Integration layer gradient prosthesis used for bone tissue engineering cartilage in experimental study
Authors: Bai Dong, Yun-Yu Hu, Yan Leping, Ren Li, Wu Gang, Li Dan, SUN
Xiao-tong, BAI Jian-ping
Abstract [Objective] To explore the collagen / chitosan / calcium phosphate nano-
integration layer gradient prosthesis used in tissue engineering of bone cartilage defect is feasible. [Methods] type ? collagen, chitosan, nano-
TCP) as the basic raw material, non-toxic cross-linking and, through freeze-drying
method forming; scanning electron microscope, determination of scaffold material pore size, porosity and transport hole situation; separation of amplified rabbit bone marrow-derived mesenchymal stem cells (BMSC), the BMSC inoculated material,
scanning electron microscopy on the cell adhesion status of the material, MTT determination of the growth of cells in the material curve; in order to induce the liquid outer cartilage scaffold complexe
weeks after the implantation of autologous thigh muscle pouches of 6 weeks drawn, HE, toluidine blue staining, ? collagen immunohistochemical identification of induced
differentiation of results. [Results] The porous materials, pore size is larger than
100μm, porosity greater than 95%. Cell adhesion on materials in good condition, and
-dimensional in vitro can be
induced by autologous ectopic differentiation of the cartilage. [Conclusion] ? collagen /
chitosan / nano-TCP Integration layer gradient prosthesis has a good pore structure and biocompatibility, is expected to become a new type of tissue engineering scaffold material for bone repair cartilage defects.
Key words Tissue engineering; bone cartilage; bone marrow mesenchymal stem cells;
Abstract: [Objective] To explore the potential of a novel collagen I / chitosan /
TCP) bilayered scaffold for being used in tissue
engineering (TE) of osteochondral repair. [Method] Bilayered scaffolds were produced with collagen ?
drying method.The pore size, porosity and interpores of the scaffold were observed by
scanning electron microscopy (SEM). Rabbit bone mesenchymal stem cells (BMSC) were isolated and amplified , then inoculated onto the scaffold.By SEM scanning, the condition of the cells adhering onto the scaffold was observed.The proliferation of the
cells on the scaffolds was examined using MTT method, and the growth curve was
le pouches 2 weeks
later.The result was observed 6 weeks later by HE staining, toluidine blue staining and type ? collagen immunohistochemistry. [Result] The scaffold possessed high porosity and proper pore size, the porosity was above 95%. BMSC could adhere onto the
scaffold well, and the proliferation rate of the cells on the scaffolds was perfectly
scaffold possesses good pore structure and biocompatibility, and will possibly become a new biomaterial of TE used for osteochondral repair.
Key words: tissue engineering (TE); osteochondral; bone mesenchymal stem cells (BMSC); collagen;
The development of tissue engineering cartilage injury and repair provide a new method, which can effectively solve the source, as well as graft donor site injury problems, and graft in vitro any shaping to meet the needs of damaged parts 〔1〕.
Repair of cartilage defects include two main areas: (1) to functional repair tissue filling defects; (2) repair tissue with the surrounding host cartilage and bone integration 〔2〕. Cartilage tissue engineering is currently a prominent issue is the existence of
graft and host tissue between the absence of a solid anchoring of relying solely on repair result 〔3〕.
To effectively solve the graft and the integration of host tissue, the authors developed
a new type of tissue-engineered osteochondral integration of materials - collagen /
is divided into upper and lower parts of the cartilage of the subchondral bone parts,
but the overall integration of cross-linked material, avoiding the cartilage and bone
repair process in the separated part. With the bone tissue integration between the fast and strong and is expected to make the prosthesis firmly anchored at an early stage in the defect area, for the repair of articular cartilage to provide good stability and mechanical support.
In this study, the integration of the cartilage tissue-engineered bone prosthesis of the
general performance and biocompatibility in vitro was studied, as well as through in vitro induction of autologous heterotopic preliminary experiments to explore its use in tissue engineering of bone cartilage injury and repair is feasible.
1 Materials and methods
1.1 Collagen / Chitosan / nano-layered gradient prosthesis TCP Integration
Scaffold of biological material from South China University of Technology developed by the Institute to provide in order to type ? collagen, chitosan as a
after freeze-drying molding. Top of collagen / chitosan, the lower for the collagen /
n gradient from the bottom to
1.2 scaffold morphology and porosity
Measuring the material to the drainage porosity, scanning electron microscopy the material pore size and pore linking the situation.
