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We created an anisotropic material based on collagen sponge and reactive polylactide structured by laser photopolymerization. The combination of collagen with reactive polylactide improves the resistance of the formed matrices to biodegradation in comparison with collagen sponge, while the existence of sites with different mechanical characteristics and cell affinity on the matrix provides directed cell growth during their culturing. It was shown that reinforcement of the collagen sponges 7-fold increased the mean Young’s modulus for the hybrid matrix without affecting its cytotoxicity. The developed matrix provides cell adhesion and proliferation along reinforcement lines and can be used for fabrication of tissue engineering constructs.
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0007 -4888/18/16510142 © 2018 Springer Science+Business Media, LLC
Reinforced Hybrid Collagen Sponges for Tissue Engineering
K. N. Bardakova
, E. A. Grebenik
, E. V. Istranova
, L. P. Istranov
Yu. V. Gerasimov
, A. G. Grosheva
, T. M. Zharikova
, N. V. Minaev
B. S. Shavkuta
, D. S. Dudova
, S. V. Kostyuk
, N. N. Vorob’eva
V. N. Bagratashvili
, P. S. Timashev
, and R. K. Chailakhyan
Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 1, pp. 31-37, January, 2018
Original article submitted December 11, 2017
We created an anisotropic material based on collagen sponge and reactive polylactide struc-
tured by laser photopolymerization. The combination of collagen with reactive polylactide
improves the resistance of the formed matrices to biodegradation in comparison with col-
lagen sponge, while the existence of sites with different mechanical characteristics and cell
afnity on the matrix provides directed cell growth during their culturing. It was shown that
reinforcement of the collagen sponges 7-fold increased the mean Young’s modulus for the
hybrid matrix without affecting its cytotoxicity. The developed matrix provides cell adhesion
and proliferation along reinforcement lines and can be used for fabrication of tissue engineer-
ing constructs.
Key Words: collagen matrix; laser photopolymerization; directed cell growth; branched
polylactide; multipotent stromal cells
1Institute of Photonics Technologies, Federal Research Centre “Crys-
tallography and Photonics”, Russian Academy of Sciences; 2Institute
of Regenerative Medicine, I. M. Sechenov First Moscow State Medi-
cal University, Ministry of Health of the Russian Federation; 3N. F.
Gamaleya National Research Center of Epidemiology and Microbio-
logy, Ministry of Health of the Russian Federation, Moscow, Russia;
4Research Institute for Physical Chemical Problems, Belarusian State
University, Minsk, Belarus. Address for correspondence: ruben- R. K. Chailakhyan
The main requirements for tissue engineering matrices
include among other things biocompatibility, ability to
promote cell adhesion and proliferation, optimal rate
of biodegradation, and mechanical properties of native
tissue [4]. The materials based on collagen, the main
organic component of the intercellular matrix, largely
meet the above requirements and are of great interest.
Collagen scaffolds can be obtained by either chemi-
cal, mechanical, or enzymatic removal of cells from
tissues (decellularization) or extraction of collagen
from different tissues followed by creation of collagen
structures from the solution [3]. The second method
is used for preparing hydrogels, lms, and sponges
that are often characterized by relatively high rate of
biodegradation and insufcient mechanical strength
for their application in surgery. These problems can be
solved by proper choice of collagen type, variation of
collagen concentration, structuring by cross-linking [5],
spatial ordering of collagen bers [7], and modication
with synthetic polymers and inorganic agents [6].
For a number of medical tasks (e.g. muscle tis-
sue repair), the use of traditional biomaterials is also
limited due to their insufcient mechanical strength
and elasticity. These properties can be improved by
different methods. For instance, a hybrid scaffold was
prepared on the basis of collagen sponge treated with
glutaraldehyde and reinforced with polylactide bers
[10] Another strategy of modication is based on in-
clusion of bio-functional materials inducing certain
biological processes and improving mechanical prop-
erties of the original collagen matrix. For instance, hy-
droxyapatite nanoparticles were used as bioactive and
reinforcing additives [8]. Collagen scaffolds formed
by different methods can also be modied by different
polymer coatings. Thus, alginate coating of the col-
lagen scaffold formed by extrusion printing allowed
increasing Young’s modulus by 9 times [9]. The use
Cell Technologies in  Biology  and  Medicine,  No.  1,  May,  2018
DOI 10.1007/s10517-018-4116-8
of polylactide mesh also improved mechanical char-
acteristics of collagen materials. The prospects of us-
ing such hybrid scaffolds based on collagen/gelatin
sponges for skin regeneration were discussed [11].
