Received: 6 December 2019 Accepted: 11 February 2020

DOI: 10.1002/jbm.b.34591

ORIGINAL RESEARCH REPORT

In vitro biocompatibility of biohybrid polymers membrane

evaluated in human gingival fibroblasts

Bin Guo1,2 | Chuhua Tang2,3 | Mingguo Wang1 | Zhongqi Zhao2 |

Hassan A. Shokoohi-Tabrizi2 | Bin Shi4 | Oleh Andrukhov2 | Xiaohui Rausch-Fan2,4

1
Department of Stomatology, Jinan Central
Hospital affiliated to Shandong University, Abstract
Jinan, Shandong, China The biohybrid polymer membrane (BHM) is a new biomaterial designed for the treat-
2
Division of Periodontology and Conservative
ment of soft periodontal tissue defects. We aimed to evaluate the in vitro biocompat-
Dentistry, University Clinic of Dentistry,
Medical University of Vienna, Vienna, Austria ibility of the membrane in human gingival fibroblasts and the capability to induce cell
3
Department of Stomatology, PLA Strategic adhesion, migration, differentiation and improving the production of the extracellular
Support Force Characteristic Medical Center,
Beijing, China matrix. BHM and Mucograft® collagen matrix (MCM) membranes were punched into
4
Department of Oral and Maxillofacial Surgery, 6 mm diameter round discs and placed in 96-well plates. Human primary gingival
The First Affiliated Hospital of Fujian Medical
fibroblasts were seeded on the membranes or tissue culture plastic (TCP) serving as
University, Fuzhou, Fujian, China
the control. Cell proliferation/viability and morphology were evaluated after 3, 7, and
Correspondence
14 days of culture by cell counting kit (CCK)-8 assay and scanning electron micros-
Oleh Andrukhov, Division of Conservative
Dentistry and Periodontology, Medical copy, respectively. Additionally, the gene expression of transforming growth factor
University of Vienna, Sensengasse 2a, A-1090
(TGF)-β1, focal adhesion kinase (FAK), collagen type 1 (Col1), alpha-smooth muscle
Vienna, Austria.
Email: [email protected] actin (α-SMA), and fibroblasts growth factor (FGF)-2 was analyzed at 3, 7, and
14 days of culture by qPCR. Cell proliferation on BHM was significantly higher than
Funding information
Fujian Science and Technology Bureau on MCM and similar to TCP. Gene expression of TGF-β1, FAK, Col1, and α-SMA
International Cooperation, Grant/Award
were significantly increased on BHM compared to TCP at most investigated time
Number: 20170003
points. However, the gene expression of FGF-2 was significantly decreased on BHM
at Day 7 and recovered at Day 14 to the levels similar to TCP. The finding of this
study showed that BHM is superior for gingival fibroblasts in terms of adhesion, pro-
liferation, and gene expression, suggesting that this membrane may promote the
healing of soft periodontal tissue.

KEYWORDS

adhesion, biohybrid polymers membrane, differentiation, extracellular matrix, gingival

fibroblasts, migration, proliferation

1 | I N T RO DU CT I O N soft tissue recovery. To date, soft tissue augmentation using autolo-

gous tissue grafts is the first choice of use in periodontal surgery
Regeneration of periodontal soft tissue is one of the main challenges (Bertl, Melchard, Pandis, Muller-Kern, & Stavropoulos, 2017; Zuhr,
of contemporary periodontology and implantology (Larsson et al., Baumer, & Hurzeler, 2014). Despite the good clinical outcome, autolo-
2016). Conservative periodontal therapy alone is often insufficient for gous tissue grafts have some obvious disadvantages, such as the

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
© 2020 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc.

2590 wileyonlinelibrary.com/journal/jbmb J Biomed Mater Res. 2020;108B:2590–2598.

