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Keywords:

  • cell carrier;
  • hydrogel;
  • bioactive glass;
  • alginate;
  • tissue engineering

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

Cell carrier is a useful biomedical tool to deliver particular kind of cells to a specific site for cell therapy or tissue regeneration. In the current study, 45S5 bioglass (BG) was introduced into alginate (ALG) to generate BG/ALG composite hydrogel beads as cell carriers. The ions releasing behavior, dimensional stability and in vitro bioactivity of the beads were investigated. Results showed that the BG/ALG beads revealed similar calcium ion releasing behavior as compared with ALG beads. In addition, silicon ion releasing was detected in BG/ALG beads. BG/ALG and ALG beads shared similar dimensional stability, and BG/ALG beads could induce apatite deposition on their surface after being soaked in stimulated body fluid. Then, the effects of ion extracts from hydrogel beads on cell behavior were investigated. Results confirmed that extracts of BG/ALG beads could simulate proliferation and osteogenic differentiation of mesenchymal stem cells as well as angiogenesis of endothelial cells. Furthermore, MC3T3-E1 cells were successfully encapsulated in hydrogel beads. BG/ALG beads enhanced the cell proliferation and stimulated osteogenic differentiation of the encapsulated MC3T3-E1 cells as compared with ALG beads. Therefore, BG/ALG composite hydrogel beads loaded with bone forming cells may be useful tools for bone regeneration and tissue engineering applications. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 42–51, 2014.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

Facing increasing demands of bone regeneration or repair as population and average lifespan of human beings continue expanding, researchers have investigated different possible solutions including biomaterial grafting, drug therapy, cells therapy and tissue engineering.2000, 2004, 2012, 2011 Among those methods, delivery of desired cells to bone defect has been widely reported,2011, 2012, 2012 which can be achieved by directly injecting cells or delivering cells within a carrier.2008 Unlike direct injection, cell encapsulation method offers cells three-dimensional (3D) environment, and avoids cells to flow away from targeted site while allows nutrients and oxygen to be diffused in and wastes to be discharged out.2012

Alginate (ALG) is a natural polysaccharide that demonstrates great benefits as a biomaterial for cell delivery and other biomedical applications.2012 ALG hydrogel can be easily formed through various approaches, such as ionic and covalent cross-linking.2010 The most convenient way to crosslink ALG is using divalent ions such as calcium ions. Chelated with calcium ions, the molecular chains of ALG will yield an egg-box structure.1973 The high water content network and structure similar to extracellular matrix (ECM) endow ALG hydrogel an excellent biocompatibility for cells.2012 Considering these advantages, ALG hydrogels were applied in tissue regeneration, cell, and drug delivery as well as wounding healing.2008 Recently, ALG hydrogels have been reported to encapsulate several types of cells for bone,2005 vascular,2008 and cartilage2010 regeneration. However, most reports on ALG cell carriers for bone repair have introduced bone forming induction factors (e.g., bone morphogenetic protein and transforming growth factor-β), which is expensive and hard to achieve controlled release.2011 In order to improve the ability of ALG cell carriers in bone regeneration application, the capabilities of them to stimulate proliferation and differentiation of encapsulated cells need to be improved. Therefore, various approaches have been studied, such as directly regulating the microstructure of ALG networks2012 and incorporating bioactive inorganic material into ALG.2010-2012, 2011 Previous reports mainly used phosphate-based bioactive inorganic materials to modify ALG,2010-2012, 2011 whereas few reports discussed modification by silicate-based bioactive materials. Studies have shown that silicate-based bioactive materials have better stimulation effect on proliferation and osteogenic differentiation of bone forming cells.1991, 2007

Bioglass is a silicate-based bioactive inorganic material with composition of SiO2, Na2O, CaO, and P2O5 in specific proportions,2006 and bioglass 45S5 (BG) is the original composition of BG,1971 which possesses excellent osteostimulatory ability and is favorable for bone regeneration application.2012 Studies have demonstrated that the ions released from BG stimulated osteogenesis and angiogenesis in vitro and in vivo.2001, 2010 Calcium and silicon ions are among the most critical ions released from BG that affect the cell behaviors, and they can regulate cell cycle by adjusting local chemical environment.2011 In bone tissue engineering, BG has been incorporated into biopolymers, such as poly(l-lactic acid-co-glycolic acid, poly(l-lactic acid-co-glycolic acid, and poly(d,l-lactic acid), to achieve a composite system with the bioactivity from BG and the plasticity from biopolymers.2006

