ORIGINAL ARTICLE
J Appl Biomater Funct Mater 2015 ; 13 (1): 10 -16 DOI: 10.5301/jabfm.5000182
Biocompatibility of MG-63 cells on collagen, poly-L-lactic acid, hydroxyapatite scaffolds with different parameters Berivan Cecen1, Didem Kozaci2, Mithat Yuksel3, Diler Erdemli1, Alper Bagriyanik4, Hasan Havitcioglu1,5 Department of Biomechanics, The Institute of Health Science, Dokuz Eylul University, Izmir - Turkey Department of Medical Biochemistry, Medical Faculty, Adnan Menderes University, Aydin - Turkey 3 Department of Chemistry Engineering, Engineering Faculty, Ege University, Izmir - Turkey 4 Department of Histology, Medicine Faculty, Dokuz Eylul University, Izmir - Turkey 5 Department of Orthopaedics and Traumatology, Medicine Faculty, Dokuz Eylul University, Izmir - Turkey 1 2
ABSTRACT Purpose: In this study, osteoblast-like MG-63 cells were cultured on 3 different scaffold types composed of (a) collagen + poly-L-lactic acid (PLLA), (b) collagen + hydroxyapatite (HA; 30ºC) or (c) collagen + hydroxyapatite (HA; 37ºC) and produced with different porosities. Methods: Biomechanical properties of the scaffolds were characterized by tensile strength measurements. Properties of the cell-seeded scaffolds were evaluated with scanning electron microscopy (SEM). Cell adhesion and proliferation capacities were evaluated. Alkaline phosphatase (ALP) levels in media were measured. Transmission electron microscopy (TEM) and histological analyses were used to assess morphological characteristics. Results: Our results showed that collagen-based PLLA and HA scaffolds have good cell biocompatibility. MTT test showed that the scaffolds exhibited no cytotoxicity. According to the force and displacement data, collagen + HA at 37ºC showed the highest mechanical strength and displacement. Conclusion: The results suggest that collagen-based PLLA and HA scaffolds might improve osteoblastic growth in vitro and have biomaterial integration potential in possible therapeutic approaches for future clinical studies. Key words: Collagen, Hydroxyapatite, MG-63, Poly-L-lactic acid, Scaffold Accpeted: June 24, 2013
INTRODUCTION The cell interaction on biomaterials is a highly dynamic process, and it depends on various parameters influencing the cell responses. In adherence-dependent cells, not only the size and shape of cell spreading area but the size, shape and distribution of focal adhesion plaques are decisive for further migratory, proliferative and differentiation behavior of the cells (1, 2). Scaffolds for bone regeneration have been composed of various materials such as glass beads, ceramics, collagens, tricalcium phosphate, hydroxyapatite and a variety of synthetic polymers (3). Panzavolta et al investigated the influence of electrospun polymer fibers on the properties of an α-tricalcium phosphate/gelatin biomimetic cement. According to their results, the crystallinity of the apatitic phase was reduced in compact microstructure (4). 10
Poly-L-lactic acid (PLLA) is one of the well-defined substrates which is in use for osteoblast adhesion. Generally, most of the PLLA substrates for osteoblast cultures are nontoxic, biodegradable materials, widely used as scaffold material in tissue engineering. Type I collagen, the major organic component of bone extracellular matrix, on the other hand, plays an active role in osteoblast phenotype expression and encourages osteogenic differentiation and mineralization in bone (5, 6). Hydroxyapatite (HA) is the main inorganic component of bone and is recognized as a good bone-filling material, because of its osteoconductivity. However, due to their differences in mechanical and biochemical properties from those of natural bone, HA-based materials, when transplanted, remain in the bone for the patients’ lifetime and induce fractures at or around the site of transplan tation. Because of its biocompatibility, bioactivity, rapid
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Cecen et al
attachment of osteoblasts and not having any requirement for follow-up surgery, HA is considered a superior choice for drug carrier system and bioactive implant design compared with polymers (7-9). To achieve effective bone remodeling and fracture healing, scaffolds for bone tissue engineering are required to replicate the functionality of the natural bone extracellular matrix, to promote cell attachment and tissue growth, interact with host tissue, and thus have an effective biodegradable, microporous and nanostructure of the bone (10-14). The objective of this study was to investigate proliferation capacities and osteogenic responses of MG-63 human osteoblast-like cells on 3D collagen-based PLLA and HA scaffolds with different porosities.
