One of the main focuses of tissue engineering is to search for tridimensional scaffold materials, complying with nature’s properties for tissue regeneration. Determining material biocompatibility is a fundamental step in considering its use. Therefore, the purpose of this study was to analyze osteoblast cell adhesion and viability on different materials to determine which was more compatible for future bone regeneration. Tridimensional structures were fabricated with hydroxyapatite, collagen, and porous silica. The bovine bone was used as material control. Biocompatibility was determined by seeding primary osteoblasts on each tridimensional structure. Cellular morphology was assessed by SEM and viability through confocal microscopy. Osteoblast colonization was observed on all evaluated materials’ surface, revealing they did not elicit osteoblast cytotoxicity. Analyses of four different materials studied with diverse compositions and characteristics showed that adhesiveness was best seen for HA and viability for collagen. In general, the results of this investigation suggest these materials can be used in combination, as scaffolds intended for bone regeneration in dental and medical fields.
Bone regeneration has increasingly turned into a very promising field, with use of resorbable biomaterials in combination with cells [
Scaffolds act as templates mimicking the functions of the extracellular matrix (ECM), where cells can interact and differentiate into their native phenotype. It is then necessary for the scaffold to meet the following conditions: the scaffold must be biocompatible, nontoxic, nonimmunogenic, easy to elaborate, and biodegradable. In addition, it must allow for proper cell survival and signaling. Moreover, cells must be capable of growth, cellular remodeling, and reorganization [
One of the most important characteristics of the scaffold is a high interconnected porosity to enable vascularization for nutrient and gas diffusion, which permits waste disposal [
Thus, a proper porosity can improve mechanical retention between the scaffold and the host’s environment, achieved as a result of ECM unspecified protein adsorption through a formed layer. This allows for a response from the cells in regard to the biomaterial they are in contact with, thus enabling new tissue formation [
Another aspect to consider is scaffold mechanical resistance to load and forces. The scaffold must provide properties as close as possible to the host’s natural environment, to be degraded only when the new tissue is properly formed [
Also, porous silicon seems promising for scaffold use in bone repair, since this material has been shown to promote osteoblast adherence and initiate maturation process [
In spite of these discoveries, the ideal scaffold for bone regeneration has not been found. Hence, the objective of this work was to analyze osteoblast attachment and viability on four different materials: bovine bone (control), hydroxyapatite (HA), porous silicon, and collagen to determine a possible scaffold fabrication candidate for bone regeneration.
Hydroxyapatite synthesis was carried out by the wet technique. In a three-necked flask, 900 mL of 0.33 M calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) was placed with 1,500 mL 0.12 M diammonium hydrogen phosphate ((NH4)2HPO4) at 1.0 mL/min. To adjust the pH to 12, 75 mL of ammonium hydroxide (NH4OH) was added. Subsequently, the solution was heated at 90°C, stirred for 1 hour, and incubated for 10 days at room temperature. The obtained precipitate was washed several times with distilled water to neutralize the pH, dried at 250°C for 1 h, and then calcinated at 1,000°C for 3 h.
The bovine bone was acquired as follows: samples of bovine femur were purchased from a local market. The bone was cleaned and disinfected to remove tissues, organic material, and microorganisms. Hot water was used for bone marrow extraction. Two percent NaOH was employed to remove fat and protein and 1% antiseptic solution (sodium hypochlorite) to prevent microorganism growth. Subsequently, the bone was rinsed with water to remove traces of cleaning and disinfecting solutions. It was then subjected to heat treatment at 1,000°C for 3 h to remove all organic materials [
XRD was performed on HA and bovine bone samples. X-ray diffractograms were obtained on a PANalytical X’Pert PRO MPD (Netherlands) device with the following parameters: measuring range between 2° and 90° (2
Porous silicon was prepared by electrolysis using HF diluted with ethanol (HF : C2H5OH) (Merck KGaA) concentration [1 : 2] and a cell specially designed for this purpose (Figure
Cell design for porous silica elaboration (Professor Díaz-Peraza). Platinum electrode cell bracket (no. 1), p-type silicon acting as a second electrode (no. 2), and covering the circular orifice (no. 3) on a flat wall. The outer portion has a thin silicon layer of aluminum, previously vaporized to improve electrical contact between the silicon and an aluminum plate secured to the cavity by four screws (no. 4). Between the two electrodes, a DC current source was added and a milliamperimeter was used to supply and measure current.