GUO ET AL. 2591

limited size, the necessity for the additional surgical site, pain to and play a crucial role in the regeneration of soft tissue defects
patients, damage risk of the palatal artery, and the differences in tex- (Andrukhov, Behm, Blufstein, & Rausch-Fan, 2019; Smith, Martinez,
ture and color with adjacent tissues (Pietruska, Skurska, Podlewski, Martinez, & McCulloch, 2019). Therefore, in the present study, we
Milewski, & Pietruski, 2019). Therefore, researchers and clinicians tested the biological behavior of commercially produced BHM on pri-
have been exploring alternative tissue renewing materials “outside the mary human gingival fibroblasts, expression of some differentiation
palate”, and periodontal gingival surgery using xenogeneic materials or markers as well as its capability to induce production of ECM. The bio-
synthetic materials instead of autologous tissue has gradually devel- logical response of gingival fibroblasts to BHM was compared with
oped (Vaquette et al., 2018). those to Mucograft, a 3D collagen matrix developed especially for soft
Modern periodontal tissue regeneration is inconceivable without tissue regeneration (Nevins, Nevins, Kim, Schupbach, & Kim, 2011).
the application of different biomaterials like scaffolds or barrier mem-
branes. The principles underlying the application of biological barrier
membranes are the physical separation of different tissue compart- 2 | M A T E R I A L S A N D M ET H O D S
ments (Sheikh et al., 2017). Such separation allows optimizing the
healing process of different tissues with distinct properties and mini- 2.1 | Specimen preparation
mizing the disturbing effects of neighboring tissues. The barrier mem-
branes could be either nonresorbable or resorbable. The advantage of BHM was designed by Freiburg Materials Research Center and Insti-
the resorbable membrane is that they are degrading with time, which tute for Macromolecular Chemistry of the Albert-Ludwigs University
allows reconstruction of natural tissue structures. Freiburg similar to the methods described previously (Strassburg et al.,
The requirements of clinicians for the barrier membrane became 2019). In the present study, commercially produced BHM (Neo Modu-
more rigorous within the last years. Modern membranes are expected lus [Suzhou] Medical, Jiangsu, China) was used. BHM and Mucograft®
not only to perform a barrier function but also to stimulate the natural collagen matrix (MCM, Geistlich Biomaterials, Wolhusen, Switzerland)
process of wound healing. This can be achieved by modification of were cut into 6 mm diameter round with the help of a hole-punch,
different properties such as the elasticity, three-dimensional and were sterilized by UV light for 45 min each side.
(3D) structure of the membrane and incorporation of various bioactive
molecules (Chen et al., 2018; Omar, Elgali, Dahlin, & Thomsen, 2019).
A recently developed nonwoven-based gelatin membrane (NBM) is a 2.2 | Cell culture
new type of synthetic material produced by electrospinning technol-
ogy from gelatin (Schulz et al., 2014). Gelatin has several advantages Human gingival fibroblasts (hGFs) were isolated from gingival tissue
such as excellent biocompatibility, easy processing, low cost, and obtained from five periodontally healthy donors during routine extrac-
appears to be a promising candidate in clinical applications (Rose tion of their third molar teeth. The research protocol was approved by
et al., 2014). NBM membranes are produced by electrospinning with the Ethics Committee of the Medical University of Vienna. All donors
in situ cross-linking resulting in the formation of gradient fibrillary were systematically healthy ranging in age from 22 to 28 years. All
structures. These 3D structures closely mimic the native extracellular participants were informed in detail before the operation and gave
matrix (ECM) to promote cell growth and differentiation following the their written consent. Cells were cultured in Dulbecco's modified
patterns similar to those found in native tissues and organs. NBM can Eagle's medium (DMEM) supplemented with 10% FBS (fetal bovine
be further modified by combining the gelatin gradient layer with the serum), streptomycin (50 μg/ml) and penicillin (100 U/ml) under
electrospun layer of polycaprolactone (PCL) and such biohybrid poly- humidified air atmosphere with 5% CO2 at 37 C. hGFs from passage
mer membranes (BHM) possess superior mechanical properties in the levels 4–7 were used in the experiments.
aqueous environment (Angarano et al., 2013; Schulz et al., 2014).
One of the important requirements of regenerative biological
material is the ability to stimulate the formation of new tissue and 2.3 | Cell proliferation/viability
repair existing defects. On the cellular level, modern biological mate-
rial should stimulate the migration of resident progenitor cells to the Cell proliferation/viability was measured using a cell counting kit
healing area, their proliferation, and differentiation into a mature (CCK-8, Dojindo Laboratories, Japan) as previously described
tissue-specific phenotype, as well as the production of new ECM (Andrukhov et al., 2015). In these experiments, 1 × 104 cells were
(Boehler, Graham, & Shea, 2011; Nisbet, Forsythe, Shen, Finkelstein, & seeded on either BHM or MCM (the porous layer facing up) mem-
Horne, 2009; Skoog, Kumar, Narayan, & Goering, 2018). Some recent brane in 200 μl DMEM. Cells seeded at the same density on tissue
preclinical and histological studies with BHM have shown the unique culture plastic (TCP) were used as control. Cell proliferation/viability
physical and chemical properties and biological compatibility in vivo was measured after 3, 7, and 14 days of culture. Then, 20 μl of CCK-8
and in vitro (Jedrusik et al., 2018), but studies highlighting the poten- reagent was added into each well and the culture plates were incu-
tial to induce cellular processes and ECM synthesis compared with bated in 5% CO2 at 37 C for 2 hr. Thereafter, 100 μl of each culture
other commercial membranes on the market are incomplete. Gingival solution was transferred to a separate 96-well plate and the optical
fibroblasts are the major resident progenitor cells of gingival tissue density (OD) was measured at 450 nm using a microplate reader
2592 GUO ET AL.