Therefore, the combination of BG and ALG hydrogel may have the potential to be used as cell carrier. The main objectives of the current study are the preparation of the BG/ALG composite hydrogel beads and the investigation of the effect of the incorporated BG in ALG on the cell behaviors. We incorporated a small amount of BG powders into ALG hydrogels to prepare BG/ALG composite hydrogel beads. The hydrogel beads were prepared by using an extrusion method during which hydrogels were cross-linked by calcium chloride. The ion releasing, swelling and in vitro bioactivity of the hydrogel beads were investigated. In addition, the effect of the ions released from BG/ALG beads on cells was investigated. Furthermore, cells were successfully loaded into the hydrogels beads, and the interactions between the materials and the encapsulated cells were evaluated.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

Preparation of hydrogel beads

Alginic acid sodium salt from brown algae (low viscosity; Sigma) was dissolved in deionized water to produce a 1.2% (w/v) ALG solution, and the solution was sterilized with a 0.22 μm filter (Millipore). BG powders (Provided by Shanghai Institute of Ceramics, Chinese Academy of Science) used in present study have an average diameter of 20.28 μm (90% <34.86 μm). BG were sterilized by ultraviolet light, and uniformly dispersed in ALG solution to generate BG/ALG solution with different content of BG. The obtained BG/ALG solution was extruded by syringe with a 23G needle into 100 mM CaCl2 solution drop-by-drop, and cross-linked for 10 min to obtain beads. Beads were collected and washed with phosphate-buffered saline (PBS) and minimal essential medium alpha (MEM-alpha; Gibico). Those beads were incubated in MEM-alpha containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P–S) at 37°C with 5% CO2, and the beads/media volume fraction is 1/1.5. After 1 day of incubation, concentrations of calcium and silicon ions in media were measured by inductively coupled plasma optical emission spectrometer (ICP-OES, Varian 715-ES). Each formulation of hydrogel beads was designated as A-BG (Table 1), where A represents the BG content (0, 0.1, 0.2, or 0.4%, w/v) in the beads.

Table 1. Ion Concentrations of Media After 1-Day Incubation of Hydrogel Beads
NameBG Content (%, w/v)Ca (mmol L−1)Si (mmol L−1)
0-BG or ALG010.06 ± 0.250.03 ± 0.01
0.1-BG0.111.05 ± 0.080.25 ± 0.01
0.2-BG0.210.99 ± 0.080.79 ± 0.02
0.4-BG0.412.25 ± 0.102.14 ± 0.03

BG/ALG beads loaded with MC3T3-E1 cells (Purchased from Cell Bank of the Chinese Academy of Science, Shanghai, China) were prepared similarly. As Figure 1(A) shows, cells were harvested by trypsin and centrifuged to a cell pellet. The cell pellet was gently mixed with BG/ALG solution and achieved a density of 3 × 105 cells mL−1. Then, the beads were generated and cultured the same as the above description.

image

Figure 1. Preparation and characterization of BG/ALG hydrogel beads. (A) Schematic diagram of the process to prepare BG/ALG beads loaded with cells; (B) digital photos and (C) microscopy images of blank beads without cells with different compositions of BG when prepared; (D) dimensional change of 0-BG and 0.2-BG blank beads, Dt is the diameter of beads in day t, and D0 is the diameter of beads when prepared; and (E) calcium ion releasing of 0-BG and 0.2-BG blank beads. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Dimensional change and calcium ions releasing of beads

Twelve images of beads were captured by Leica-ICC50 at different time points (days 0, 1, 2, 4, 6, 8, 10, 12, and 14) with same magnification (40×). Images were analyzed by Image J software to measure the diameters of beads. Dimensional change was indicated by Dt/D0, where Dt stands for the average diameter of beads in day t, and D0 is the average diameter of freshly prepared beads.

As calcium ions served as cross-linking agent of ALG in present study, the change in calcium ions of MEM-alpha in which hydrogel beads cultured was detected. Media were collected at days 1, 2, 4, and 7, and calcium ion concentrations of the collected media were measured by ICP-OES.