Reinforcement of the collagen sponge intended for
epithelial tissue repair with commercial polylactide
meshes was reported to increase scaffold resistance to
in vivo biodegradation [1].
Our aim was to create an anisotropic material
based on collagen sponge and reactive polylactide
structured by laser photopolymerization. Reinforce-
ment with polylactide is assumed to reduce the rate
of biodegradation [13,14] and stimulate directed cell
proliferation along the polylactide bers followed by
uniform population of the matrix.
Preparation of the collagen sponge. The collagen
sponge was prepared from collagen solution (0.7 mg/ml;
pH 2.9) by the method of freeze-drying. For preparing
collagen solution, the middle layer of cattle dermis
was cut into 5×5 cm pieces that were incubated in
2.5 M NaOH with 0.85 M Na2SO4 for 48 h at 20oC
with periodic mixing. After 6-h washing in 0.85 M
Na2SO4, the fragments were incubated with 4% boric
acid until complete alkali neutralization on section
(qualitatively assessed by the reaction with phenol-
phthalein). Neutralized samples were washed with
distilled water until negative reaction for the pres-
ence of sulphate ions, after which they were placed in
0.5 M acetic acid to complete dissolution. The resultant
solution was puried by collagen precipitation with
12% NaCl followed by centrifugation at 3000 rpm for
20 min; the pellet was then dissolved in 0.25 M acetic
acid. To remove salts, the solution was dialyzed for
24 h against 0.25 M acetic acid. The collagen solution
brought to a concentration of 0.7 mg/ml was trans-
ferred to cuvettes with (layer thickness 3 mm), frozen
at -30oC, and lyophilized.
Preparation of photosensitive composition.
To obtain photoactive material, 800 µl of 50 wt%
branched polylactide solution (M(NMR)=749 g/mol
for one beam; proportion of reagents during synthesis:
[D,L-lactide]/[PE]=20/1; detailed synthesis procedure
described in [12]) was mixed with 3600 µl dichloro-
methane and 40 mg of photoinitiator 4,4’-bis (diethyl-
amino)benzophenone, the solution was mixed for 24 h
and then used for laser structuring.
Experiment on laser photopolymerization. The
collagen sponge (a square fragment with a side of 15
mm and thickness 2-4 mm) was evenly coated with
photosensitive composition (500 µl). In 10 min, the
excess of photoactive liquid was removed from super-
cially dry collagen sponge (to this end, the sponge
was squeezed for 2-3 sec between the coverslips);
then, a metal mold with a square window was placed
on top of the collagen sponge to x the scaffold on
the slide.
The reinforcement was carried out using a UV di-
ode laser at λ=405 nm and maximum power 100 mW.
Laser radiation was focused directly on the sample, the
diameter of the laser spot was 60-80 μ. Reinforcing
lines were formed at the following radiation param-
eters: laser power from 50-70 mW, scanning speed
on the surface of the sample 3-5 mm/sec, the distance
between the centers of neighboring lines varied from
200 to 250 μ. This laser treatment allowed reinforcing
the collagen lm without damaging its structure.
Removal of polymer excess. Immediately af-
ter structuring, the reinforced collagen scaffold was
placed in dichloromethane for 2-3 days (the solvent
was changed 3-4 times) and then washed with ethyl
alcohol (95 vol%) and deionized water for 7-10 days.
Characterization of hybrid scaffolds. The scaf-
fold morphology was studied by scanning electron
microscopy Phenom Pro X (Phenom). Collagen struc-
ture integrity after laser exposure was studied by re-
ectance IR spectroscopy on a Spectrum 100 FT-IR
spectrometer (Perkin Elmer).
Nanoindentation. The local mechanical proper-
ties of the prepared collagen sample were determined
using a Piuma nanoindenter (Optics11) that included
a controller, optical ber, and spherical probe used
for construction of the force—displacement curves.
The probe was xed to a exible beam of the canti-
lever. The contact of the cantilever with the sample
surface led to its deection that was measured inter-
ferometrically using an optical ber. To determine
Young’s modulus of the collagen scaffolds, the probe
was forced 5 µ deep into the sample at each measure-
ment point. The Young’s modulus was calculated from
the force—displacement curve using Piuma applied
software and Hertzian model for a sphere in contact
with a plane.
To study mechanical characteristics of colla-
gen scaffolds, a cantilever with a spring constant of
0.45 N/m and tip radius 26.5 μ was used. The samples
were xed on the bottom of a Petri dish. All mea-
surements were carried out in distilled water at 37oC.
