Biomaterials Functionalized with Insulin can enhance Osteoblasts
Activity
Elena Mavropoulos1, Moema Hausen1, Jéssica Dornelas da Silva1, Suzana Azevedo dos
Anjos1-2, Gutemberg Alves2, Alexandre Malta Rossi1
1
Depto. de Física Aplicada, Laboratório de Cultura Celular, CBPF, Rio de Janeiro (RJ), Brasil
2
Depto. de Biologia Celular e Molecular, HUAP, Universidade Federal Fluminense, Niterói (RJ), Brasil.
E-mail: elena@cbpf.br
Abstract. Hydroxyapatite (HA), zinc substituted hydroxyapatite (ZnHA) and carbonated hydroxyapatite
(CHA) are already studied as implanted biomaterial in bone grafts due to its similar chemical
composition with bone and due to their biocompatibility, bioresorption and bioactivity properties. HA,
ZnHA and CHA powders were synthesized from wet method at 37° C and the DRX patterns showed that
the samples presented low crystallinity compared with materials produced at higher temperature. FTIRATR analysis revealed that insulin is present to samples disks after adsorption. Cell attachment assays
were performed by fluorescence microscopy and showed a strong increase in attachment in insulin
coated samples. The results here obtained showed that insulin coatings on biomaterials could be a
promising alternative to rapidly mobilize cells to favor osteointegration.
Keywords: Hydroxyapatite, Insulin, surface interaction, osteointegration
1.
INTRODUCTION
Calcium phosphates are the major mineral constituent of bone and teeth and they
are commonly used as a filler of bone defects or as a coating to promote bone formation
into orthopedic implants. More recently the focus is to design “smart” biomaterials by
modifying the biomaterials surfaces through adsorbing proteins or peptide sequences
obtaining a desired surface to control cell response and enhance tissue regeneration [1].
Protein–surface interactions are the major responsible for the biocompatibility of
medical devices, since cell settlement to the implanted biomaterial surface, will directly
interact with the adsorbed protein layer. According to the surface, a positive approach in
the bone cells is expected: the cytoskeleton prompt mobilization and the establishment
of new focal contacts. In in vivo models, one negative consequence for delay in the
cells adhesion process of bone grafts is unattachment followed by cell death and the
formation of a fibrous encapsulation of the material. So on, in vitro models of materials
that could generate increased cell attachment are potencial candidates for tests in vivo.
Insulin is a polypeptide hormone that acts as a growth-stimulating factor for
some cells types in culture, it travels around the bloodstream and is a small,
predominantly α-helical protein consisting of 51 residues. The insulin monomer
assembles as dimmers (diffused in the blood neutral pH) and in the presence of zinc
ions, as hexamers. Some authors [2] showed that bone cells express insulin receptors
which are directly correlated to osteoblast proliferation and differentiation. Also it is
known that diabetic’s high insulinemia can cause atherosclerosis in patients in a role
that is directly linked to an increase in leukocytes adhesion to blood vessels wall [3].
There is a noticeable lack in literature of the insulin interaction in cell adhesion and
apparently there is no previous evaluation in any biomaterial.
Herein we first report the effects of three different apatite compounds
funcionalized with human insulin to interact with osteoblastic cells. A qualitative
evaluation of each apatite sample through fluorescence microscopy was performed. Cell
attachment analysis of early adhesion was carried out.
2. MATERIALS AND METHODS
2.1 Samples preparation
HA and ZnHA powder samples were prepared by dropwise addition of
Ca(NO3)2.4H2O and Zn(NO3)2 solution to (NH4)2HPO4 aqueous solution, at 37°C and
pH was kept around 9 by addition of KOH. After 2 hours the precipitated was separated
by filtration and repeatedly washed with cold water. CHA powder was synthesized from
calcium nitrate tetrahydrate, ammonium di-hydrogen phosphate and ammonium
carbonate extra pure (Merck) salts dissolved in aqueous solutions. The reagents were
mixtured and maintained at 37°C for 2 hours and pH maintained at 13.0 with KOH. All
the suspensions were lyophilized and the dried powders were manually grounded, with
the < 210 µm particles separated by sieving. For the in vitro assays the powder was uniaxially pressed at 2 Ton to form discs with diameter of 10mm and thickness of 1 mm.
The obtained disks were washed three times with ethanol 70% and Milli-Q water and
sterilized by gamma radiation. Each apatite disk (HA, ZnHA or CHA) was adsorbed for
24h with 20µg/mL of human insulin (Ins). The insulin-free groups were adsorbed with
saline buffer only (PBS).
2.2 Samples Characterization
The characterization of the synthesized powders was performed by powder Xray diffraction (XRD) and Fourier-transformed infrared spectroscopy (FTIR). XRD
analysis was carried out to determine the crystallinity of the samples and to identify
phase composition. The analysis was performed using a SEIFERT-FPM GmbH
diffractometer operating with CuKα radiation (1.5418 Å) at 40 kV and 40 mA with a
graphite monochromator in the primary bunch. The XRD patterns were obtained in the
interval of 2θ step interval from 10 to 100°. Fourier Transformed Infrared (FTIR) was
performed using a Shimatzu IR- Prestige-21/AIM-880 operating from 400 to 4000 cm-1.