In vitro bioactivity

Stimulated body fluid (SBF) was prepared as described in the previous report,2006 and used to evaluate the in vitro bioactivity of hydrogel beads. Freshly prepared hydrogel beads were soaked in SBF with a beads/SBF volume fraction of 1/5 for 10 days at 37°C. After removing water by a freeze dryer (Christ BETA 1-8), the structure and composition of collected beads were analyzed by scanning electron microscopy (SEM, Hitachi S-4800), Fourier transform infrared spectroscopy (FTIR, Nicolet), and X-ray diffraction (XRD, D8 Advance).

Effects of ions released from the composite beads on cell behavior

Preparation of ion extracts of hydrogel beads

To investigate the effect of the ions released from the BG/ALG composite hydrogel beads on cell behaviors, ion extracts of beads were applied to culture rat bone marrow mesenchymal stem cells (rBMSCs, kindly provided by Renji Hospital Clinical Stem Cell Research Center, Shanghai, China) and human umbilical vein endothelial cells (HUVECs, isolated from human umbilical cord vein according to the previous report1973). Ion extracts of the beads were prepared according to the previous report2013 with small modification. Briefly, beads were soaked in serum-free Dulbecco's modified eagle medium–low glucose (DMEM; Gibico) or endothelial basal medium-2 (EBM-2; Lonza) with a beads/media volume fraction of 1/1.5 and incubated in 5% CO2 at 37°C for 24 h. The mixture was centrifuged, and the supernatant was collected and sterilized with a 0.22-μm filter (Millipore). For the proliferation and osteogenic differentiation assay of rBMSCs, ion extracts prepared from DMEM were supplemented with 10% FBS and 1% P–S, and diluted with DMEM + 10% FBS + 1% P–S at ratios of 1, 1/2, and 1/4. For angiogenesis assay of HUVECs, ion extracts prepared from EBM-2 were supplemented with 1% FBS, and diluted with EBM-2 + 1% FBS at ratios of 1 and 1/2. Concentrations of calcium and silicon ion in each extract group were measured by ICP-OES. Each extract was designated as “X–Y” (Table 2), where X represents the beads (0-BG and 0.2-BG), and Y stands for dilution ratio.

Table 2. Ion Concentrations of Hydrogel Beads Extracts at Different Dilution Ratios
Derived FromGroupCa (mmol L−1)Si (mmol L−1)
  1. a

    DMEM containing 10% FBS and 1% P–S.

  2. b

    EBM-2 containing 1% FBS.

DMEMDMEM controla1.90 ± 0.02<0.01
0-BG-18.09 ± 0.040.04 ± 0.01
0-BG-1/25.07 ± 0.050.02 ± 0.01
0-BG-1/43.54 ± 0.07<0.01
0.2-BG-18.62 ± 0.140.71 ± 0.01
0.2-BG-1/25.39 ± 0.060.36 ± 0.01
0.2-BG-1/43.60 ± 0.040.18 ± 0.01
EBM-2EBM-2 controlb1.62 ± 0.010.02 ± 0.01
0-BG-111.65 ± 0.050.03 ± 0.01
0-BG-1/26.65 ± 0.080.02 ± 0.01
0.2-BG-110.37 ± 0.030.89 ± 0.01
0.2-BG-1/26.06 ± 0.100.45 ± 0.01
Proliferation of rBMSCs cultured in beads extracts

For proliferation assay, rBMSCs were seeded into 96-well plates at a density of 3 × 103 cells per well and cultured in different beads extracts. Cells cultured with DMEM +10% FBS +1% P–S were served as a control. At days 0, 1, 3, 5, and 7, CCK-8 (Beyotime) assay was undertaken to determine the relative cell number according to the manufacture's instruction.

Osteogenic differentiation of rBMSCs cultured in beads extracts

For differentiation assay, rBMSCs were seeded into 6-well plates at a density of 3 × 104 cells per well and cultured in different beads extracts. Cells cultured with DMEM +10% FBS + 1% P–S were served as a negative control, and cells cultured with osteogenic medium (OM, DMEM + 10% FBS + 1% P–S supplemented by 100 nM dexamethasone, 10 mM β-glycerol phosphate, and 0.05 mg mL−1 ascorbic acid) were served as a positive control. After 1 week of culture, alkaline phosphatase (ALP) staining was undertaken using Fast blue RR salt (Sigma) and Naphthol AS-MX phosphate alkaline solution (Sigma) according to the previous reports.1968 Quantitative ALP activity analysis was undertaken by the similar method in section “ALP activity assay of encapsulated cells”, except 384-well plates were used.