During measurements, the cantilever probe always re-
mained in the liquid medium to minimize errors due to
adhesion forces at the air—water interface. The area for
Young’s modulus distribution maps was 1000×500 µ
with 25 µ increments along X and Y axes. Based on
the results of measurements, the effective Young’s
modulus was determined and the maps of Young’s
modulus distribution on the surface were constructed.
MTT test. 3T3 mouse broblasts were seeded
on a 96-well plate (5000 cells per well) and cultured
K. N. Bardakova,  E. A.  Grebenik, et al.
in a CO2 incubator at 37oC in DMEM/F-12 (PanEco)
supplemented with glutamine and 10% fetal calf serum
(FCS; HyClone). In 24 h, the culture medium was
replaced with successive dilutions of extracts of the
original collagen sponge and hybrid scaffold prepared
in a culture medium containing 5% FCS. The extracts
were obtained by incubating the samples (total area
6 cm2 each) in culture medium in a CO2 incubator at
37oC for 24 h. Sodium dodecyl sulfate in a concentra-
tion range of 0.013-0.500 mg/ml was used as the posi-
tive control. The sponges were preliminary sterilized
in 70% ethanol for 30 min at constant stirring, then
washed three times.
Isolation of human bone marrow mesenchymal
stromal cells (MSC). Human bone marrow was ob-
tained from a maxillofacial surgical clinic; informed
consent of the patient was obtained. The bone mar-
row specimen was transferred to a vial with fresh nu-
trient medium, suspended with a syringe, and ltered
through a 4-layer capron lter; the total number of
cells was counted. The cells were seeded into 80-cm2
plastic asks (Nunc) with 15 ml complete culture me-
dium containing 80% α-MEM (Sigma-Aldrich), 20%
FCS (HyClone), and antibiotics (100 U/ml penicillin
and 100 mg/ml streptomycin); seeding density 3.5-
4.0×104 cells/cm2. The cells were cultured at 37oC
and 5% CO2. The cells were rst passaged on days
12-14 of culturing, when discrete colonies of stro-
mal broblasts appeared, and then, upon attaining
MSC culturing in scaffolds. For studying cell
adhesion and growth on the hybrid scaffold, passage
4 human bone marrow MSC were used. The scaffold
(reinforced collagen) was preliminary sterilized and
placed in the medium. Fragments 3×3 mm were dried
with a tampon for complete medium removal. Then,
the fragments were treated with FCS for 15 min and
then soaked with the tampon. The fragments were
placed onto the bottom of 25 cm2-culture ask tightly
to each other. The total area of this construction was
~1 cm2. The scaffold was lled with cells suspension
(concentration 106 in 0.5 ml culture medium) using a
syringe. The cells adhered to plastic for 1.5 h at 37oC
and 5% CO2. Then, the total volume of the medium
supplemented with serum was added to the culture
ask and the cells were cultured for 2 months; the
medium was changed regularly.
Confocal and light microscopy. Population
of the hybrid scaffold by cells was assessed using
a Nikon A1 Multiphoton confocal microscope and
Nikon Eclipse TS 100) light inverted microscope.
For confocal microscopy, the cells were fixed with
4% formalin and the nuclei were poststained with
ethidium bromide homodimer (Thermo Fisher Sci-
At the rst stage of the study, the regimens for form-
ing reinforcing strips of reactive polylactide on the
collagen sponge were chosen. The parameters of laser
exposure were as follows: power 70 mW, laser beam
speed 5 mm/sec, density 4 lines per mm. These condi-
tions prevented overheating of the collagen material
under the action of laser radiation and reinforced colla-
gen scaffolds after structuring retained exibility typi-
cal of collagen sponge. Reinforcement did not change
porosity of the collagen scaffold, but the proportion of
free pores in the material decreased (Fig. 1, a, b). At
the same time, the structured scaffolds retained their
conguration and manipulative properties after 2-week
incubation in PBS, in contrast to the original collagen
sponge that considerably swelled and disintegrated to
separate bers during this time.
This change in the physical properties of the scaf-
fold (reduced swelling in the solvent and reduced per-
meability) attests to increased concentration of cross-
links and reorganization of the 3D network. The length
of polylactide lines was 70-80 μ and the distance be-
tween them 140-150 µ.
Possible changes in the collagen structure after
laser exposure were assessed by IR spectroscopy. On
both IR spectra of collagen (Fig. 1, c), we observed
the appearance of typical oscillation frequencies of
hydroxyl groups (3200-3700 cm—1; not shown on our
spectra) and peptide bonds, amides I, II and III (1200-
1700 cm—1). During collagen denaturation, its triple
helix becomes unwound and forms random peptide
coils, which should affect uctuations of amides I
and III most sensitive to such conformational transi-
tions. Unchanged oscillation frequency can indicate
the absence of transformation of collagen bers dur-
ing laser heating. Insignicant shift of amide II band
towards the low-frequency region after reinforcement
and washing procedure (from 1546 to 1538 cm—1)
can indicate weakening of hydrogen bonds due to in-
creased humidity of the collagen scaffold. After rein-
forcement, the mean Young’s modulus for the collagen
scaffold increased by ~7 times: to 520.75±201.23 vs.