(Synergy HTX; BioTek) at 450 nm. The experiments were repeated 2.6 | Statistical analysis
five times with the cells isolated from five different donors.
Cell viability was further visualized using the LIVE/DEAD cell The statistical differences between different groups were analyzed by
Staining Kit (Enzo Life Sciences AG, Lausen, Switzerland). Then, one-way analysis of ANOVA's statistic or Wilcoxon-Test. All statistical
1 × 104 cells were seeded on either BHM or MCM (the porous layer analysis was performed using the statistic program SPSS 21.0 (SPSS,
faces up) membrane in 200 μl of DMEM. After 1, 3, and 7 days of cul- Chicago, IL). Data are expressed as means ± SEM. Differences were

ture, the cells were stained with 100 μl of staining solution at 37 C considered to be statistically significant at p < .05.
for 15 min and observed under a fluorescence microscope immedi-
ately after the staining.
3 | RE SU LT S

2.4 | Scanning electron microscopy analysis 3.1 | Proliferation/viability of hGFs grown on

BHM and MCM
The morphology and microstructure of hGFs grown on BHM and
MCM were analyzed using scanning electron microscopy (SEM; Shi Proliferation/viability of primary hGFs grown on BHM, MCM, and
et al., 2017). hGFs were seeded on the BHM at a density of 1 × 104 in TCP group after different culture times are summarized in Figure 1. At
96-well-plate and cultured at 37 C as described above. Specimens in all investigated time points (3, 7, and 14 days), proliferation/viability
each group were scanned under SEM at 3, 7, and 14 days. For SEM, of hGFs grown on MCM was significantly lower compared to those
the specimens were fixed with 4% formaldehyde at least 24 hr and grown on BHM and TCP (p < .05). No statistically significant differ-
washed three times with PBS to remove unattached cells. Then, the ence in proliferation/viability was observed between hGFs grown on
specimens were dehydrated by rinsing with gradually increased etha- BHM and TCP (p > .05). Proliferation/viability of hGFs grown on
nol. Afterward, ethanol was exchanged by hexamethyldisilazane BHM and TCP was gradually increased with prolonged culture time.
(HMDS, Sigma-Aldrich), the specimens were coated with gold and In contrast, hGFs grown on MCM exhibited similar OD values after all
observed under the scanning electron microscope (SEM; JEOL-JSM IT observation time points suggesting no proliferation.
300, JEOL, Tokyo, Japan) at an accelerating voltage of 15 kV. The Live/dead staining of hGFs grown on showed that most cells
SEM images of cross-sectional and surface views were acquired. were viable, dead cells were not observed (Figure 2).

