Bone graft substitutes and cancellous biomaterials have been widely used to heal critical-size long bone defects due to trauma, tumor resection, and tissue degeneration. In particular, porous hydroxyapatite is widely used in reconstructive bone surgery owing to its biocompatibility. In addition, the in vitro modification of cancellous hydroxyapatite with osteogenic signals enhances the tissue regeneration in vivo, suggesting that the biomaterial modification could play an important role in tissue engineering. In this study, we have followed a tissue-engineering strategy where ultrasonically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix inside a porous hydroxyapatite scaffold. The ultrasonic stimulus had the following parameters: average power equal to 149 mW and frequency of 1.5 MHz. In comparison with control conditions, the ultrasonic stimulus increased the cell proliferation and the surface coating with bone proteins (decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen). The mechanical stimulus aimed at obtaining a better modification of the biomaterial internal surface in terms of cell colonization and coating with bone matrix. The modified biomaterial could be used, in clinical applications, as an implant for bone repair.
One of the key challenges in reconstructive bone surgery is to provide living constructs that possess the ability to integrate in the surrounding tissue. Bone graft substitutes, such as autografts, allografts, xenografts, and porous biomaterials have been widely used to heal critical-size long bone defects due to trauma, tumor resection, and tissue degeneration. The biomaterials used to build 3D scaffolds for bone tissue engineering are, for instance, the hydroxyapatite [
The preceding osteoinductive and osteoconductive biomaterials are ideal in order to follow a typical approach of the tissue engineering, an approach that involves the seeding and the in vitro culturing of cells within a cancellous scaffold before the implantation.
The tissue-engineering method is of great importance. In order to overcome the drawbacks associated with the standard culture systems in vitro, such as limited diffusion and inhomogeneous cell-matrix distribution, several bioreactors have been designed to provide different physical stimuli: a rotating vessel bioreactor [
Gorna and Gogolewski [
In this study, following the preceding “golden rules” of Gorna and Gogolewski, we have elected porous hydroxyapatite [
Hydroxyapatite is widely used in reconstructive bone surgery owing to its biocompatibility. The in vitro modification of porous hydroxyapatite, with osteogenic signals of the transforming growth factor-
As consequence, aiming, in a future work, at accelerated and enhanced bone regeneration in vivo, in the present study of tissue engineering, we show a particular “biomimetic strategy” that consists in the in vitro modification of porous hydroxyapatite with proliferated osteoblasts and their extracellular matrix produced in situ. In other words, applying an ultrasonic wave [
Porous Orthoss bovine hydroxyapatite disks (diameter, 8 mm; height, 4 mm) were kindly provided by Geistlich Pharma AG (Wolhusen, Switzerland) [
SEM image of unseeded hydroxyapatite, bar equal to 100
The human osteosarcoma cell line SAOS-2 was obtained from the American Type Culture Collection (HTB85, ATCC, Rockville, MD). The cells were cultured in McCoy’s 5A modified medium with
In order to anchor the hydroxyapatite disks to two standard well-plates, 3% (w/v) agarose solution was prepared and sterilized in autoclave, and during cooling, at
The well-plates with the biomaterial disks were sterilized by ethylene oxide at
A cell suspension of
An ultrasound stimulus [
The ultrasonic culture was placed into a standard cell culture incubator with an environment of
The static culture was placed into a standard cell culture incubator. The duration of the static culture was 22 days and the culture medium was changed on days 4, 7, 10, 13, 16, and 19.
At the end of the culture period, the disks were fixed with 2.5% (v/v) glutaraldehyde solution in 0.1 M Na-cacodylate buffer (pH = 7.2) for 1 hour at
At the end of the culture period, the cells were lysed by a freeze-thaw method in sterile deionized distilled water and the released DNA content was evaluated with a fluorometric method (PicoGreen, Molecular Probes, Eugene, OR). A DNA standard curve [
Fisher et al. (
Decorin [
At the end of the culture period, the disks were fixed with 4% (w/v) paraformaldehyde solution in 0.1 M phosphate buffer (pH = 7.4) for 8 hours at room temperature and washed with PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM
L. Fisher’s antidecorin, antiosteocalcin, antiosteopontin, anti-type-I collagen, and anti-type-III collagen rabbit polyclonal antisera were used as primary antibodies with a dilution equal to 1 : 1000 in PAT. The incubation with the primary antibodies was performed overnight at
At the end of the incubation, the disks were washed in PBS, counterstained with Hoechst solution (2
At the end of the culture period, in order to evaluate the amount of the extracellular matrix constituents over the internal and external hydroxyapatite surfaces, the disks were washed extensively with sterile PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Calibration curves to measure decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen were performed. Microtiter wells were coated with increasing concentrations of each purified protein, from 1 ng to 2
The wells were finally incubated with 100
The disks number was 24 in each repeated experiment (12 disks in the control culture and 12 disks in the ultrasonic culture). The experiment was repeated 4 times. Results are expressed as mean ± standard deviation. In order to compare the results between the two culture systems, one-way analysis of variance (ANOVA) with
The human SAOS-2 osteoblasts were seeded onto porous hydroxyapatite disks, and then cultured without or with an ultrasonic stimulus for 22 days. These culture methods permitted the study of the SAOS-2 cells as they modified the biomaterial through the proliferation and the coating with extracellular matrix. The cell-matrix distribution was compared between the two culture systems.