Angiogenesis of HUVECs cultured in beads extracts

In vitro angiogenesis assay was processed using ECMatrix™ (Millipore, ECM625). The 96-well plates were coated with ECMatrix™ according to the manufacture's instruction. 104 HUVECs were seeded and cultured on ECMatrix™ with different beads extracts for 3, 6, and 12 h. Cells cultured on ECMatrix™ with EBM-2 + 1% FBS were used as a control. At each time point, cells were observed and photographed with five random microscopic fields by a microscope (Leica DMI 3000B). The numbers of branch points in HUVECs lines (nodes), mesh-like circles (circles), and tube-like parallel cell lines (tubes) were quantified according to manufacture's instruction. The formation of nodes, circles and tubes are parameters of the dynamic regeneration process of angiogenesis, counting them can be served as a qualitative assay of angiogenesis.

Effects of beads on encapsulated cells

Preosteoblast MC3T3-E1, a commercial cell line, has been used for evaluation of various cell carrier systems for bone regeneration applications.2012, 2012 In present study, as a preliminary evaluation of the cell encapsulation ability of BG/ALG composite beads, MC3T3-E1 has also been chosen to be loaded in BG/ALG hydrogel beads.

Viability analysis of encapsulated cells

A LIVE/DEAD staining kit (Invitrogen) was applied to detect the viability of cells within the beads according to the supplier's procedure. The live cells exhibited green fluorescence color and the dead cells produced red fluorescence color after being stained with the kit. The beads were observed and photographed with a fluorescent microscope (Leica DMI 3000B).

Total protein of encapsulated cells

The amount of total protein of hydrogel beads has been measured to determine the proliferation of cells within the beads. Briefly, 10 beads were collected after 1 day and 1 week of culture and washed with PBS for at least three times. Those collected beads were then lysed and homogenized with 300 μL of lysis buffer (AnaSpec). Total cellular protein of lysate was measured by BCA protein assay kit (Pierece) according to supplier's protocol.

ALP activity assay of encapsulated cells

Hydrogel beads loaded with MC3T3-E1 cells were collected after 1 week of culture and lysed with lysis buffer supplied by the ALP assay kit (AnaSpec). ALP activity was detected according to manufacture's protocol. Briefly, samples were added to 96-well plates with 3,6-fluorescein diphosphate reaction mixture supplied by the kit, and the mixtures were then incubated for 30 min at 37°C. Finally, stop solution was added to each sample to end the reaction, and the fluorescence intensity was measured using Synergy 2 microplate reader (BioTek) by detecting emission at 528 nm with excitation at 485 nm. Readings are recorded in relative fluorescence units (RFUs). Total cellular protein was measured by BCA protein assay kit (Pierece) according to supplier's protocol. ALP activity was finally normalized by total protein of each sample.

Statistical analysis

Results are present as mean ± standard deviation. One-way analysis of variance was used to determine statistical significance for all data with StatPlus software (AnalystSoft).

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

Preparation and characterization of BG/ALG hydrogel beads

Preparation of BG/ALG hydrogel beads

BG/ALG composite hydrogel beads with different contents of BG were prepared by the procedures presented in Figure 1(A). The beads showed a round shape with uniform diameter [Figure 1(B)]. BG in the beads appeared as dark dots under microscopy observation [Figure 1(C)]. The opacity of beads increased with the increase in BG due to the light impermeability of BG.

In order to choose the composition of beads for further experiment, calcium and silicon ion concentrations of media were measured by ICP-OES after 1-day incubation of hydrogel beads. Results showed that the calcium ion concentrations were similar in all groups (Table 1). Silicon ions were detected in 0.1-BG, 0.2-BG, and 0.4-BG beads groups, whereas no silicon ions were found in ALG group. Silicon ion concentration increased with the increase in the BG content. According to previous reports on optimal silicon ion concentrations for cells,2009, 2013 0.2-BG beads were chosen to undertake further experiment as compared with pure ALG beads, since the silicon ion concentration in 0.2-BG group had a close value to previous reports.