2.3 Cell culture
For the testing of biocompatibility the murine preosteoblast cell line MC3T3-E1
subclone 14 was chosen as these cells are highly differentiated and behave most like
osteoblasts. Cells were cultured with α-MEM with 10% fetal bovine serum (FBS) and
incubated at 37°C at 5% CO2 atmosphere. Confluent low passages were trypsinized,
counted in a Neubauer chamber and used in experiments. The control group of each
assay was seeded on Thermanox coverslips (Th) previously coated with 0.1% porcine
gelatin (Gel) or 20µg/ml of insulin (Ins).
2.4 Cell attachment and proliferation by fluorescence microscopy
The osteoblastic cells were seeded over disks of each material at a density of
0.5x10 cells and incubated at 37ºC/5% CO2. After incubation of 4h, cells were fixed
with PFA 4% for 20min, washed in PBS, and permeabilized with Triton 0.1%. After
three PBS washes, each disk was immersed in 4% albumin for 30min. Cells were
stained for fluorescence with DAPI 1:1000 for 15min and phalloidin-TRITC 1:100 for
1h at RT. Disks were immersed on antifading solution, mounted on coverslips and
observed in an inverted fluorescence microscope (AxioObserverA1, Zeiss, Germany).
The cell quantification was performed by Image Pro Plus 6.0 software (Media
Cybernetics Inc., USA).
4
3. RESULTS AND DISCUSSION
3.1 HA, ZnHA and CHA samples characterization
The peaks position and peaks linewidths for all powder samples corresponded to
a well-crystallized hydroxyapatite (JCPDS 09-0432), as shown in Fig. 1. Comparing the
XRD patterns of HA, CHA and ZnHA powder produced at 37°C no significant changes
in respect to the crystallinity were observed. The HA and CHA samples presented the
main characteristics peaks for hydroxyapatite phase according to data Powder
Diffraction File 89-4405 JCPDS (211, 112 and 300 planes) indexed as shown in figure
1. Broader peaks for ZnHA sample causing the overlap of 211, 112 and 300 planes.
Figure 1. XRD pattern of CHA 37°C, HA 37°C and ZnHA 37°C.
Fourier Transformed Infrared Attenuated Total Reflectance Microscopy (FTIRM-ATR)
spectroscopy is a suitable tool to detect protein conformational changes after adsorption
experiments [4]. The FTIRM-ATR analysis of HA, ZnHA and CHA disks before and
after insulin adsorption showed additional bands after insulin adsorption that were
attributed to protein presence (data not shown).
3.2 Cell attachment by fluorescence microscopy
The insulin adsorbed materials in all samples increased the cell spreading after
4h of incubation (Figure 2). The total cell count by nuclei quantification was the same
in all samples after 4h (data not shown). The stress fibers were observed mainly by Th +
Ins and CHA + Ins groups. A striking difference in the cell spreading by the thermanox
(Th) with and without insulin was evident. Comparing the morphological presentation
of the three analyzed ceramics, the ZnHA + Ins group showed the lower cell spreading
while CHA + Ins showed the higher spreading.
Is well known that integrins are directly related to cell adhesion. According to
some authors [5] the integrins play a role in bone formation. These authors showed that
when cell were exposed to insulin-like growth factor 1 (IGF-1), the osteoblast cells
increased activity and changed morphology. Others authors [6] also showed that IGF-1
stimulated osteoblast adhesion. According to these authors, several matrix proteins in
bone contain the RGD sequence and may be involved in the binding of the osteoblast to
osteoid through integrins, so on when cells were treated with IGF-1 they became flatted
and increased the binding to many matrix proteins. It´s possible that the increase in cell
spreading observed here in insulin coatings rapidly increased the integrin expression
outcoming in higher adhesion. The insulin molecule has no binding site to the
osteoblastic cell integrin although the cells posses insulin receptors. Thus, the increased
adhesion here observed is probably not related to a direct adhesion to the insulin itself,
but due to an increase in the recruitment of integrin expression, outcoming the higher
adhesion to the materials. The insulin may have acted as an IGF-1 as previously
reported by these authors.
Regarding the concept of smart materials to induce a rapid accession to favor
osseointegration, the coating materials with insulin seems to be a promising alternative
to bone grafts. Futher analysis of specific bone signaling molecules are to be
investigated in order to full understand the role of insulin in bone cells adhesion,
proliferation and differentiation.
Th + Gelatin
Th + Ins
HA + PBS
HA + Ins
ZnHA + PBS
ZnHA + Ins
CHA + PBS
CHA + Ins
Figure 2. The cell adhesion after 4h in each material adsorbed with insulin (right
column) and with PBS (left column) showed that cell spreading is highly increased
when in the presence of insulin. Typical stress fibers usually observed in late adhesion
times were observed in Th + Ins and the general polygonal morphology of this lineage
was also observed.
4. ACKNOWLEDGMENTS
The authors would like to thank CNPq, CAPES and FAPERJ for financial support and
Luciana Consentino for technical support in cell culture.
5. REFERENCES
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homeostasis. J Bone Miner Res. 2010
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AM Adsorption and bioactivity studies of albumin onto hydroxyapatite surface.
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