Bovine tendon from the tail was selected for type I collagen extraction using 0.05 M acetic acid and continuous stirring for 72 h at 4°C. The obtained solution was subsequently filtered through a sterile gauze and centrifuged at 3,000 rpm for 2 h at 4°C. Finally, type I collagen solution was lyophilized, extracted, and stored at 4°C for subsequent 3D matrix construction.
Primary osteoblasts from the knee trabecular bone were obtained from the tissue obtained from joint replacement surgery, after informed signed consent by the patient. Surgery was performed at San Ignacio Hospital (Department of Orthopedics of Pontificia Javeriana University in Bogotá, Colombia) and approved by Research Ethics Committee at the Pontificia Universidad Javeriana act 002 of 2009.
Osteoblasts were isolated, following the method reported by Ducheyne and Qiu [
Primary human osteoblasts seeded into 12-well plates at a cell density of 50,000 cells/well on HA, silicon, bovine bone, and collagen matrices were evaluated for biocompatibility. To this end, manufactured porous matrices were sterilized with ethylene oxide and 1 × 106 osteoblasts were seeded on each material. Cells were maintained in DMEM supplemented with 10% FBS and 200
After one week, culture matrices with cells were fixed with 3% buffered glutaraldehyde solution, followed by dehydration with increasing ethanol concentrations (50, 60, 70, 80, 90, and 100%), and dried at a critical point ending with gold coating. Morphology was evaluated using SEM on a JEOL Model JSM-6490 LV operated under high vacuum mode, equipped with sensors allowing for scattered electron imaging.
Cell viability was assessed on matrices (
Hydroxyapatite sample X-ray diffractgrams are illustrated in Figure
X-ray diffractograms of synthetic HAp and bovine bone. Qualitative identification of the crystalline phase established by comparison between reflections of the profile measured with diffraction profile reflections, reported by the International Center for Diffraction Data (ICDD) (powder diffraction file) using search-match software illustrates (a) hydroxyapatite components synthesized by the wet technique and (b) bovine bone analysis after treatment for cell seeding.
Scaffolds prepared from four different materials were synthesized according to established methods, obtaining three-dimensional structures with different porosities and textures (Figures
SEM micrographs of scaffolds used for osteoblast seeding. (a) Bovine bone (sintered at 1,000°C). Particles varied from smooth to rough, with asymmetric distribution. Shapes were round, irregular, short, or long. Other particles were polyhedral in configuration. Microporosity ranged in size range from 0.1
The results of SEM images showed osteoblast capability of cell attachment and interaction with other cells (Figures
Osteoblasts’ cell morphology on different scaffolds. SEM micrographs of osteoblasts seeded on different scaffolds and cultured for one week. (a) Osteoblasts seeded on bovine bone scaffold after seven days of culture at 3,000x. Cytoplasmic processes of osteoblast-substrate interactions and cell adhesion with filopodia and lamellipodia. (b) Osteoblasts seeded on bovine bone scaffold after seven days of culture at 1,500x. (c) Osteoblasts seeded on a HA scaffold after seven days in culture at 3,000x. (d) Osteoblasts seeded on a porous silicon scaffold after seven days of culture at 800x. Osteoblasts spreading on surface with filopodia in the substrate contact.
The results of fluorescence microscopy showed osteoblast viability in all four scaffolds after seven days of culture (Figures
Primary osteoblast viability evaluation. (a) Osteoblasts seeded on collagen type I after seven days of culture. Fluorescence micrograph depicting viable cells as bright green dots immersed at multiple collagen sites. (b) Osteoblasts seeded on HA matrix after seven days in culture. (c) Osteoblasts seeded on bovine bone matrix after seven days in culture. (d) Fluorescence micrograph of osteoblasts on porous silicon after seven days in culture.