TABLE I - COMPARATIVE ION CONCENTRATIONS OF Tris-ABF AND Lac-ABF USED IN THIS STUDY Ion
Kokubo- Lac-ABFx1 Lac-ABFx2.5 ABF (mM) (mM) (mM) 142.0
142.0
142.0
142.0
142.0
147.8
103.0
103.0
103.0
103.0
HCO3-
4.2
27.0
27.0
27.0
27.0
K
5.0
5.0
5.0
5.0
5.0
+
1.5
1.5
1.5
1.5
3.0
Ca2+
2.5
2.5
6.25
2.5
5.0
HPO42-
1.0
1.0
2.5
1.0
2.0
SO
0.5
0.5
0.5
0.5
1.0
Mg
2+
24
-
22
26.5
Lactic acid (1 M)
-
36 mL
40 mL
Scaffold preparation and characterization
Tris
50
-
-
PLLA solution (4%) was prepared using PLLA (Purasorb Poly-L-Lactide, molecular weight 800.000 Da. Purac Biochem, Gorinchem, Holland), dissolved in chloroform. Gelfix, a 3D scaffold collagen material (Gelfix® collagen; Euroresearch s.r.l., Italy) was soaked in preprepared PLLA solution, and collagen fibrils were allowed to moisten. Excess solution was then removed, and collagen with PLLA was soaked in absolute alcohol (ethanol). Alcohol was changed regularly to allow PLLA to deposit on collagen fibrils, which was then dried at 50ºC in a vacuum. Preparation of artificial body fluid (ABF) Artificial body fluid was prepared as in Tab. I. The list of chemicals used in preparation of ABF was as follows: (i) CaCl2.2H2O, (ii) MgCl2.6H2O, (iii) KCl, (iv) NaCl, (v) Na2HPO4.2H2O, (vi) Na2SO4, (vii) NaHCO3, (viii) lactate and (ix) lactic acid. In ABF × 2.5; Ca+2 and PO4-3 concentrations were 2.5 times higher compared with the concentrations in Tab. I. To prepare ABF, the chemicals numbered 3-8 were added in a container with 900 mL of distilled H2O, and then the pH was adjusted to 8.05 while the solution was stirred on a heated magnetic mixer. Seven milliliters of lactic acid (1 M) was quickly added, the temperature was increased to 37ºC quickly and pH was adjusted to 6.95. Salts with numbers 1 and 2 were dissolved in 10 mL of distilled H2O and added to the first solution
(mEq/L)
Cl-
Lactate
Collagen and PLLA
(mM)
Na
+
MATERIALS AND METHODS
In this study, 3 different types of scaffolds were assessed: (a) collagen + PLLA, (b) collagen + HA at 30ºC and (c) collagen + HA at 37ºC.
Blood plasma
(+)ion: 155 mEq/l (-)ion: 133 mEq/l + 22 mEq/l organic anions -
-
ABF = Artificial body fluid.