2.5 | Gene expression analysis 3.2 | SEM analysis

hGFs were seeded on the BHM at a density of 1 × 104 in 96-well- SEM images showing structural features of two membranes are
plates in 200 μl DMEM. Cells seeded at similar density on TCP were shown in Figure 3. Although MCM and BHM are both multilayered
used as control. After 3, 7, and 14 days of culture, the total mRNA
was isolated using Cells-to-CT Bulk Lysis Reagents (Invitrogen, Carls-
bad, CA) according to the manufacturer's instructions as previously
described (Behm et al., 2019; Blufstein et al., 2019). mRNA samples
were reversely transcribed into cDNA using the Cells-to-CT Bulk RT
reagent (Ambion/Applied Biosystems, Foster City, CA). Quantitative
real-time PCR (qRT-PCR) was performed in ABI StepOnePlus device
(Applied Biosystems, Foster City, CA) using the Taqman gene expres-
sion assays (Applied Biosystems, Foster City, CA) with the following
ID numbers: GAPDH, Hs99999905_m1; Type I collagen (Col1),
Hs00164004_m1; Transforming growth factor β1 (TGF-β1),
Hs00998133_m1; Focal adhesion kinase (FAK), Hs00169444_m1;
Fibroblast growth factor-2 (FGF-2), Hs00266645_m1; α-smooth mus-
cle actin (α-SMA), Hs00909449_m1. qRT-PCR reactions were per-
formed in 96-well plates using the following thermocycling F I G U R E 1 Proliferation/viability of primary human gingival
conditions: 95 C for 10 min, 50 cycles, each for 15 s at 95 C and at fibroblasts grown on different membranes. Human gingival fibroblasts
60 C for 60 s. The point at which the PCR product was first detected were seeded on biohybrid polymer (BHM), Mucograft® collagen
matrix (MCM), and TCP and their proliferation/viability were tested
above a fixed threshold (termed cycle threshold, Ct) was determined
using CCK-8 test at 3, 7, and 14 days. Data are presented as means ±
for each sample. Changes in the expression of target genes were cal-
SD of OD values (450 nm) measured in five independent experiments
culated by a 2−ΔΔCt method using the following formula: with cells of five different donors. One-way ANOVA (n = 5),
ΔΔCt = (Ct target
− CtGAPDH)sample − (Cttarget − CtGAPDH)control. Difference between groups are indicated by: *p < .05, ** p < .01
GUO ET AL. 2593

structures, obvious differences were detected between them. The layer of MCM. The pore size of BHM surface is visibly smaller than
outer two layers of BHM are gelatin prepared by electrospinning, and that of MCM (Figure 3c,d).
the middle layer is PCL layer (Figure 3a). MCM is composed of a com- Representative SEM pictures of hGFs grown on BHM and MCM
pact layer and a spongy layer (Figure 3b). Compared to BHM, the after different periods are presented in Figure 4. On BHM, after
double-layer matrix of MCM is markedly thicker and upholds a nearly 3 days hGFs were attached to the membrane and exhibited typical
three-fold volume. The BHM surface is smoother than the spongy fibroblast morphology (Figure 4a). After 7 days of culture, the cells
were nearly confluent and covered almost the whole membrane sur-
face (Figure 4b). After 14 days, some multilayered cell structures could
be observed (Figure 4c). On MCM, cells were hardly observed
throughout the whole observation period (Figure 4d–f).