77.60±55.30 kPa for original scaffold (Fig. 1, d).
The obtained result was primarily determined by
the formation of photocured polylactide strips on the
collagen surface, which was conrmed by distribution
of the Young’s modulus values on the surface of the
reinforced scaffold (Fig. 1, d). The highest values of
Young’s modulus showed periodicity corresponding to
that of polylactide strips applied to the collagen scaf-
fold. Only one of the reinforcing lines is clearly seen
(Fig. 1, d). The non-uniform mechanical properties of
the obtained strips are determined by anisotropy of
the initial collagen scaffold, which leads to the sec-
Cell Technologies in  Biology  and  Medicine,  No.  1,  May,  2018
Fig. 1. Microscopic, spectral, and mechanical characteristics of the original and reinforced matrix. a) Original collagen sponge; b) imme-
diately after laser structuring; c) IR spectra of reinforced (1) and original (2) collagen matrix; d) surface distribution of Young’s modulus
for the original and reinforced collagen sponge. Polylactide strips formed by laser irradiation on the surface of the collagen sponge (b, d).
K. N. Bardakova,  E. A.  Grebenik, et al.
Fig. 2. Biocompatibility of collagen sponges. a) MTT-analysis of cytotoxicity of extracts of the original and reinforced collagen sponge. So-
dium dodecyl sulfate (SDS) was used as the positive control. b, c) MSC cultured for 1 month (b) and 2 months (c). Staining with ethidium
bromide homodimer. Confocal microscopy.
Fig. 3. Vital light microscopy of human bone marrow MSC cultured on reinforced collagen for 1 week (a) or 4 weeks (b), ×20.
Cell Technologies in  Biology  and  Medicine,  No.  1,  May,  2018
ond reason for Young’s modulus growth for reinforced
scaffold — the formation of spatial cross-linking in the
collagen material as a result of laser exposure. Con-
siderable macroscopic heterogeneity of the original
collagen scaffold is the cause of partial reection and
scattering of laser radiation and energy absorption by
the collagen scaffold because of diffuse scattering of
photons leads to the formation of cross-links in the
polymer network. The existence of areas with different
mechanical properties in the reinforced collagen scaf-
fold is very important for cell culturing on its surface.
The cytotoxicity of the reinforced sponge was as-
sessed by the MTT analysis (Fig. 2, a).
Addition of the polylactide component during re-
inforcement did not increase the cytotoxicity of the
collagen sponges. The observed decrease in prolifera-
tive activity of cells can be explained by high swell-
ing capacity of collagen structures and, consequently,
depletion of the growth medium with FCS present in
the scaffold.
The human bone marrow MSC were stained with
ethidium bromide homodimer (orange) (Fig. 2, b, c).
Blue color corresponds to scaffold autouorescence,
this parameter reects colocalization of cells and sites
modied by polylactide.
Vital light microscopy of human bone marrow
MSC on reinforced collagen showed active prolifera-
tion of cells by week 4; they populated the central part
of the scaffold fragment both on the surface and in the
depth (Fig. 3, a, b).
Thus, reinforcement of the collagen sponges
7-fold increased the mean Young’s modulus for the
hybrid scaffold without affecting its cytotoxicity. The
developed scaffold provides cell adhesion and proli-
feration along the reinforcement lines and can be used
for fabrication of tissue engineering constructs.
The study was supported by the Russian Science
Foundation (grant No. 16-15-00042; Development of
hybrid structures, cell experiments), Russian Founda-
tion for Basic research (grant No. 16-02-00248; physi-
cochemical and mechanical characterization of materi-
als, analysis of the cell morphology and distribution
by the method of confocal microscopy), and Federal
Agency for Scientic Organizations (FASO Russia)
(Agreement No. 007-GZ/Ch3363/26; analysis of cell
distribution by the method of confocal microscopy).
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... Similarly, fabricated scaffolds (2PP technique) were also used for supporting of Schwann cells growth and thus, as neural scaffolds in nerve repair [70]. Laser-induced crosslinked star-shaped methacrylate-terminated oligo(d,l-lactide)s (M n = 2400) were used as a reinforcement of collagen materials [74,75]. The material exhibited improved resistance to biodegradation, while the direct multipotent stromal cell growth during their culture was observed. ...