quickly, and pH was adjusted to 7.37. The final volume was completed to 1 L. In the literature, an incubation period of 1-2 months has been suggested for the preparation of collagen + HA using artificial body fluid (ABF). When ABF is used as 1 × blood plasma product, this duration is decreased to 3 weeks. In our study, by using ABF × 2.5 and by increasing the HPO and Ca concentrations 2.5 times, we achieved 6.5 mM and 2.5 mM final concentrations for Ca and HPO, respectively, which was enough to have HA deposition in 24 hours. This is one of the novel findings of our study (Tab. I). Preparation of collagen and HA at 30ºC, using ABF Gelfix collagen scaffold (5×5×1 cm) was soaked in the prepared ABF solution at 30ºC in a closed container. After 24 hours, the pH was measured as 7.04. No change was observed in the size of the collagen scaffold after 24 hours at this temperature. The scaffold was then washed with distilled water and dried at room temperature. Preparation of collagen and HA at 37ºC, using ABF Gelfix collagen scaffold (5×5×1 cm) was soaked in ABF solution and kept at 37ºC for 24 hours. This procedure caused a decrease in size of the scaffold to 2.5×2.5×0.5 cm. The scaffold was then washed with distilled H2O and dried at 40ºC. All scaffolds were sterilized at 90ºC with ethylene oxide prior to use in tissue cultures.
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
11
MG-63 cells on bio-absorbable scaffolds
Cell culture MG-63 Osteosarcoma Human Cell Line, obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) (ATCC-CRL 1427 Lot number: 57840088) was used. The cells were seeded in a culture flask (25 cm2) with MEM-Eagle (MEM, 03-025-1A; Biological Industries) medium which contained 10% fetal calf serum. L-glutamine (200 mM, G7513; Sigma) and Pen-Strep-Ampho (03-0331B; Biological Industries) and was cultured at 37°C in a humidified 5% CO2 atmosphere. The medium was changed on alternate days. After that, the cells (5×105 cells/µL) were seeded on scaffolds (size: 8×6×3 mm; L×W×H; n = 3).
sured according to manufacturer’s recommendations for 7 days in culture medium using a commercially available kit (Enzyline PAL Optimise; Biomerieux, France) by spectrophotometer (Cary 50 UV-Vis). MTT assay Cellular growth of MG-63 cells on collagen-based PLLA and HA scaffolds was verified by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using a commercially available kit according to manufacturer’s recommendations in 4-day cultures. Histological analysis
Scanning electron microscopy Scaffolds were analyzed by scanning electron microscopy (SEM) prior to cell seeding to evaluate their porosity sizes. When a confluent monolayer was achieved, cells were trypsinized and centrifuged, and supernatant was discarded. After 21-day cultures on scaffolds, the scaffolds were washed with phosphate-buffered saline (PBS). Subsequently, the cells were fixed with 5% glutaraldehyde (pH 7.2), 7% sucrose and 2% osmium tetroxide in sodium cacodylate buffer (0.1 M). The specimens were dehydrated using graded ethanol changes and gold splattered in vacuum (Polaran SC7620) at 10 kV, and examined using SEM (JEOL JSM–6060). Transmission electron microscopy The scaffolds were fixed on day 21 with Karnovsky solution (2.5% buffered glutaraldehyde + 2% paraformaldehyde in 0.1 M sodium phosphate buffer [Sorensen buffer]). The following day, scaffolds were postfixed with 0.1-M sodium cacodylate buffer (pH 7.4). After that, the scaffolds were dehydrated in acetone serial. The samples were then embedded in Epon solution. Sections were cut with an ultramicrotome, set to 50- to 100-nm section thickness and examined with an EFTEM (Carl Zeiss Libra 120, Germany) at 60 kV.