3.3 | Gene expression analysis

Since MCM did not support the growth of HGFs, gene expression anal-
ysis was limited only to BHM group. The expression of several genes in
hGFs grown on BHM in comparison with TCP after 3, 7, and 14 days of
culture is shown in Figure 5. We focused on the expression of FGF-2,
TGF-β1, FAK, Col1, and α-SMA, which are involved in the process of
soft tissue formation. The expression of FGF-2 in BHM group was
lower compared to TCP group after 7 days (p < .05, Figure 5a), but after
14 days FGF-2 expression was similar in both groups. The expression
of other investigated genes was generally higher in hGFs grown on
F I G U R E 2 Live-dead staining of human gingival fibroblasts grown BHM compared to those grown on TCP at all time points. Significant
on BHM for 7 days differences were observed for TGF-β1 after 7 and 14 days (p < .01,

F I G U R E 3 SEM analysis of BHM

and MCM. Microscopic structural
characteristics of MCM and BHM
observed at different projections.
Panels (a) and (b) show the cross-
sections of the analyzed materials
(a = BHM) and (b = MCM) and panels
(c) and (d) show the spongy surface of
BHM and MCM surface separately.
Scale bar is given for each picture
2594 GUO ET AL.

F I G U R E 4 Scanning electron microscopy analysis of hGFs grown on BHM and MCM. HGFs after (a, d) 3, (b, e) 7, and (c, f) 14 days of culture.
Scale bar = 200 μm

Figure 5b), for FAK at all time points (p < .01, Figure 5c), for α-SMA and physicochemical properties might be useful for the achievement of
Col1 after 3 and 7 days (p < .01, Figure 5d, e). the regeneration of highly specific gingival soft tissue through stimula-
tion of the resident progenitor cells such as gingival fibroblasts.
In our study, we compared the surface characteristics of BHM
4 | DISCUSSION and MCM and their effects on the functional properties of primary
hGFs. BHM is a newly developed membrane composed of gelatin and
The purpose of this study was to evaluate the in vitro biocompatibility polycaprolactone. On the one hand, it serves as a barrier membrane
of BHM in hGFs and the capability to support cell adhesion and prolif- but on the other hand, its surface has a 3D structures composed of
eration as well as the ability to influence the expression of some fac- cross-linked nanofibers serving as scaffold (Jedrusik et al., 2018).
tors involved in gingival tissue regeneration and ECM production. MCM is one of the types of resorbing porcine xenogeneic materials,
hGFs are the most common gingival progenitor cells and play an which is a pure collagen type I and type III matrix obtained with a
important role in repairing tissue damage and maintain periodontal standardized, controlled manufacturing process without any cross-
health (Andrukhov et al., 2019; Smith et al., 2019). Modern biomate- linking or chemical treatment. It consists of two functional layers: one
rials designed for periodontal soft tissue regeneration are expected to thin, smooth, low-porosity compact layer and one thicker, porous, 3D
stimulate the adhesion, migration, and proliferation of hGFs as well as spongy layer (Ghanaati et al., 2011). The matrix is a resorbable 3D
production of specific factors involved in the processes of gingival matrix designed specifically for soft tissue regeneration in the oral
connective tissue formation and remodeling (Cao, Wang, Pu, Tang, & cavity and for the replacement of autologous grafts (Carter et al.,
Meng, 2018). Physical and mechanical features of scaffold such as 2017; Menceva et al., 2018; Ramachandra, Rana, Reetika, & Jithendra,
material, topography, rigidity, and porosity substantially influence dif- 2014; Schmitt & Moest, 2016). In clinical application, the compact
ferent cellular characteristics and subsequently the regenerative pro- layer contacts the epithelial cells outward, and the porous layer con-
cesses (James, Levene, Parsons, & Kohn, 1999; Stevens & George, tacts the fibroblasts inward, so we choose the spongy layer of MCM
2005; Yeung et al., 2005). Therefore, specific modifications of scaffold membrane as the surface for cell culture.
GUO ET AL. 2595

