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Artificial Cells, Nanomedicine, and Biotechnology
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ISSN: 2169-1401 (Print) 2169-141X (Online) Journal homepage: https://www.tandfonline.com/loi/ianb20
Sequential VEGF and BMP-2 releasing PLA-PEG-PLA
scaffolds for bone tissue engineering: I. Design and
in vitro
tests
Sinan Eğri & Numan Eczacıoğlu
To cite this article: Sinan Eğri & Numan Eczacıoğlu (2017) Sequential VEGF and BMP-2 releasing PLA-PEG-PLA scaffolds for bone tissue engineering: I. Design and in�vitro tests, Artificial Cells, Nanomedicine, and Biotechnology, 45:2, 321-329, DOI: 10.3109/21691401.2016.1147454
To link to this article: https://doi.org/10.3109/21691401.2016.1147454
Published online: 25 Feb 2016.
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http://dx.doi.org/10.3109/21691401.2016.1147454
Sequential VEGF and BMP-2 releasing PLA-PEG-PLA scaffolds for bone tissue
engineering: I. Design and in vitro tests
Sinan Eg˘riaand Numan Eczacıog˘lub
a
Department of Bioengineering, Faculty of Engineering and Natural Sciences, Gaziosmanpasa University, Tokat, Turkey;bDepartment of Bioengineering, Faculty of Engineering, Karamanog˘lu Mehmetbey University, Karaman, Turkey
ABSTRACT
Biodegradable PLA-PEG-PLA block copolymers were synthesized with desired backbone structures and molecular weights using PEG20000. Rectangular scaffolds were prepared by freeze drying with or without using NaCl particles. Bone morphogenetic protein (BMP)-2 was loaded to the matrix after the scaffold formation for sustained release while vascular endothelial growth factor (VEGF) was loaded within the pores with gelatin solution. VEGF release was quite fast and almost 60% of it was released in 2 d. However, sequential – sustained released was observed for BMP-2 in the following few months. Corporation of VEGF/BMP-2 couple into the scaffolds increased the cell adhesion and proliferation. Neither significant cytotoxicity nor apoptosis/necrosis were observed.
ARTICLE HISTORY Received 25 January 2016 Accepted 25 January 2016 Published online 24 February 2016 KEYWORDS Biodegradable/biocompat-ible; BMP-2 and VEGF con-trolled release; bone tissue engineering; freeze drying; in vitro tests; PLA-PEG-PLA block copolymers; salt leaching; scaffolds
Introduction
Various type of biodegradable scaffolds has been designed and utilized in bone tissue engineering together with healthy cells – recently mainly stem cells (Bo¨lgen et al.2014, Chen et al.2002, Do Kim et al.2004, Wang et al.2015, Zhang et al.2012). Poly-L-lactide (PLA) – as a synthetic bio-based polymer has attracted a huge interest and many different forms – also copolymers have already being used in the production biodegradable polymers for diverse applications including tissue engineering scaffold (Aydin et al.2011, Lee et al.2006, Temenoff and Mikos 2000, Wan et al.2003, Xiao et al.2010) which was also used in this study as the main matrix polymer for the preparation of cryogel-scaffolds. Polyethylene glycols (PEGs) are one of the few water soluble polymers and have been already used in many years in or on the surfaces to improve biocompatibilities of diverse biomaterials (Xiao et al.2010). In this study we have attempted to prepare PLA-PEG-PLA block copolymer not only to increase the biocompatibility but also to have more hydrophilic materials which will allow cryogelation and increase the degradation rate.
Biodegradable polymers are processed into 3D structures – having highly porous structure with interconnected pore morphologies by using different techniques (Hou et al.2003, Lannutti et al.2007, Liu and Ma2004). Cryogelation and freeze drying with particle leaching are among them – porous matrices are mainly formed in frozen conditions – which are very simple and inexpensive techniques and create excellent pore structures allowing 3D cell growth (Bo¨lgen et al. 2007,
Hou et al. 2003, Lozinsky et al. 2002, Reignier and Huneault
2006). They do adsorb quite high amount of water – surrounding aqueous phase after implantation – very fast which is an important point contributing in 3D cell growth in vivo (Bo¨lgen et al.2014).