... Investigation of mechanical properties showed that with increasing nano-HAP content the elasticity modulus of the composite PDLLA/nano-HAP network materials increased. Some authors applied a two-photon polymerization technique (2PP) as the type of stereolithography [109] for the preparation of UV crosslinked PLA-based materials designed for tissue scaffolds [70,71,[73][74][75]. 2PP is a computer-aided microfabrication method by which it is possible to produce biomimetic synthetic scaffolds with high precision and reproducibility. ...
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The growing applications of tissue engineering technologies warrant the search and development of biocompatible materials with an appropriate strength and elastic moduli. Here, we have extensively studied a collagenous membrane (GSCM) separated from the mantle of the Giant squid Dosidicus Gigas in order to test its potential applicability in regenerative medicine. To establish the composition and structure of the studied material, we analyzed the GSCM by a variety of techniques, including amino acid analysis, SDS-PAGE, and FTIR. It has been shown that collagen is a main component of the GSCM. The morphology study by different microscopic techniques from nano-to microscale revealed a peculiar packing of collagen fibers forming laminae oriented at 60-90 degrees in respect to each other, which, in turn, formed layers with the thickness of several microns (a basketweave motif). The macro-and micromechanical studies showed high values of the Young's modulus and tensile strength. No significant cytotoxicity of the studied material was found by the cytotoxicity assay. Thus, the GSCM consists of a reinforced collagen network, has high mechanical characteristics, and is non-toxic, which makes it a good candidate for the creation of a scaffold material for tissue engineering.
... The created dermal scaffolds should contribute to the improvement of healing indicators (reduction of the inflammatory response, formation of granulation tissue, stimulation of angiogenesis, acceleration of wound epithelization, etc.) and reduction of complications. (1,2) Currently, there is no doubt that the effectiveness of artificial extracellular matrices in stimulating tissue regeneration is associated with providing sufficient temporary mechanical support for the formation of a new fibrous backbone. (3)(4)(5) To assess the prospects of scaffolds, morphological studies of tissue response to the implantation of scaffolds in vivo and the study of the features of the formation of the collagen framework are necessary. ...
Background: the use of various scaffolds allows us to model the future fibrous framework of the newly formed regenerate, and also serves as a substrate for the settlement of the cellular component. The development of tissue engineering in regenerative medicine demands an understanding of the more specific mechanisms of the formation of the connective framework at the site of the defect. The aim of this research was to study the morphofunctional rearrangement of the fibrous structures of the rat dermis in response to the implantation of a 3D scaffold based on polyprolactone Methods and Results: The experiment was performed on 30 white male Wistar rats. The object of the study was a skin fragment together with an implantable 3D scaffold based on polyprolactone, taken on Days 3, 7 and 14 after implantation. Biomaterial with implantable scaffold was studied using light and scanning electron microscopy. The results of the study indicate that the 3D scaffold based on polyprolactone has good biocompatibility, causing a weak inflammatory reaction, and contributes to the formation of the connective tissue framework by Day 14. Conclusion: The results of the study can be used to develop new scaffolds or modify existing ones, as a "framework" for populating the cellular component and creating tissue-engineering structures.
... Collagen (type I) was obtained from cattle dermis as reported earlier. 38,39 All solvents were purchased from Acros Organics (Belgium) as analytical grade and were used without further purication. ...
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Graft-copolymers based on bioresorbable synthetic (oligo-/polylactide) and natural (chitosan and collagen/gelatin) components were synthesized through solid-state reactive co-extrusion and used for fabrication of fibrous non-woven mats via the electrospinning technique. The effect of the macromolecular features of the initial components on the copolymer characteristics was evaluated using FTIR-spectroscopy, differential scanning calorimetry and elemental analysis. Dynamic light scattering analysis showed that the copolymers have a tendency to form stable ultra-fine dispersions with a mean size of macromolecular aggregates of 150 nm within chlorinated solvents. The copolymer-containing non-woven fibrous mats were fabricated via an electrospinning procedure using chloroform as a solvent. An effect of the copolymer composition on the casting solution's viscosity, conductivity and surface tension was evaluated. Scanning electron microscopy showed that the obtained mats consist of randomly distributed fibers with a mean size of ∼5 μm and a more complex morphology than mats fabricated from neat polylactide. The proposed mechanochemical approach to obtain hybrid copolymeric compositions differs from typical liquid-phase methods in terms of high efficiency, simplicity and cleanness.