F I G U R E 5 Gene expression analysis of hGFs grown on BHM. Expression of FGF-2, TGF-β1, FAK, Col1, α-SMA in hGFs grown on BHM at
3, 7, and 14 days: (a) FGF-2; (b) TGF-β1; (c) FAK; (d) Col1; (e) α-SMA. Y-axis represents the n-fold expression levels of the target gene in relation
to cells in TCP group (control) calculated by 2−ΔΔCt method. Data are presented as mean ± SEM of five independent experiments with hGFs of
five different donors (n = 5), *significant difference between groups as tested by Wilcoxon test (p < .05)

The results of CCK-8 showed that the cell viability/proliferation Although in our study hGFs did not grow on MCM membrane, clinical
of hGFs grown on BHM and TCP was gradually increased up to studies suggest its positive effect on soft tissue regeneration (Carter
14 days of culture. As can be seen on the SEM images, the density of et al., 2017; Menceva et al., 2018; Ramachandra et al., 2014; Schmitt &
cells growing on BHM was gradually decreased until the membrane Moest, 2016). Therefore, we can conclude that the biological effects
was fully covered with hGFs monolayer. Live/Dead staining showed of MCM are explained by other mechanisms that stimulate progenitor
that BHM had no cytotoxic effect on the hGFs during the whole cul- cells proliferation.
ture period. Thus, we can conclude that BHM shows excellent bio- Ideal bioresorbable material for oral surgery should provide not
compatibility and supports the proliferation of primary hGFs. These only cell attachment and proliferation, but also influence the synthe-
observations are in agreement with previous reports, showing that sis, transport, and secretion of extracellular matrix protein and growth
nonwoven-based gelatin membranes support attachment and viability factors. Immediately after surgical procedure, resident progenitor cells
of different cell types, and particularly hGFs (Schulz et al., 2014; are attracted by various clot-derived factors into the wound area,
Strassburg et al., 2019). where they adhere to temporary scaffold and start to proliferate.
In contrast, the viability/proliferation of cells on MCM was not HGFs start to produce different growth factors and ECM proteins
changed during 14 days. Surprisingly, we observed that collagen- required for new tissue formation. The process of new tissue forma-
based Mucograft membrane does not support the proliferation of pri- tion is complicated and largely determined by the continuous dynamic
mary hGFs. This observation seems to contradict to a former study interaction of HFGs with ECM. ECM influences the behavior and
showing a gradual proliferation of hGFs in the presence of MCM activity of HGFs, which secrete new proteins participating in ECM for-
(Lima et al., 2015). The reasons for the contradictory observations mation and remodeling. The contacts of cells with ECM are mediated
could be some differences in the protocols between these studies. In by focal adhesion (FA) complex, which is an assembly of different pro-
our study, the pieces of membranes fit the dimensions of the cell cul- teins involved in the adhesion to ECM and subsequent activation of
ture well, so that that seeded hGFs grew mainly on membranes and various intracellular signaling pathways FAK, a nonreceptor protein
not on TCP. In contrast, the size of the membrane used in a previous tyrosine kinase, localizes to integrin-rich cellular FA sites (Fischer,
study was <50% of the well diameter (Lima et al., 2015), suggesting Wong, Baruth, & Cerutis, 2017). The autophosphorylation of FAK
that the cells grew without direct contact with the membrane. is critical for the regulation of adhesion enhancement (Michael,
2596 GUO ET AL.