Different bioactive agents – mostly growth factors are incorporated/used together with hard tissue repair materials including tissue engineering scaffolds for triggering auto-inductive capacity of bone. Bone morphogenetic proteins (BMPs) are among the most widely studied – even with commercial products to apply clinically in most bone regen-eration strategies. It has been well documented that they do have high osteoinductive potential and trigger bone formation processes, including the migration of mesenchymal stem cells and their differentiation into osteoblasts (Kang et al. 2004, Li et al.2003, Sampath et al.1992, Wang et al.1990, Wozney2002, Wozney et al.1988). Vascular endothelial growth factor (VEGF) is another important bioactive agent which is one of the most essential elements in angiogenesis (Eckardt et al. 2005, Gerstenfeld et al. 2003, Kaigler et al.2006, Ozturk et al.2013, Peng et al. 2005, Street et al. 2002). Many related studies suggested that BMP-2 and VEGF works together by coupling osteogenesis and angiogenesis for proper bone regeneration (Deckers et al. 2002, Huang et al. 2005, Kaigler et al. 2005, Kozawa et al.2001, Patel et al.2008, Peng et al.2002). It should be noted that – in the natural bone regeneration process – VEGF is expressed in the early phases of bone formation while BMP expression comes later (Cho et al. 2002, Groeneveld and
CONTACTSinan Eg˘ri sinan.egri@gop.edu.tr Department of Bioengineering, Faculty of Engineering and Natural Sciences, Gaziosmanpasa University, 60150 Tokat, Turkey
Burger 2000, Komatsu and Hadjiargyrou 2004, Niikura et al.
2006, Pufe et al.2002, Uchida et al.2003). Therefore, in many related studies this natural process is mimicked to use this couple together in a sequential order for bone repair (Huang et al. 2005, Kanczler et al. 2010, Kempen et al. 2008a, 2008b,
2009) – which was also the main rational that we have designed in this study.
From the previous and ongoing studies in bone tissue engineering applications using bioactive agents it can be concluded that – not only the type of cells (primary cells, stem cells, and so forth) and scaffold properties (the material, its biocompatibility, degradation rate, pore morphology, and so forth) but also growth factor type, loading/release characteristics play crucial role in proper bone formation. In this study we have focused on the scaffolds and their BMP-2 and VEGF-loaded forms and conducted the following experi-ments before we carry this hybrid system to further animal model studies. Following our previous experience, we synthesized PLA-PEG-PLA triblock copolymers, process them into highly porous structures by freeze drying/particle leaching, load BMP-2 and VEGF to reach the desired sequential release kinetics, an investigated their interaction with model cell lines in vitro.
Materials and methods
L-lactide dimer (L-LA) (Purac, Amsterdam, The Netherlands) was recrystallized from dry toluene before further use. PEG (Fluka, Munich, Germany) with 20000 Da molecular weight was dried under vacuum before copolymerization reactions. 1,4-dioxane (Merck, Germany), tin(II) 2-ethylhexanoate (Stannous octoate, Sn(Oct)2) (Aldrich, Germany), recombinant human BMP-2
(Sigma, Germany), recombinant human VEGF 165 (Sigma, Germany), gelatin from bovine skin (Sigma, Germany) were reagent grade and they were used as received. MC3T3-E1 mouse osteoblast cell line and Roswell Park Memorial Institute medium (RPMI-1640) were purchased from ATCC, cell culture flasks and all other plastic materials from Corning, USA, Dubelco modified medium (DMEM-F12), l-glutamine, fetal bovine serum (FBS), penicillin-streptomycin antibiotic and Trypsin-EDTA, Hoechst 33342, propidium iodide (PI), RNase from Biological Industries Ltd. Kibbutz Beit Haemek, Israel, and WST-1 (2–(4-iodophenyl)-3(4-nitrophenyl)-5–(2,4-disulfophe-nyl)-2 h-tetrazolium), E-plate 96 were purchased from Roche, Germany.