The cultures were cultivated for 21 days on scaffolds for histological observations (n = 3). Scaffolds were stained with hematoxylin and eosin (H&E), and semi-thin sections of 1.5 µm in thickness were cut and stained with 1% toluidine blue for transmission electron microscopy (TEM) fixation. Statistical analysis The data of biomechanical tests of force data and MTT assay were analyzed with a nonparametric Kruskal-Wallis test. The statistical analysis was carried out using “SPSS”, version 15 (SPSS Inc., Chicago, IL, USA). A p value of
J Appl Biomater Funct Mater 2015 ; 13 (1): 10 -16 DOI: 10.5301/jabfm.5000182
Biocompatibility of MG-63 cells on collagen, poly-L-lactic acid, hydroxyapatite scaffolds with different parameters Berivan Cecen1, Didem Kozaci2, Mithat Yuksel3, Diler Erdemli1, Alper Bagriyanik4, Hasan Havitcioglu1,5 Department of Biomechanics, The Institute of Health Science, Dokuz Eylul University, Izmir - Turkey Department of Medical Biochemistry, Medical Faculty, Adnan Menderes University, Aydin - Turkey 3 Department of Chemistry Engineering, Engineering Faculty, Ege University, Izmir - Turkey 4 Department of Histology, Medicine Faculty, Dokuz Eylul University, Izmir - Turkey 5 Department of Orthopaedics and Traumatology, Medicine Faculty, Dokuz Eylul University, Izmir - Turkey 1 2
ABSTRACT Purpose: In this study, osteoblast-like MG-63 cells were cultured on 3 different scaffold types composed of (a) collagen + poly-L-lactic acid (PLLA), (b) collagen + hydroxyapatite (HA; 30ºC) or (c) collagen + hydroxyapatite (HA; 37ºC) and produced with different porosities. Methods: Biomechanical properties of the scaffolds were characterized by tensile strength measurements. Properties of the cell-seeded scaffolds were evaluated with scanning electron microscopy (SEM). Cell adhesion and proliferation capacities were evaluated. Alkaline phosphatase (ALP) levels in media were measured. Transmission electron microscopy (TEM) and histological analyses were used to assess morphological characteristics. Results: Our results showed that collagen-based PLLA and HA scaffolds have good cell biocompatibility. MTT test showed that the scaffolds exhibited no cytotoxicity. According to the force and displacement data, collagen + HA at 37ºC showed the highest mechanical strength and displacement. Conclusion: The results suggest that collagen-based PLLA and HA scaffolds might improve osteoblastic growth in vitro and have biomaterial integration potential in possible therapeutic approaches for future clinical studies. Key words: Collagen, Hydroxyapatite, MG-63, Poly-L-lactic acid, Scaffold Accpeted: June 24, 2013
INTRODUCTION The cell interaction on biomaterials is a highly dynamic process, and it depends on various parameters influencing the cell responses. In adherence-dependent cells, not only the size and shape of cell spreading area but the size, shape and distribution of focal adhesion plaques are decisive for further migratory, proliferative and differentiation behavior of the cells (1, 2). Scaffolds for bone regeneration have been composed of various materials such as glass beads, ceramics, collagens, tricalcium phosphate, hydroxyapatite and a variety of synthetic polymers (3). Panzavolta et al investigated the influence of electrospun polymer fibers on the properties of an α-tricalcium phosphate/gelatin biomimetic cement. According to their results, the crystallinity of the apatitic phase was reduced in compact microstructure (4). 10
Poly-L-lactic acid (PLLA) is one of the well-defined substrates which is in use for osteoblast adhesion. Generally, most of the PLLA substrates for osteoblast cultures are nontoxic, biodegradable materials, widely used as scaffold material in tissue engineering. Type I collagen, the major organic component of bone extracellular matrix, on the other hand, plays an active role in osteoblast phenotype expression and encourages osteogenic differentiation and mineralization in bone (5, 6). Hydroxyapatite (HA) is the main inorganic component of bone and is recognized as a good bone-filling material, because of its osteoconductivity. However, due to their differences in mechanical and biochemical properties from those of natural bone, HA-based materials, when transplanted, remain in the bone for the patients’ lifetime and induce fractures at or around the site of transplan tation. Because of its biocompatibility, bioactivity, rapid
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
Cecen et al
attachment of osteoblasts and not having any requirement for follow-up surgery, HA is considered a superior choice for drug carrier system and bioactive implant design compared with polymers (7-9). To achieve effective bone remodeling and fracture healing, scaffolds for bone tissue engineering are required to replicate the functionality of the natural bone extracellular matrix, to promote cell attachment and tissue growth, interact with host tissue, and thus have an effective biodegradable, microporous and nanostructure of the bone (10-14). The objective of this study was to investigate proliferation capacities and osteogenic responses of MG-63 human osteoblast-like cells on 3D collagen-based PLLA and HA scaffolds with different porosities.