Dimensional stability and calcium ion releasing of the beads

ALG hydrogel possesses 3D high water content network, and this structure usually alters in a liquid environment, which results in dimensional change. Though having different compositions, 0-BG and 0.2-BG beads showed a similar tendency on diameter changes [Figure 1(D)]. All hydrogel beads experienced a diameter decrease after 1 day of incubation in media. After that, diameters of all beads began to rise until 1 week when the diameters of beads became stable. Both 0-BG and 0.2-BG beads demonstrated a final diameter increase rate around 140%.

Since ALG hydrogels are cross-linked by calcium ions in the present study, calcium ion content becomes a critical factor that determined the structure of the hydrogel. The change of calcium ion content with time was measured after incubation of hydrogel beads in MEM-alpha medium. It was found that the 0-BG and 0.2-BG beads shared a similar calcium ion releasing behavior [Figure 1(E)]. The calcium ion concentrations in media of both 0-BG and 0.2-BG beads showed a much higher value (around 10 mmol L−1) at day 1 than other days (<6 mmol L−1), and the calcium ion concentration at day 1 is similar to the data of undiluted group in the extracts experiment (Table 2). After 2 days culture, the calcium ion concentration in media of both 0-BG and 0.2-BG beads dropped by about half of that of the first day. After 1 week of culture, the calcium ion concentration in media of both 0-BG and 0.2-BG beads became about a quarter of that of the first day.

In vitro bioactivity of hydrogel beads

SBF soaking experiment was used to determine the in vitro bioactivity of hydrogel beads. Before being soaked in SBF, both 0-BG and 0.2-BG beads possessed a smooth morphology under SEM observation [Figure 2(A,B)], and showed similar FTIR [Figure 2(F.a,F.c)] and XRD [Figure 2(G.a,G.c)] pattern. In 0.2-BG beads, BG powders can be seen uniformly embedded in ALG matrix and demonstrated a gel-cover appearance [Figure 2(A)]. After being soaked in SBF for 10 days, the surface of 0-BG beads remained smooth with some microcracks [Figure 2(E)], whereas 0.2-BG beads revealed a coarse morphology. Apatite-like structure can be seen on the surface of 0.2-BG beads [Figure 2(C,D)]. In the FTIR pattern of 0.2-BG beads [Figure 2(F.d)], the bending mode of the phosphate group is observed at 603.28 and 564.55 cm−1, and a peak at 1384.85 cm−1 is detected due to the stretching mode of the carbonate group.2011 The XRD pattern of 0.2-BG beads [Figure 2(G.d)] showed sharp peaks at 32 and 46° (2θ), which attributed to (211) and (222) plane of apatite.2011, 2000 Above results indicate that the BG/ALG composite hydrogel beads can induce the deposition of apatite-layer on their surface, the formed apatite supposed to be hydroxyl-carbonate apatite, which equivalent to the inorganic mineral phase of bone.2000, 2002, 2005

image

Figure 2. SEM images (A–E), FTIR patterns (F), and XRD patterns (G) of 0-BG and 0.2-BG beads before and after being soaked in SBF for 10 days. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Effects of ions released from the composite beads on cell behaviors

The effects of ions released from composite hydrogel beads on the proliferation, differentiation, and angiogenesis of cells were determined by culturing cells in the presence of beads extracts in different concentrations (Table 2).

Ion concentration of extracts

To elucidate the effects of ions from beads extracts on cell behaviors, the calcium and silicon ion concentrations of different extracts were determined by ICP-OES, and the results are displayed in Table 2. It can be seen that the calcium ion contents in both undiluted 0-BG and 0.2-BG extracts (8–12 mmol L−1) are higher than that in the control groups (<2 mmol L−1). With the increase in dilution ratio, calcium ion content in 0-BG and 0.2-BG groups decreased but still higher than that of the control groups. However, silicon ion concentration in 0.2-BG extracts (0.2–0.9 mmol L−1) is much higher than that in both 0-BG extracts (<0.05 mmol L−1) and DMEM control group (<0.01 mmol L−1).