PLA-PEG-PLA copolymers
Copolymers were synthesized in bulk by ring-opening poly-merization of L-LA dimer in the presence of PEG using stannous octoate as the catalyst with a selected ratio of 1000:1 (w/w) – selected considering our previous experience (Eg˘ri2009) and in the preliminary studies (Eczac{og˘lu 2015). Polymerization reactions were conducted in a sealed glass tube which was saturated with pure nitrogen, at 120C for 24 h. After
polymerization the bulk copolymer was dissolved in chloroform and precipitated into cold methanol. The precipitate was then washed with distilled water, filtered and dried under vacuum
for 48 h. The average molecular weights and the molecular weight distributions were obtained with a gel permeation chromatography system (GPC) (Malvern Viscotek, UK) in which the column temperature was 35C, tetrahydrofuran was used
as the mobile phase with a flow rate of 1.0 mL/min. Chemical structure analysis were conducted with Fourier Transform Infrared Spectroscopy (FTIR) (FT-IR-430 JASCO, Japan) and1 H-NMR (Bruker UltraShield 400, Germany) working at 400 Mhz and 13C using CDCl3as solvent.
Scaffolds
The scaffolds made of the PLA-PEG-PLA triblock copolymers synthesized in the previous step were produced by freeze drying only and also including salt particles loading and leaching protocol. Briefly, the copolymer was dissolved in 1,4-dioxane with concentrations of 3.0%, 5.0%, and 8.0% (w/v). The NaCl particles with 100–250 mm size range – selected in the preliminary studies (Eczac{og˘lu 2015) were mixed with the solution with a ratio of 1 g/5 mL and the solution was molded below the freezing temperature of 1,4-dioxane in order to obtain frozen matrix. The frozen matrix was stored at18C
overnight and then treated repeatedly with fresh ethanol at temperatures ranging between18 and +4C for thawing and
solvent extraction processes followed by washing with distilled water until the complete removal of NaCl particles in the matrix. The matrix was dried under vacuum. Pieces with 6 53 mm dimensions were cut out using a spatial apparatus. Morphology and the pore structure of the scaffolds were determined with Environmental Scanning Electron Microscope (FEI QUANTA, Hillsboro, OR) at 5.0 kV.
Growth factor loading and release
A release system was designed in order to provide sequential release of the growth factors – fast release of VEGF in about 1 week and slower release of BMP-2 almost constant from the scaffold. The amounts of growth factors (both BMP-2 and VEGF) in each scaffold were 375 ng considering for slow release BMP-2 was loaded to the matrices with the copolymer solution in dimethyl sulfoxide in which BMP-2 was dispersed. We assumed that the scaffold after all these processes – ready to use – were carrying about the same amount of BMP-2 (375 g). These scaffolds were dried at room temperature under nitrogen atmosphere before VEGF loading. VEGF was then loaded to the scaffolds with a very simple approach. VEGF was mixed with 1 mL 1% gelatin solution in water/ethanol (50:50) which was then dropped slowly with a micropipette onto the scaffolds to assure that all the liquid was sucked properly, and then dried under vacuum. It was very fast and easy due to pore capillary effect.
In vitro release kinetics were studied as follows: The growth factor loaded scaffolds were placed in bottles tightly closed containing 5 mL of phosphate-buffered solution (PBS) at 37C and the medium was replaced with same amount of fresh PBS every day for 14 d, and every 2 weeks after the first 14 d for total 3 months using ELISA kits (Sigma, Germany) by following the instructions of the producer.
Interaction of scaffolds with cells Cells on the scaffolds
In order to observe/compare the cell behavior of the MC3T3-E1 mouse osteoblast cells were interacted with the scaffolds – four different types: (i) the scaffolds without any growth factor loading (as the control); (ii) the VEGF-loaded; (iii) the BMP-2-loaded; and (iv) the scaffold loaded with both VEGF and BMP-2. Thin slices of scaffolds (6 51 mm) – three from each type – sterilized by ethanol evaporation were placed in 48 well plates. The same amount of cells (2 105) were added onto the
scaffolds in each well and then 5.0 mL culture medium (DMEM-F12 containing 1.0% antibiotics/antimicotics, 8.0% FBS, 8.0% ascorbic acid, and 1.0% L-glutamine) was added to the wells. On the 7th and 14th d, sections with 3 mm thickness were sliced in the cryostat from the scaffolds that were previously fixed with 4% paraformaldehyde (4%). Cells attached on the scaffolds were displayed morphologically by a microscope with fluores-cent attachment, after treating with double staining followed by additional 10 min of incubation. Scaffolds incubated with MC3T3-E1 mouse osteoblast cells were fixed and freeze-dried with a lyophilizer (Christ Alpha 1–2 LD plus, Germany) and coated with gold-palladium (Au-Pd) for scanning electron microscopy (SEM).