Dumbauld, Burns, Hanks, & Garcia, 2009). We have observed that We can assume that FGF-2 expression can play a more important role
FAK expression in hGFs on BHM was significantly increased com- in the later phases of the regenerative process such as tissue matura-
pared to TCP at the initial phase of culture, which can be explained by tion and remodeling. Therefore, decreased expression of FGF-2 during
the fact that BHM is a scaffold with a 3D structure. This assumption the first proliferative phase could be physiologically important, but this
is in line with a former observation that the expression of the proteins question needs to be further explored. One study shows that at higher
implicated in the FA in gingival fibroblast grown on 3D gelatin amounts, FGF-2 might inhibit the proliferation of hGFs (Ma et al.,
nanofibrous scaffold is significantly increased compared to the 2D 2012). Besides, there is a reciprocal relationship between FGF-2 and
plastic surface (Sachar et al., 2014). One recent study implies that TGF-β1 signaling (Liguori, Liguori, Moreira, & Harmsen, 2018). Further-
hGFs exhibited lower expression of FAK compared to dermal fibro- more, FGF-2 decreases the expression of α-SMA and inhibits
blasts, and this was associated with the decreased spreading and myofibroblast differentiation (Akasaka et al., 2007, 2010), which is in
adhesion ability (Guo, Carter, Mukhopadhyay, & Leask, 2011). Thus, agreement with the expression pattern observed in our study.
our data suggest that BHM membrane due to its structural and mate- There are still some limitations in our experiments. First, we only
rial characteristics support adherence of hGFs. The expression of FAK chose HGFs as experimental cells, but the gingival soft tissue is a
in hGFs on BHM was decreased after 14 days. At this time point, cells mixture of cells. As shown by recent reports, gelatin-based mem-
that grew on BHM already reached confluence and started to form brane affects epithelial cells as well as their communication with gin-
some multilayered structures. A decreased direct contact of hGFs gival fibroblasts (Jedrusik et al., 2019; Strassburg et al., 2019).
with BHM after prolonged culture and confluence could be associated Second, we could not imitate the complex oral microenvironment
with a decreased FAK expression. in vitro study. Therefore, when trying to translate our results to
Previous studies have shown that elevated FAK expression finally clinical application, it should be quite cautious and further
induces the expression of α-SMA, which plays an important role in studies are still necessary.
mechanotransduction and tissue remodeling (Guo, Carter, & Leask,
2014). Furthermore, the expression of Col1, which is the most impor-
tant component of the ECM, is also be enhanced by FAK (Cheung, 5 | CONC LU SIONS
McCulloch, & Santerre, 2014). The expression of both α-SMA and Col1
in hGFs grown on BHM was substantially increased after 3 and 7 days, In summary, BHM stimulates HGFs adhesion, migration, and differen-
which could be associated with an increased FAK expression. High tiation in vitro. It decreases the gene expression of FGF-2, increases
expression of α-SMA and Col1 suggests an increased production of new the gene expression of FAK and TGF-β1, then enhances the gene
ECM and improved ECM remodeling. After 14 days, when FAK expres- expression Col1 and α-SMA. These data provide the first scientific
sion was decreased, we did not observe any significant differences in evidence to support the BHM as a potential material could be used in
α-SMA and Col1 expression between BHM and TCP. This observation soft tissue augmentation.
supports our assumption about the association between FAK expression
on the one hand and α-SMA and Col1 expression on the other hand. ACKNOWLEDG MENT
The other important read-outs of our study were TGF-β1 and a The authors are grateful to Mrs. Phuong Quynh Nguyen for her excel-
member of fibroblast growth factors family protein FGF-2. These lent technical assistance. All BHM investigated in this study were
growth factors play an important role in the regeneration of periodontal manufactured and provided by the Amor (Suzhou) Medical Sci-Tech.
tissue, particularly bone defect (Kitamura et al., 2011; Maeda, Wada, The study was supported by the Fujian Science and Technology
Tomokiyo, Monnouchi, & Akamine, 2013; Ripamonti, 2019). Interest- Bureau International Cooperation project Nr. 20170003.
ingly, these growth factors were differently regulated by in hGFs grown
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