Proliferation of rBMSCs cultured in beads extracts

CCK-8 assay was used to evaluate the proliferation of rBMSCs by determining the relative cell number. At days 0 and 1, the cell number showed no difference between each group (Figure 3). After 3 days culture, the number of cells cultured with 0.2-BG extracts was significantly higher than that of cells cultured with 0-BG extracts with same dilution ratio. In addition, after 5 days culture, cells cultured with 0.2-BG-1/2 and 0.2-BG-1/4 extracts showed significantly higher cell numbers than cells cultured with DMEM control group. After 1 week of culture, the numbers of cells cultured with 0.2-BG-1 and 0.2-BG-1/2 extracts are still higher than that of cells cultured with 0-BG-1 and 0-BG-1/2 extracts, respectively. Besides, cells cultured with 0.2-BG-1/2 extracts showed a significantly higher number than that of cells cultured with DMEM control group. All of these results demonstrated that the incorporation of BG stimulated the proliferation of rBMSCs.

image

Figure 3. Proliferation of rBMSCs cultured in beads extracts. 0-BG: ion extracts of 0-BG beads; 0.2-BG: ion extracts of 0.2-BG beads; 1, 1/2, and 1/4 stands for the dilution ratio; DMEM: pure culture medium without extracts; * denotes statistical significance higher than DMEM group in the same day, p <  0.05; # denotes statistical significance higher than 0-BG group with same dilution ratio, p <  0.05.

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Differentiation of rBMSCs cultured in beads extracts

According to the proliferation assay results, we selected undiluted and half diluted extracts for cell differentiation assay. After 1 week of culture, the rBMSCs were fixed and underwent ALP staining before being observed under microscope, under which ALP appeared a purple color. Cells cultured with 0.2-BG-1 extracts and OM group expressed the deepest purple color [Figure 4(A,F)], whereas 0-BG-1 extracts and DMEM group showed a light purple color [Figure 4(C,E)]. 0.2-BG-1/2 and 0-BG-1/2 extracts group demonstrated a mid-level staining [Figure 4(B,D)]. More details can be seen from the quantitative assay of ALP activity [Figure 4(G)]. The results show that 0.2-BG-1 extracts group displayed similar ALP activities as OM group, which was significantly higher than all other groups. Meantime, ALP activity of cells cultured in 0-BG-1, 0-BG-1/2, and 0.2-BG-1/2 extracts group were lower than that of cells cultured with OM group, although there is no statistical significance between them and DMEM group. Above results indicated that the ions released from the composite hydrogel resulted in the stimulation of osteogenic differentiation of rBMSCs.

image

Figure 4. Alkaline phosphatase activity of rBMSCs cultured in beads extracts. (A–F) Microscopy images of rBMSCs after ALP staining; (G) quantitative ALP activity (RFU, in 384 wells) that normalized by total protein. 0-BG: extracts of 0-BG beads; 0.2-BG: extracts of 0.2-BG beads; 1 and 1/2 stands for the dilution ratio; DMEM: pure culture medium without extracts; * denotes statistical significance higher than all groups expect the OM group, p <  0.01; scale bar in (A)–(F) is 400 μm. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Angiogenesis of HUVECs cultured in beads extracts

An in vitro angiogenesis assay kit was used to evaluate the angiogenesis ability of HUVECs cultured with ionic extracts of composite hydrogel beads. According to manufacturer's instruction, branch points (nodes), mesh-like structure (circles), and tube-like parallel lines (tubes) can be formed by activated endothelial cells when they contacted the ECMatrix™ gel. The progression of angiogenesis can be quantified by counting numbers of those patterns formed by endothelial cells.

After 3 h of culture on ECMatrix™, cells began to migrate, align themselves, and formed some short lines and branch points [Figure 5(A)]. A higher degree of angiogenesis of HUVECs cultured in 0.2-BG extracts was observed, which was demonstrated by the formation of more nodes, circles and tubes than that in 0-BG extracts [Figure 5(A,B)]. Although average numbers of these patterns in 0.2-BG extracts are higher than those in control medium, no statistically significance difference has been found.

image

Figure 5. In vitro angiogenesis assay of HUVECs cultured on ECMatrix™ in beads extracts. 0-BG: extracts of 0-BG beads; 0.2-BG: extracts of 0.2-BG beads; 1 and 1/2 stands for the dilution ratio; Cont: pure culture medium without extracts; # denotes the numbers of nodes, circles or tubes are statistical significance higher than that of Cont group in the same time point, p <  0.05; * denotes the numbers of nodes, circles or tubes are statistical significance higher than that of 0-BG group with same dilution ratio in the same time point, p <  0.05.

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When the culture was maintained for 6 h, cells in all extracts developed more complex structure than 3 h culture, where more nodes and circles were observed. Simultaneously, more parallel cell lines can be found with extended length [Figure 5(A)]. Quantitative assay indicated that more nodes, circles and tubes were formed by HUVECs cultured in 0.2-BG extracts than in 0-BG extracts and control group [Figure 5(B)].