In recent years, there has been enormous search and demand for naturally sourced polymers and excipients in the healthcare sector. Chitosan is a very important marine-derived natural polymer being a current topic of interest as the anti-infective for wound care. It is obtained through the process of deacetylation having β-(1–4)-linked D-glucosamine and N-acetyl-D-glucosamine units and considered as a linear polysaccharide derived from chitin. Few studies showed that the chitosan exhibits nontoxic, biocompatible, low immunogenic, and biodegradable properties which ultimately favors pharmaceutical dosage delivery. Chitosan-based formulations are successfully being used in the cosmetic industry, antimicrobial activity, biomedical tissue engineering, and gene delivery for wound care. Researchers successfully fabricated chitosan-based sponges utilizing suitable techniques for biomedical applications. Sponges made from chitosan are lightweight and capable of soaking extra liquid and drug solution on topical application. Additionally, osteoblastic differentiation and bone regeneration are successfully observed in the application of the chitosan matrix scaffold. Moreover, this chapter discusses various biomedical applications of marine-based chitosan and in drug delivery for anti-infective activities.
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Collagen materials are widely applied in medicine due to optimal handling characteristics, biocompatibility, controlled biodegradation, the ability to form complexes with drugs and facilitate regeneration. Researchers from Sechenov University developed, studied in experiments and introduced into medicine a variety of materials based on collagen — a protein of connective tissue. At the same time, new collagen materials were launched into clinical practice abroad. In this review of Russian and world literature, we described how scientific and applied studies of collagen materials developed over time and tried to illustrate the current state and trends of collagen application for a variety of medical purposes — from hemostatic sponges to tissue-engineered constructs. The range of available collagen-based medical products and the emergence of new collagen materials indicate the keen interest in this biomaterial from the medical community and the potential of future discoveries.
Bioprosthetic materials based on mammalian pericardium tissue are the gold standard in reconstructive surgery. Their application range covers repair of rectovaginal septum defects, abdominoplastics, urethroplasty, duraplastics, maxillofacial, ophthalmic, thoracic and cardiovascular reconstruction, etc. However, a number of factors contribute to the success of their integration into the host tissue including structural organization, mechanical strength, biocompatibility, immunogenicity, surface chemistry, and biodegradability. In order to improve the material's properties, various strategies are developed, such as decellularization, crosslinking, and detoxification. In this review, the existing issues and long-term achievements in the development of bioprosthetic materials based on the mammalian pericardium tissue, aimed at a wide-spectrum application in reconstructive surgery are analyzed. The basic technical approaches to preparation of biocompatible forms providing continuous functioning, optimization of biomechanical and functional properties, and clinical applicability are described. Abstract The review describes the milestones in the development of bioprosthetic materials based on mammalian pericardium tissue. The basic technical approaches to preparation of biocompatible forms including decellularization, crosslinking, detoxification, and application range of pericardial biomeshes in reconstructive surgery are discussed.
We studied the effect of non-thermal argon plasma on proliferative activity of bone marrow multipotent stromal cells in vitro. Treatment of stromal cell suspension with pure argon did not affect their proliferation. The cells treated with non-thermal argon plasma and explanted in the treatment medium demonstrated growth inhibition by 30-40% in comparison with the control. Multipotent stromal cells treated with plasma and after centrifugation explanted in normal medium within 12 min demonstrated accelerated growth. The total cell growth from the pellet and supernatant significantly exceeded the control values. We also analyzed adhesion and proliferative activity of multipotent stromal cells treated with non-thermal plasma on bioresorbable carriers. The cells adhered and proliferated on all types of studied samples. Adhesion properties of scaffolds differed. Caprolactone was found to be the most suitable material for adhesion and proliferation of multipotent stromal cells.
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Urethral strictures are a pressing issue in modern medicine. Substitution urethroplasty is considered one of the most effective treatment methods. However, despite the surgery showing good results, many problems remain unresolved, one being substitute material deficiency in extensive or recurrent strictures, as well as in cases requiring multistage surgeries, including those used to treat hypospadias. Graft removal also leaves the donor area prone to diseases and increases the length of surgery leading to a higher risk of intra- and postoperative complications. Tissue engineering (namely tissue-engineered products comprised of scaffolds and cells) may be a useful tool in dealing with these issues. The authors assessed the characteristics of a novel hybrid scaffold created from “reconstructed” collagen and a poly(lactic-co-glycolic acid) mesh. The resulting composite product showed good mechanical properties and functional performance. The hybrid scaffold was non-cytotoxic and provided an adequate base for cell adhesion and proliferation. Biodegradation resulted in the scaffold being replaced by urothelium and urethral mucosa. The newly formed tissues possessed adequate structural and functional properties. Only one rabbit out of 12 developed urethral stricture at the site of scaffold implantation. The abovementioned facts suggest that the novel hybrid scaffold is a promising tissue-engineered product with potential implication in substitution urethroplasty.