TABLE I - COMPARATIVE ION CONCENTRATIONS OF Tris-ABF AND Lac-ABF USED IN THIS STUDY Ion
Kokubo- Lac-ABFx1 Lac-ABFx2.5 ABF (mM) (mM) (mM) 142.0
142.0
142.0
142.0
142.0
147.8
103.0
103.0
103.0
103.0
HCO3-
4.2
27.0
27.0
27.0
27.0
K
5.0
5.0
5.0
5.0
5.0
+
1.5
1.5
1.5
1.5
3.0
Ca2+
2.5
2.5
6.25
2.5
5.0
HPO42-
1.0
1.0
2.5
1.0
2.0
SO
0.5
0.5
0.5
0.5
1.0
Mg
2+
24
-
22
26.5
Lactic acid (1 M)
-
36 mL
40 mL
Scaffold preparation and characterization
Tris
50
-
-
PLLA solution (4%) was prepared using PLLA (Purasorb Poly-L-Lactide, molecular weight 800.000 Da. Purac Biochem, Gorinchem, Holland), dissolved in chloroform. Gelfix, a 3D scaffold collagen material (Gelfix® collagen; Euroresearch s.r.l., Italy) was soaked in preprepared PLLA solution, and collagen fibrils were allowed to moisten. Excess solution was then removed, and collagen with PLLA was soaked in absolute alcohol (ethanol). Alcohol was changed regularly to allow PLLA to deposit on collagen fibrils, which was then dried at 50ºC in a vacuum. Preparation of artificial body fluid (ABF) Artificial body fluid was prepared as in Tab. I. The list of chemicals used in preparation of ABF was as follows: (i) CaCl2.2H2O, (ii) MgCl2.6H2O, (iii) KCl, (iv) NaCl, (v) Na2HPO4.2H2O, (vi) Na2SO4, (vii) NaHCO3, (viii) lactate and (ix) lactic acid. In ABF × 2.5; Ca+2 and PO4-3 concentrations were 2.5 times higher compared with the concentrations in Tab. I. To prepare ABF, the chemicals numbered 3-8 were added in a container with 900 mL of distilled H2O, and then the pH was adjusted to 8.05 while the solution was stirred on a heated magnetic mixer. Seven milliliters of lactic acid (1 M) was quickly added, the temperature was increased to 37ºC quickly and pH was adjusted to 6.95. Salts with numbers 1 and 2 were dissolved in 10 mL of distilled H2O and added to the first solution
(mEq/L)
Cl-
Lactate
Collagen and PLLA
(mM)
Na
+
MATERIALS AND METHODS
In this study, 3 different types of scaffolds were assessed: (a) collagen + PLLA, (b) collagen + HA at 30ºC and (c) collagen + HA at 37ºC.
Blood plasma
(+)ion: 155 mEq/l (-)ion: 133 mEq/l + 22 mEq/l organic anions -
-
ABF = Artificial body fluid.