At the 12 h observation, most cells began to undergo apoptosis, which is consistent with the instruction of the angiogenesis assay kit. Mesh-like structures started to collapse and break, branch points decreased dramatically and tube-like structure shrunk. However, HUVECs cultured in 0.2-BG extracts still maintained a higher numbers of nodes, circles and tubes than that in 0-BG extracts.

Viability and proliferation of encapsulated MC3T3-E1 cells

MC3T3-E1 cells were successfully encapsulated into the beads, which displayed as green dots under florescent microscopy observation after LIVE/DEAD staining [Figure 6(A)]. The viability of encapsulated cells in 0-BG and 0.2-BG beads was compared in a 12-day culture, and the fluorescent density showed no significant difference between cells in 0-BG and 0.2-BG beads during the first 4 days culture. However, more green dots appeared in 0.2-BG beads than those in 0-BG beads after 1 week of culture, which indicates higher viability of cells in 0.2-BG beads as compared to that of cells in 0-BG beads.

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Figure 6. Viability, proliferation, and alkaline phosphatase activity of MC3T3-E1 cells encapsulated in hydrogel beads. (A) LIVE/DEAD staining of MC3T3-E1 cells encapsulated in 0-BG and 0.2-BG beads after different days of culture (green: live cells; red: dead cells; bar = 200 μm); (B) total protein concentration of hydrogel beads after 1 day and 1 week of culture; (C) alkaline phosphatase activity (RFU in 96 wells) of MC3T3-E1 encapsulated in 0-BG and 0.2-BG beads after 1 week of culture that normalized by total protein. (* denotes statistical significance between 0-BG and 0.2-BG group, p level <0.05). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Total cellular protein in hydrogel beads showed no significant difference between 0-BG and 0.2-BG after 1 day of culture [Figure 6(B)]. However, total cellular protein in 0.2-BG beads became significant higher than that in 0-BG beads after 1 week of culture, which indicates 0.2-BG enhanced the proliferation of MC3T3-E1 beads.

ALP activity of encapsulated MC3T3-E1 cells

The change of ALP activity is involved in a variety of physiological events such as bone development, and ALP is usually served as an early marker of osteogenic differentiation. In this study, after 7 days culture, proteins of encapsulated cells were extracted, and ALP activity of cells was detected and normalized by total protein. The results showed that relative ALP activity in cells encapsulated in 0.2-BG beads is higher than that in 0-BG beads [Figure 6(C)], which indicates that the incorporation of a small amount of BG in ALG hydrogel beads can stimulate the osteogenic differentiation of encapsulated cells.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

In this study, 45S5 BG was incorporated into ALG hydrogel to prepare bioactive BG/ALG composite hydrogel beads as cell carriers for bone regeneration and tissue engineering applications. First, it is necessary to confirm whether the 3D structure and hydrogel properties of ALG as well as the bioactivity of BG can be maintained in the composite system. Results in present study showed that the incorporation of BG did not change the 3D structure of ALG, and BG/ALG beads shared similar macro and micro appearance with ALG beads. Also, the diameter of BG/ALG beads remained <2 mm after preparation.

Swelling property is among the most fundamental properties of hydrogels, and proper swelling of cell carriers is very useful when the carriers are delivered into the living body. A previous study proposed that this property would allow hydrogel to have a tighter contact with surrounding tissues.2008 In the current study, BG/ALG beads demonstrated similar swelling property as compared with ALG beads. All beads displayed shrinkage during the first day in the present study, which may be caused by the remaining unreacted calcium ions in hydrogel networks after preparation. The presence of residual calcium ions can be proved from the ion concentration measurement, which showed that the calcium ion concentration of media in the first day was the highest, and decreased rapidly when medium was replaced by fresh medium. Those residual calcium ions served as cross-linking agent and caused the shrinkage of ALG and BG/ALG beads after preparation. After that, both ALG and BG/ALG beads began swell, which may caused by ion exchange between calcium ions in the hydrogel networks and dechelation ions, such as sodium ions, from surrounding media.2012