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Objective: to perform a comparative morphological study of biocompatibility, biodegradation, and tissue response to implantation of collagen matrices (scaffolds) for tissue engineering in urology and other areas of medicine. Material and methods: Nine matrix types, such as porous materials reconstructed from collagen solution; a collagen sponge-vicryl mesh composite; decellularized and freeze-dried bovine, equine, and fish dermis; small intestinal submucosa, decellularized bovine dura mater; and decellularized human femoral artery, were implanted subcutaneously in 225 rats. The tissues at the implantation site were investigated for a period of 5 to 90 days. Classical histology and nonlinear optical microscopy (NLOM) were applied. Results: The investigations showed no rejection of all the collagen materials. The period of matrix bioresorption varied from 10 days for collagen sponges to 2 months for decellularized and freeze-dried vessels and vicryl meshes. Collagen was prone to macrophage resorption and enzymatic lysis, being replaced by granulation tissue and then fibrous tissue, followed by its involution. NLOM allowed the investigators to study the number, density, interposition, and spatial organization of collagen structures in the matrices and adjacent tissues, and their change over time during implantation. Conclusion: The performed investigation could recommend three matrices: hybrid collagen/vicryl composite; decellularized bovine dermis; and decellularized porcine small intestinal submucosa, which are most adequate for tissue engineering in urology. These and other collagen matrices may be used in different areas of regenerative medicine.
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Here, we describe a porous 3-dimensional collagen scaffold material that supports capillary formation in vitro, and promotes vascularization when implanted in vivo. Collagen scaffolds were synthesized from type I bovine collagen and have a uniform pore size of 80 μm. In vitro, scaffolds seeded with primary human microvascular endothelial cells suspended in human fibrin gel formed CD31 positive capillary-like structures with clear lumens. In vivo, after subcutaneous implantation in mice, cell-free collagen scaffolds were vascularized by host neovessels, whilst a gradual degradation of the scaffold material occurred over 8 weeks. Collagen scaffolds, impregnated with human fibrinogen gel, were implanted subcutaneously inside a chamber enclosing the femoral vessels in rats. Angiogenic sprouts from the femoral vessels invaded throughout the scaffolds and these degraded completely after 4 weeks. Vascular volume of the resulting constructs was greater than the vascular volume of constructs from chambers implanted with fibrinogen gel alone (42.7±5.0 μL in collagen scaffold vs 22.5±2.3 μL in fibrinogen gel alone; p<0.05, n = 7). In the same model, collagen scaffolds seeded with human adipose-derived stem cells (ASCs) produced greater increases in vascular volume than did cell-free collagen scaffolds (42.9±4.0 μL in collagen scaffold with human ASCs vs 25.7±1.9 μL in collagen scaffold alone; p<0.05, n = 4). In summary, these collagen scaffolds are biocompatible and could be used to grow more robust vascularized tissue engineering grafts with improved the survival of implanted cells. Such scaffolds could also be used as an assay model for studies on angiogenesis, 3-dimensional cell culture, and delivery of growth factors and cells in vivo.
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Collagen is the main structural protein of most hard and soft tissues in animals and the human body, which plays an important role in maintaining the biological and structural integrity of the extracellular matrix (ECM) and provides physical support to tissues. Collagen can be extracted and purified from a variety of sources and offers low immunogenicity, a porous structure, good permeability, biocompatibility and biodegradability. Collagen scaffolds have been widely used in tissue engineering due to these excellent properties. However, the poor mechanical property of collagen scaffolds limits their applications to some extent. To overcome this shortcoming, collagen scaffolds can be cross-linked by chemical or physical methods or modified with natural/synthetic polymers or inorganic materials. Biochemical factors can also be introduced to the scaffold to further improve its biological activity. This review will summarize the structure and biological characteristics of collagen and introduce the preparation methods and modification strategies of collagen scaffolds. The typical application of a collagen scaffold in tissue engineering (including nerve, bone, cartilage, tendon, ligament, blood vessel and skin) will be further provided. The prospects and challenges about their future research and application will also be pointed out.