Cytotoxicity
The MC3T3-E1 mouse osteoblast cell line were placed in 24 well plates (10 105 cells in each well) and incubated for
24 h at 37C in 5% CO2 in the culture medium The scaffolds
(the plain and VEGF/BMP-2-loaded) were grinded and the resulting powder was dispersed in the same culture medium homogenously to reach 1 mg/mL concentration and 0.5 mL of the final dispersions were delivered to wells containing 1 d culture of the MC3T3-E1 cells in triplicate. The plates were incubated for another 24 h at 37C in 5% CO2. WST-1
solution was added on each well and incubated for additional 4 h. After incubation yellow colored solution over the cells was taken to new well plate and read out at
420 nm with the ELISA plate readder (Biotek, Gene 5 Power Wave XS2, USA)
Apoptosis and necrosis
A double staining protocol was applied to study the possible effects the scaffolds produced on the extent of apoptosis and necrosis of the MC3T3-E1 cells. Hoechst 33342 (emits blue/cyan fluorescence) and PI (emits red fluorescence) were used. Note that Hoechst stains apoptotic cells, PI stains necrotic cells and RNase A avoids staining of RNA. 400 mL of suspension of scaffold in the medium were added on the cells seeded on 24 well plates and incubated for 24 h at 37C in 5% CO
2. Then
70 mL of double staining solution (100 mL of RNase A, 100 of PI, and 500 mL of Hoechst in 10 mL of PBS) was added and incubation was continued about 10 min in dark. Samples were observed/evaluated using a fluorescence microscope (Leica, Germany).
Results and discussions
Synthesis and characterization of PLA-PEG-PLA copolymers
PLA-PEG-PLA triblock copolymers were synthesized by ring-opening polymerization of the L-LA dimer in bulk by using stannous octoate as the catalyst in which terminal hydroxyl (–OH) groups of PEG 20000 at both ends acts as a macro-initiator and forms the central block of the triblock copolymer (Srivastava and Albertsson2006). In the initial studies we have used different PEGs with different molecular weights, change the recipe (relative amounts of L-lactide and the catalyst) and conducted polymerization conditions and prepared scaffolds from the copolymers produced (Eczac{og˘lu 2015, Eg˘ri 2009). Several scaffolds prepared and tested then we have decided to use the recipe and conditions given in the previous section.
The weight average molecular weight (Mw), number average
molecular weight (Mn), and polydispersity index (PI: Mw/Mn) of
the copolymer synthesized were obtained by GPC were 84,007, 65,564, and 1.28, respectively.1H-NMR spectrum confirmed the
Figure 3. Representative SEM micrographs of PLA-PEG-PLA scaffolds: (A) Prepared without using salt particles copolymer conc. is 50 mg/mL; (B), (C), and (D) prepared with salt (with a conc. of 1 g NaCl/0.25 g polymer) with different amounts of the copolymer, 30, 50, and 80 mg/mL, respectively.
Figure 2.FTIR spectrum of PLLA-PEG copolymer. 324 S. EG˘RI AND N. ECZAC_IOG˘ LU
basic chemical structure of the PLA-PEG-PLA triblock copoly-mers (Figure 1). Peaks at ¼ 3.6 and ¼ 5.2 correspond to the protons of CH of the PLA block and CH2 of the PEG block,
respectively. Relatively insignificant chemical shift between 1.3 and 1.2 ppm corresponding to methyl protons of hydroxylated lactyl end units and between 4.3 and 4.1 ppm corresponding to -methylene protons of these units indicate that ratio of repeating lactyl units to hyrdoxylated lactyl units at both ends is high enough to hinder significance of these chemical shifts (Rashkov et al.1996). FTIR spectrum (Figure 2) confirmed the successful copolymerization. Peak at 1758 cm1corresponds to stretching of C¼ O stretching vibration of PLA, peak at 2881 cm1 corresponds to C–H stretching of PEG, and the peak around 1095 cm1 can be attributed to stretching vibration of C–O–C of the PEG block (Angelopoulou et al.
2015, Zhang et al.2009).
Scaffold production
Freeze drying/cryogelation is a technique in which highly porous matrices can be produced at subzero temperatures quite easily by rather straight forward protocols. It is possible to produce these materials from aqueous or organic solutions of polymeric precursors and/or polymers (Mattiasson et al.2005). They have various applications in literature as tissue engineer-ing scaffolds (Bhat and Kumar2012). In this study we aimed to produce PLA-PEG-PLA copolymer scaffolds by combining freeze drying technique with particulate leaching method to increase the porosity and also interconnectivity between pores that are usually limitations in solvent casting and particulate leaching (Bhat and Kumar2012).