The reserved 3D structure and swelling properties of ALG are important for practical applications, and one explanation why the BG/ALG beads had similar swelling property as compared with ALG beads is that only very small amount of BG (0.2%, w/v) was incorporated into the ALG hydrogel, which was significantly lower than that usually used for preparation of BG/polymer composite scaffolds (>10%, w/v).2012, 2005 However, even at such a low amount of BG (0.2%, w/v), the composite hydrogel beads still retain the ability to induce apatite deposition on their surface after being soaked in SBF while pure ALG hydrogel beads lack this ability. The formed apatite layer indicated that BG/ALG beads have a better in vitro bioactivity than pure ALG beads.2006, 2011 The mechanism of the formation of apatite layer is generally believed to be a result of a series of reactions on the surface of the BG, which involve condensation of a SiO2-rich layer with the increase in silicon ions concentration.2012

Some studies showed that the ions released from BG would change the local chemistry environment, and stimulate osteogenesis as well as angiogenesis in vitro and in vivo by regulating several genes, which are related to bone formation and vascularization.2010, 2011 Some researches also confirmed that the activities of cells could be regulated by controlling the concentration range of silicon or calcium ions in the culture media.2013, 2012 So it is important to evaluate whether the composite hydrogel beads will also release ionic products and stimulate osteogenesis and angiogenesis. In the present study, the BG/ALG beads extracts showed a beneficial effect on both bone forming cells and endothelia cells, which confirmed that small amount of BG in BG/ALG beads still released bioactive ionic products. The positive regulation effect of BG/ALG beads extracts on cells may be caused by silicon ions, since only the BG/ALG beads released silicon ions while ALG beads did not, and no obvious difference was detected in calcium ions releasing between groups with same dilution rate. The silicon ion has been shown to be a critical ion for metabolic processes related to bone formation.2011 Previous reports suggested that the ionic products of BG with silicon ion concentrations ranging from 15 to 20 μg mL−1 (0.54–0.71 mmol L−1) were optimal for fetal osteoblast proliferation and differentiation.2009 A recent study2013 suggested that silicon ions at a concentration of 0.625 mmol L−1 significantly enhanced the proliferation and osteogenic differentiation of BMSCs suggesting that silicon-containing biomaterials can enhance bioactivity at desired concentration. Those reported silicon ion concentrations are very similar to those in the BG/ALG beads extracts in the present study. Meantime, calcium ions releasing from the hydrogel beads may also have an impact on cells. Low calcium ion concentration (<8 mmol L−1) is reported to stimulate proliferation and differentiation of osteoblast cells, whereas high calcium concentration (>10 mmol L−1) may be unfavorable to osteoblast cells.2005 This may further explain the positive effects of BG/ALG beads extracts and the inhibition effects of 0-BG-1 extracts on proliferation of BMSCs and angiogenesis of HUVECs.

The aim of the present study was to prove that the incorporation of a small amount of BG into ALG matrix could enhance the function of ALG beads as cell carrier system. Results from the current study showed that BG/ALG beads could serve as cell carriers and had positive impact on the encapsulated cells. Small amount of BG incorporated in ALG did no obvious change on cell encapsulation ability of ALG. MC3T3-E1 cells shared the same dot-like appearance in both BG/ALG and ALG beads, and this appearance is similar to that in previous report.2012 In addition, BG/ALG beads showed a stimulatory effect on proliferation and osteogenic differentiation of encapsulated MC3T3-E1 cells, and this effect can be attributed to the beneficial effect of ionic products from BG/ALG beads. The results of the present study confirmed our assumption that the incorporation of small amount of BG into the ALG hydrogel would result in the formation of a bioactive hydrogel system, which might be used for bone regeneration and tissue engineering applications, although further in vitro and in vivo studies are required to proof the practical applicability of the composite hydrogels.

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES

In the current study, BG/ALG composite hydrogel beads with different BG content were prepared. BG/ALG beads released silicon ions, and showed a favorable in vitro bioactivity by inducing apatite formation on the surface of the composite hydrogels. In addition, ion extracts of BG/ALG beads stimulated proliferation and differentiation of rBMSCs as well as angiogenesis of HUVECs. Furthermore, MC3T3-E1 cells were successfully encapsulated in BG/ALG beads. BG/ALG beads enhanced the cell proliferation and stimulated osteogenic differentiation of the encapsulated MC3T3-E1 cells. Above favorable properties endow the BG/ALG composite hydrogels with great potentials in bone regeneration and tissue engineering applications.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIAL AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSIONS
  8. REFERENCES