectin were observed in this study. It was concluded that an optimum concentration of adsorbed fibronectin on Ti surfaces plays an important role in governing cell attachment.

Osteoblast Precursor Cell Attachment on Heat-treated Calcium Phosphate Coatings

The influence of properties of calcium phosphate (CaP) coatings on bone cell activity and bone-implant osseointegration is not well-established. This study investigated the effects of characterized CaP coatings of various heat treatments on osteoblast response. It was hypothesized that heat treatments of CaP coatings alter the initial osteoblast attachment. The 400°C heat-treated coatings were observed to exhibit poor crystallinity and significantly greater phosphate or apatite species compared with as-sputtered and 600°C heat-treated coatings. Similarly, human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, seeded on 400°C heat-treated coatings, exhibited significantly greater cell attachment compared with Ti surfaces, as-sputtered coatings, and 600°C heat-treated coatings. The HEPM cells on Ti surfaces and heat-treated coatings were observed to attach through filopodia, and underwent cell division, whereas the cells on as-sputtered coatings displayed fewer filopodia extensions and cell damage. Analysis of the data suggested that heat treatment of CaP coatings affects cell attachment.