Three different scaffolds were produced by using three different concentrations of copolymer solutions (3.0%, 5.0%,
and 8.0% (w/v) in dioxane) while keeping the copolymer salt mass ratio the same at 0.2 g/mL. SEM micrographs (Figure 3) of these scaffolds are given at two magnifications. Scaffolds prepared without using NaCl particles resulted with pores often having diameters between 10 and 30 mm (Figure 3A). Space left by NaCl particles created the large pore along the matrix and tiny pores on the walls of the matrix were the result of the solvent crystals. These tiny pores maintained very good interconnectivity along the matrix. As the concentration of the solution increased, the walls became denser but it provided a stronger matrix. Therefore, the scaffold produced from 5% solution were selected to be used in the further studies because of good porosity, high interconnectivity, and good mechanical stability.
Growth factor release
VEGF plays a very important role in neovascularization stage during bone healing (Harwood et al.2010). It has already been known that BMPs are important factors of bone healing especially have regulatory effect on osteogenic differentiation of pluripotent stem cells to osteblasts (Schliephake2002). It has been demonstrated that better both angiogenesis and osteo-genesis are observed by using VEGF and BMP-2 couple sequentially, which depends on also biomaterial/scaffold both material biodegradability and initial pore morphology (Kanczler et al. 2010). It was generally agreed that optimization of biomaterial design and site-specific pharmacological release of growth factors with desired kinetics has also crucial role in bone regeneration but remain challenging in translational bone regeneration studies. These are the main rational of this study. We have loaded both VEGF and BMP-2 in our PLA-PEG-PLA scaffolds to design a sequential releasing system. Our main Figure 4. Release rates of VEGF and BMP-2 from scaffolds prepared by freeze drying plus salt leaching using 1 g NaCl/0.25 g polymer from copolymer solutions with 5% copolymer in diaxone.
Figure 5.MC3T3-E1 mouse osteoblast cells on the scaffolds prepared by from 5% the copolymer solutions by freeze drying and salt leaching, plain and growth factor’s loaded–representative images: (A) and (B) SEM and fluorescence images of the plain scaffolds after 7 d culture, respectively; (C) and (D) SEM and fluorescence images of the plain scaffolds after 14 d; (E) and (F) SEM and fluorescence images of the scaffolds loaded with VEGF/BMP-2 after 7 d culture, respectively; and (G) and (H) SEM and fluorescence images of the scaffolds loaded with VEGF/BMP-2 after 14 d, respectively.
objective was scaffolds were loaded with the growth factors to reach a fast – a rather ‘‘burst’’ release of VEGF in the first week or so, and slow/prolonged release of BMP-2.
Figure 4shows in vitro release curves of both VEGF and BMP-2. Almost 60% of VEGF has been released within the first few days which was actually aimed in this study as mentioned above the aim of burst release. BMP-2 exhibited a slow release upto 100 d (the limits of the test period). These were the main objective of this study – and it seems that they are achieved.
Cell on scaffolds
Behavior of cells (MC3T3-E1 mouse osteoblast cell line here) onto the PLA-PEG-PLA scaffold – prepared by using 5% copolymer by using freeze drying and salt leaching – carrying no growth factor (scaffold alone) or loaded with the VEGF and BMP-2 couple – were investigated in cell culture studies as described in the previous sections for 14 d. Note that the pictures presented in Figure 5 shows the cells in the central part of the scaffold were also healthy indicating the very good mass transfer in the highly porous also – interconnected – pore structure. Both SEM and fluorescence images showed that there were a quite high number of healthy cells even at the Day 7, confirming that the rough and porous surface and excellent interconnectivity between pores through the small pores on the surfaces allow/supported the osteoblasts attachment and proliferation, which was even better in the case of the scaffold loaded with growth factors. The differences between the plan and growth factor loaded matrices – there were more cells around as expected – were even more noticeable at the Day 14 – and the effects of growth factors were more visible. It was concluded that – these results are promising most probably the effects the release of VEGF/BMP-2 couple will be much clear in the in vivo use due to possible angiogenesis/osteogenesis.