In comparison to control condition, SEM images revealed that, due to the ultrasound stimulus, the osteoblasts proliferated and built their extracellular matrix over the available internal hydroxyapatite surface (Figures
SEM image of the static culture, bar equal to 30
SEM image of the ultrasonic culture, bar equal to 30
The immunolocalization of type-I collagen and decorin with the counterstaining of the cellular nuclei showed the stimulation effects in terms of higher cell proliferation and more intense building of the extracellular matrix (Figures
Immunolocalization of type-I collagen (panels a and c, green) and cellular nuclei (panels b and d, blue) in the static culture (panels a and b) and in the ultrasonic culture (panels c and d), bars equal to 80
Immunolocalization of decorin (panels a and c, green) and cellular nuclei (panels b and d, blue) in the static culture (panels a and b) and in the ultrasonic culture (panels c and d), bars equal to 80
These observations were confirmed by the measure of the DNA content at the end of the culture period: in the static culture, the cell number per disk grew to
In order to evaluate the amount of bone extracellular matrix inside the hydroxyapatite disks, an ELISA of the extracted matrix was performed: at the end of the culture period, in comparison with the static culture, the ultrasound stimulation significantly increased the internal surface coating with decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen
Amount of extracellular matrix constituents inside hydroxyapatite.
Matrix protein total coating after 22 days of culture in fg/(cell | |||
Static culture | Ultrasonic culture | Ultrasonic /Static | |
Decorin | 5.58 ± 0.22 | 15.25 ± 0.42 | 2.73-fold |
Osteocalcin | 1.79 ± 0.33 | 5.76 ± 0.39 | 3.22-fold |
Osteopontin | 1.75 ± 0.73 | 3.04 ± 0.47 | 1.74-fold |
Type-I collagen | 3.72 ± 0.49 | 16.85 ± 0.95 | 4.53-fold |
Type-III collagen | 4.59 ± 0.13 | 11.04 ± 0.71 | 2.40-fold |
Table note:
The aim of this study was the in vitro modification of a porous hydroxyapatite with extracellular matrix and osteoblasts to make the biomaterial more biocompatible for the bone repair in vivo.
A discussion about the concept of “biocompatibility” is necessary. When a biomaterial is implanted in a biological environment, a nonphysiologic layer of adsorbed proteins mediates the interaction of the surrounding host cells with the material surface. The body interprets this protein layer as a foreign invader that must be walled off in an avascular and tough collagen sac. Therefore, the biomedical surfaces must be developed so that the host tissue can recognize them as “self”. Castner and Ratner think the “biocompatible surfaces” of the “biomaterials that heal” as the surfaces with the characters of a “clean, fresh wound” [
To enhance the coating of the biomaterial internal surface, an ultrasonic wave was applied to the seeded biomaterial [
The preceding results could be explained with a signaling model. The ultrasound stimulation raises the net
Consistent with Pavalko’s model, mechanically stimulated osteoblasts produce autocrine and paracrine prostaglandin signal for cell proliferation; the same mechanically stimulated osteoblasts produce bone extracellular matrix. Prostaglandins are released in the culture medium, whereas the proteins are deposited onto the biomaterial. Even if prostaglandins and proteins have partially common biochemical pathways [
In this study, the ultrasonic stimulus was a physical method to obtain the biomimetic modification of the material, whose internal surface was coated by osteoblasts and by a layer of bone matrix. The use of a cell line showed the potential of the ultrasound stimulation; nevertheless, appropriately tuning the parameters of the ultrasonic wave, the stimulus duration, and the culture time, a better result could be obtained with autologous bone marrow stromal cells instead of SAOS-2 osteoblasts for total immunocompatibility with the patient. In addition, after the in vivo implantation of the cultured cancellous hydroxyapatite, an ultrasound therapy could be applied with the same wave parameters [
In conclusion, we theorize that the cultured “self-surface” could be used fresh, that is, rich in autologous cells and matrix, or after sterilization with ethylene oxide, that is, rich only in autologous matrix. In future work, we intend to use our constructs, which are rich in autologous matrix, as a simple, storable, tissue-engineering product for the bone repair [
The authors thank L. W. Fisher, K. Martin, S. Setti, R. Cadossi, A. Mortara, D. Picenoni, and P. Vaghi. Hydroxyapatite disks were kindly provided by Geistlich Pharma (Wolhusen, Switzerland). The FAST ultrasound generator was a gift from Igea (Carpi, Italy). This work was supported by Fondazione Cariplo Grants (2004.1424/10.8485 and 2006.0581/10.8485) to Francesco Benazzo, by PRIN Grant (2006) from Italian Ministry of Education, University and Research to Livia Visai, and by FIRB Grant (RBIP06FH7J) from Italian Ministry of Education, University and Research to Maria Gabriella Cusella De Angelis.