quickly, and pH was adjusted to 7.37. The final volume was completed to 1 L. In the literature, an incubation period of 1-2 months has been suggested for the preparation of collagen + HA using artificial body fluid (ABF). When ABF is used as 1 × blood plasma product, this duration is decreased to 3 weeks. In our study, by using ABF × 2.5 and by increasing the HPO and Ca concentrations 2.5 times, we achieved 6.5 mM and 2.5 mM final concentrations for Ca and HPO, respectively, which was enough to have HA deposition in 24 hours. This is one of the novel findings of our study (Tab. I). Preparation of collagen and HA at 30ºC, using ABF Gelfix collagen scaffold (5×5×1 cm) was soaked in the prepared ABF solution at 30ºC in a closed container. After 24 hours, the pH was measured as 7.04. No change was observed in the size of the collagen scaffold after 24 hours at this temperature. The scaffold was then washed with distilled water and dried at room temperature. Preparation of collagen and HA at 37ºC, using ABF Gelfix collagen scaffold (5×5×1 cm) was soaked in ABF solution and kept at 37ºC for 24 hours. This procedure caused a decrease in size of the scaffold to 2.5×2.5×0.5 cm. The scaffold was then washed with distilled H2O and dried at 40ºC. All scaffolds were sterilized at 90ºC with ethylene oxide prior to use in tissue cultures.
© 2014 Società Italiana Biomateriali - eISSN 2280-8000
11
MG-63 cells on bio-absorbable scaffolds
Cell culture MG-63 Osteosarcoma Human Cell Line, obtained from American Type Culture Collection (ATCC; Manassas, VA, USA) (ATCC-CRL 1427 Lot number: 57840088) was used. The cells were seeded in a culture flask (25 cm2) with MEM-Eagle (MEM, 03-025-1A; Biological Industries) medium which contained 10% fetal calf serum. L-glutamine (200 mM, G7513; Sigma) and Pen-Strep-Ampho (03-0331B; Biological Industries) and was cultured at 37°C in a humidified 5% CO2 atmosphere. The medium was changed on alternate days. After that, the cells (5×105 cells/µL) were seeded on scaffolds (size: 8×6×3 mm; L×W×H; n = 3).
sured according to manufacturer’s recommendations for 7 days in culture medium using a commercially available kit (Enzyline PAL Optimise; Biomerieux, France) by spectrophotometer (Cary 50 UV-Vis). MTT assay Cellular growth of MG-63 cells on collagen-based PLLA and HA scaffolds was verified by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using a commercially available kit according to manufacturer’s recommendations in 4-day cultures. Histological analysis
Scanning electron microscopy Scaffolds were analyzed by scanning electron microscopy (SEM) prior to cell seeding to evaluate their porosity sizes. When a confluent monolayer was achieved, cells were trypsinized and centrifuged, and supernatant was discarded. After 21-day cultures on scaffolds, the scaffolds were washed with phosphate-buffered saline (PBS). Subsequently, the cells were fixed with 5% glutaraldehyde (pH 7.2), 7% sucrose and 2% osmium tetroxide in sodium cacodylate buffer (0.1 M). The specimens were dehydrated using graded ethanol changes and gold splattered in vacuum (Polaran SC7620) at 10 kV, and examined using SEM (JEOL JSM–6060). Transmission electron microscopy The scaffolds were fixed on day 21 with Karnovsky solution (2.5% buffered glutaraldehyde + 2% paraformaldehyde in 0.1 M sodium phosphate buffer [Sorensen buffer]). The following day, scaffolds were postfixed with 0.1-M sodium cacodylate buffer (pH 7.4). After that, the scaffolds were dehydrated in acetone serial. The samples were then embedded in Epon solution. Sections were cut with an ultramicrotome, set to 50- to 100-nm section thickness and examined with an EFTEM (Carl Zeiss Libra 120, Germany) at 60 kV.
The cultures were cultivated for 21 days on scaffolds for histological observations (n = 3). Scaffolds were stained with hematoxylin and eosin (H&E), and semi-thin sections of 1.5 µm in thickness were cut and stained with 1% toluidine blue for transmission electron microscopy (TEM) fixation. Statistical analysis The data of biomechanical tests of force data and MTT assay were analyzed with a nonparametric Kruskal-Wallis test. The statistical analysis was carried out using “SPSS”, version 15 (SPSS Inc., Chicago, IL, USA). A p value of