Cytotoxicity, apoptosis and necrosis
For cytotoxicity tests (that is, the ‘‘WST-1 cytotoxicity assess-ment’’) we have used MC3T3-E1 mouse osteoblast cell line grown in 24 well plate for 24 h. 0.1 mL of the dispersions containing the grinded scaffolds (the plain and VEGF/BMP-2-loaded) with a concentration of 1 mg/mL scaffolds were added in the wells and incubated for another 24 h at 37C in 5% CO
2.
Note that the wells containing no scaffold was the control. After incubation completed the absorbance were measured with ELISA reader at 420 nm and cell viabilities were determined the results are summarized inTable 1.
Comparing with the control, the cell viability with the scaffold decrease little – not significant showing that scaffold material is highly compatible – it should be noted the very positive effect of PEG in the structure as expected (Xiao et al.
2010). Interestingly, – even the culture period was short – an increase in the cell number (increase the cell viability value) clearly observed which was considered as a positive/promising result for further animal studies.
Effects of both plain and VEGF/BMP-2-loaded scaffolds onto MC3T3-E1 osteoblast cells were also observed in the apoptosis and necrosis studies applying a double staining protocol described in the pervious sections. As it can be seen inFigure 6, both the plain scaffold and VEGF/BMP-2-loaded scaffold did not show any necrotic effect on MC3T3-E1 osteoblast cells, since healthy cells shine in blue color (stained with the Hoechst 33342 dye) and necrotic cells shine in red color (stained with propidium iodate). However, VEGF/BMP-2-loaded scaffold exhibited relatively better viability and higher number of healthy MC3T3-E1 osteoblast cells than plain scaffold. The apoptotic index was obtained as 2 ± 1%, therefore it can be concluded that scaffolds did not posses apoptotic effect.
Conclusion
One of the most attractive approaches in bone repair is using tissue engineering hybrid materials composed of scaffolds and cells carrying also bioactive agents such as growth factors triggering angiogenesis and osteogenesis. It is well-known that VEGF and BMP-2 take important role in natural bone regener-ation – therefore this couple has been proposed/used in preparation of bioactive hybrid materials. The important issue
Figure 6.Representative fluorescence microscopy images of MC3T3-E1 on; (A) the plain scaffolds; (B) VEGF/BMP-2-loaded scaffolds.
Table 1.WST-1 cytotoxicity test results.
Scaffold Absorbance (420 nm) % cell viability No scaffold 0.084 ± 0.01 100 Plain PLA-PEG-PLA 0.082 ± 0.02 97.6 ± 2.3 PLA-PEG-PLA loaded with VEGF/BMP-2 0.092 ± 0.03 109.5 ± 3.6
here is their sequential release – release of VEGF in early phase but rather slow and long-term release of BMP-2 – which would biomimic the natural process in a proper way. The main rational of this study is to develop biodegradable/biocompatible scaffolds loaded with this growth factor couple having release profile to reach desired kinetics.
Following our previous experience, we synthesized triblock copolymers of PLA and PEG which are among the most successful biodegradable and biocompatible polymers – there-fore have been widely used in the production of diverse biomaterials including tissue engineering scaffolds. One of the most critical property of scaffolds is their 3D pore morphologies – they should be highly porous with interconnected pores to reach desired 3D cell growth within the matrix. Note that degradation if scaffold is one of the main requirements to allow tissue formation and regeneration. Therefore, we have used well-known polymers PLA and PEG for the construction of our scaffolds. The scaffolds were produces by freeze drying/particle leaching which allowed us to reach both high porosity and pore-interconnectivity. Especially addition of salt particles and leaching of them after formation by leaching significantly improved the pore morphology.
We have followed also simple but an effective protocol to load the growth factors that would release with the desired kinetics. We have observed quite fast release of VEGF – almost 60% was released in the first 2 d and the release of BMP-2 was quite slow and almost constant – zero order kinetics.
The in vitro release curves approved this release behavior. In the material – cell interaction studies which were conducted in cell culture media, it was found that the scaffolds are quite biocompatible – almost no significant adverse effect were detected in contrast including growth factors affected cell behavior quite positively. Therefore, we concluded that it would be very worthy to carry these scaffolds to animal model studies for bone regeneration.
Disclosure statement
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
Funding information
This study has been partly supported by Turkish Scientific and Technological Council – Project No: 112S489.
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