Attractive alternatives to bone grafting have been achieved through recent advances in biomaterial development [
In this study, scaffolds were prepared with four different materials to evaluate
Hydroxyapatite has been one of the most used bioceramics in dental reconstruction and bone tissue regeneration due to its biocompatibility, osteoconductivity, and lack of cytotoxicity [
Romanelli et al. developed a nanofibrous gel scaffold to which titanium nanoparticles and nanocrystals were added to simulate bone tissue, achieving osteoblast differentiation [
It is important to note that HA porosity can increase when combined with materials, such as gelatins, which supply HA ions improving cell viability but in turn decreasing its crystallinity and grain size [
Another relevant aspect to consider is scaffold biodegradability. Several studies indicate use of HA mixtures with different materials to improve their slow degradation. Among these, Kasuya et al. used a mixture of phosphate cement gelatin/HA achieving improved mechanical properties, degradability, and bone formation [
Recent studies evaluated the biodegradability and mechanical properties of a hydroxyapatite/chitosan/magnetite scaffold, showing that a higher hydroxyapatite content in the mixture and increased nucleation sites. This allowed increased mineral apposition by chitosan and magnetite, therefore increasing HA mechanical strength. On the other hand, biodegradability increased, with decreased HA content, due to decreased nucleation sites covering the surface, thus increasing the material’s porosity. Collectively, a correlation between surface degradation and pore formation was observed [
In contrast, collagen has biodegradable properties and is a good alternative. Collagen type I has been considered as a promising scaffold material, since it is one of the most prevalent components of the ECM [
Won et al. [
Porous silicon results showed osteoblast adhesion (Figure
Osteoblast adhesion on material surfaces is a long-term phenomenon, involving interactions among a host of biological molecules to induce signal transduction and cell response. The reactions of osteoblasts on material surfaces are exteriorized in a series of different time-related phenomena: protein adsorption at the material’s surface, followed by a sequence of rapid short-term events constituting the attachment phase and subsequent adhesion phase, followed by proliferation, migration, and phenotypic differentiation (Figure
Osteoblast adhesion to substrates is mediated via adhesion molecules. The cell-matrix adhesions mechanically interlink internal actin filaments and matrix, leading to the formation of focal adhesions, focal plaque, or focal contact. Such focal contacts are tenacious adhesion structures, which remain attached to the substratum, even after forceful cell detachment (Figure
Collectively, all materials assayed provided different properties that need further optimization to achieve a substrate mimicking a natural environment. The best material providing a compatible surface for cell adhesion was bovine bone, followed by HA, porous silicon, and collagen. In contrast, after one week of culture, collagen sustained the most number of viable cells.
In this study, osteoblast cell morphology, adhesiveness, and viability were evaluated as a possible biocompatible indicator. We observed a favorable cellular response between the cells seeded onto the prepared scaffold. Depending on the substrate, different responses were observed. Adhesiveness was best seen for HA and viability for collagen. These materials have been widely reported as alternatives for 3D scaffold development [
Bone tissue engineering still holds many challenges. Scaffold material characteristics are key to reach the ultimate goal: cellular recognition, tissue specific commitment, repair, and regeneration. Therefore, it is of special interest to focus on using a combination of materials to provide mutual benefits. This interaction will tackle the weaknesses of one material by complementing with the strengths of the other material, leading to an ideal preparation for future scaffold establishment in tissue regeneration.
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.
The authors thank Vice-Rectory for Research at Pontificia Universidad Javeriana for financial support (Projects 6387 and 3263). In addition, they express their gratitude to the School of Dentistry Dental Research Center and the School of Science for logistical support.
The graphic abstract is included and is titled “SEM micrographs of structure and the CFM (confocal fluorescent Microscopy) images of cell viability, on different scaffolds after 7 days of osteoblastic cells culture.”