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A novel composite scaffold based on chitosan-collagen/organomontmorillonite (CS-COL/OMMT) was prepared to improve swelling ratio, biodegradation ratio, biomineralization and mechanical properties for use in tissue engineering applications. In order to expend the basal spacing, montmorillonite (MMT) was modified with sodium dodecyl sulfate (SDS) and was characterized by XRD, TGA and FTIR. The results indicated that the anionic surfactants entered into interlayer of MMT and the basal spacing of MMT was expanded to 3.85 nm. The prepared composite scaffolds were characterized by FTIR, XRD and SEM. The swelling ratio, biodegradation ratio and mechanical properties of composite scaffolds were also studied. The results demonstrated that the scaffold decreased swelling ratio, degradation ratio and improved mechanical and biomineralization properties because of OMMT. © 2015 Higher Education Press and Springer-Verlag Berlin Heidelberg
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The aim of the investigation was to develop a novel biodegradable material based on chitosan synthesized by solid-state technoogy, and to create based on biocompatible three-dimensional cell-carrying scaffolds using laser stereolithography. Materials and Methods. Reactive systems were developed based on chitosan grafted with allyl, polyethylene glycol diacrylate, and the photoinitiator Irgacure 2959. The structures were obtained using laser stereolithography setting LS-120 (Institute on Laser and Information Technologies, Russian Academy of Sciences, Russia). Results. Partial replacement of chitosan amino groups by allyl groups (CТ-А) and the introduction of polyethylene glycol diacrylate (PEG-DA) as a crosslinking agent were found not to reduce the material biocompatibility. The metabolic activity determination of NCTC L929 cells using MTT assay showed that the samples under study to contain none water-soluble components toxic to mammalian cells. The samples based on CT-A and CT-A with a crosslinking agent PEG-DA are biocompatible and are able to support adhesion, spreading and proliferative activity of human mesenchymal stromal cells, but have significant differences in the extent and nature of the expression activation of gene markers for osteogenic differentiation path.
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A review about design, manufacture, and mechanobiology of biodegradable scaffolds for bone tissue engineering is given. First, fundamental aspects about bone tissue engineering and considerations related to scaffold design are established. Second, issues related to scaffold biomaterials and manufacturing processes are discussed. Finally, mechanobiology of bone tissue and computational models developed for simulating how bone healing occurs inside a scaffold are described.
Aim: To assess the properties of 3D biodegradable scaffolds fabricated from novel star-shaped poly(D,L-lactide) (SSL) materials for bone tissue regeneration. Materials & methods: The SSL polymer was synthesized using an optimized synthetic procedure and applied for scaffold fabrication by the two-photon polymerization technique. The osteogenic differentiation was controlled using human adipose-derived stem cells cultured for 28 days. The SSL scaffolds with or without murine MSCs were implanted into the cranial bone of C57/Bl6 mice. Results: The SSL scaffolds supported differentiation of human adipose-derived stem cells toward the osteogenic lineage in vitro. The SSL scaffolds with murine MSCs enhanced the mineralized tissue formation. Conclusion: The SSL scaffolds provide a beneficial microenvironment for the osteogenic MSCs' differentiation in vitro and support de novo bone formation in vivo.
Biomedical scaffolds have recently evolved into various functional materials, including drug delivery systems (DDS). Here, we report the development of a new, highly porous scaffold based on a layer-by-layer collagen scaffold coated with an alginate polymer, which shows improved mechanical properties and controllable drug release without loss of the original biological function of the collagen scaffold. In particular, the scaffold (75 vol % alginate in a collagen scaffold with a porosity of 88%) attained a Young’s modulus of 30 MPa, which is 9 times the value for the pure collagen scaffold (porosity = 98%). Although the scaffolds are highly porous, the drug release and initial burst were well-controlled with an appropriate volume fraction of alginate. Osteoblast-like cells (MG63) readily proliferated and migrated into the interior of the scaffolds, and calcium and phosphate on the cell surfaces were well-formed, similarly on pure collagen and alginate/collagen scaffolds, within only 7 days of culture. The alginate/collagen scaffolds with a drug delivery function have potential as biomedical scaffolds for clinical use in soft and hard tissue regeneration.
Porous scaffolds play important roles in tissue engineering. Biodegradable synthetic polymers such as poly(ε-caprolactone) (PCL) are frequently used in the preparation of porous scaffolds. In this study, a reinforcing composite scaffold was fabricated using the freeze-drying technique by embedding porous PCL core within collagen shell. Controls of pure collagen and PCL scaffolds were also investigated for comparison in this study. Microstructure and mechanical properties of the scaffolds were investigated and related to their morphologies examined by SEM. Fracture micromechanism under tension was also characterized on the basis of microstructural deformation behavior. It was found that the composite scaffold possessed an appropriate mechanical property, which was greater than that of pure collagen scaffold. This study demonstrated that the reinforcing technique could modulate the mechanical properties and porous structure of the composite scaffold, which could be potential factors in ligament tissue engineering.