Measuring 1.3 MTT cells in the scaffold material on the proliferative activity
Take 3-month-old Japanese white rabbits, weighing 2.1 kg, general anesthesia after disinfection of the tibial plateau from 0.5 cm punishable under the puncture needle 18, each side of the bone marrow 2 ~ 3 ml, carefully adding pre-loaded with an equal
volume of percoll working solution (density 1.073) in centrifuge tubes, centrifuge 25 min to 2 000r/min nucleated cell layer were collected, re-hanging with 10% newborn
calf serum (GIBCO) in high glucose DMEM culture medium, were inoculated on 100
ml culture bottles, at 37 ?, the volume fraction of 5% CO2 in the culture of the
incubation box. 3 d after the first exchange of medium, after 2 ~ 3 d for each fluid, to be covered with 80% of primary cells when subcultured bottom of the bottle.
Take the first two generation of cells, trypsin digestion produced 3.0 × 105A/ ml of cell suspension. Placed in 48-well plates 20 the same type of material to drip cell
suspension was inoculated into the material, each piece of material 150 μl; the other
five will develop a simple droplet injection material to be used as controls. Put it aside for 4 h inside the incubator, each hole by adding 700 μl culture medium in order to fully cover material, 18h to be fully adherent cells, will be combined with cell material
transferred to the new holes, each hole plus culture medium 1.5 ml, each subsequent 2 d given for liquid. Turn hole at 2,4,6,9, and 12 d after three randomly selected composite materials and a cell control material, each hole by adding 700 μl culture
medium and 70 μl 5 mg / ml solution of MTT in culture Box 4 h after abandoning the suction hole liquid, and in each hole by adding 700 μl DMSO, micro-oscillator
oscillation 10 min later, 150 μl per well absorb the liquid by adding 96-well plates used
for detection. Select 492 nm wavelength in order to control hole-zero, the instrument
was determined by enzyme-linked immunosorbent assay absorbance of Hole. Abscissa
of time points, MTT at different time points, the mean growth curve drawn for the
1.4 Scanning electron microscopy of the adhesion of cells in the material state of
The cell suspension to 3.0 × 105A/ ml was inoculated into the density of scaffold material, and placing 24-well plates in culture 12 d after a 3% glutaraldehyde fixed, gradient alcohol dehydration, tert-butanol replacement, vacuum drying, plasma jet
Miriam spray gold, scanning electron microscopic observation cells in the adhesion of surface and internal morphology.
1.5 MSCs in the scaffold material on the induced differentiation
Take 3-month-old Japanese white rabbits, weighing 2 ~ 2.5 kg, according to the above-mentioned methods for separating amplified rabbit BMSC. Take the first two generation of cells, digestion dubbed in 2 × 106A/ ml of cell suspension, the cell suspension was inoculated in 24-well plates within the scaffold material, each material 150 μl, put it aside in the incubator within 4 h after the , add 2 ml culture medium continued to train. 24 h after material transfer to the new hole, and replace the culture
medium with high glucose DMEM prepared as cartilage inducing medium (including rhTGFβ1 10μg / L, dexamethasone 1 × 107 mol / L, VitC 50 μg / L, new bovine serum 100 ml / L), thereafter every 2 d for liquid. 14d sustained induction culture, will be general anesthesia disinfection experimental animals, respectively, shares skin incision, subcutaneous and fascia along the muscle fiber blunt manufacturing muscle Traveling bag. Implanted on the left side of the blank vector as control; right after implantation-
induced cell - scaffold for the experimental side. Harvested after 6 weeks after operation, made of paraffin OK HE, toluidine blue staining and type ? collagen
immunohistochemical identification of cartilage differentiation.
2.1 The materials, shape and porosity
SEM can be observed with a porous scaffold structure, pore structure, rules, pore size of about 100 ~ 150 μm, material within the hole and good linking between holes
(Figure 1); nano-TCP wrapped in the repair surface, the two are linked closely together, uniform particle distribution (Figure 2). Determination of the material through the drainage porosity greater than 95%. Reposted elsewhere in the paper for
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2.2 Cell adhesion on the material state and the proliferation curve
SEM shown by BMSC in the material on the growth of three-dimensional culture in
good condition after 14 d, into the existence of discrete cell adhesion on the stent
surface and pore wall, and visible cell processes connected to each other through into the tablets (Figure 3).
Figure 1 supports the internal pore structure (SEM × 100) Figure 2 parcels β-TCP
scaffold surface (SEM × 2 000) Figure 3, the adhesion of cells in the surface
morphology (SEM × 500) BMSC proliferation in the material quickly, three-
dimensional culture 12 d, MTT values from 2 d of 0.255 up to 1.557 when the cells showed logarithmic growth of the basic trend, 4 ~ 6 d, 9 ~ 12 d period of the
proliferation of the most significant (P <0.05); but the 6 ~ 9 d period of the proliferation of non - significantly (P> 0.05) (Figure 4).
2 weeks after animal parts can be touched on the quality tough nodules, nodules gradually hardening after the experimental side, while no significant change in the control side. After the experimental side scaffold materials in vitro cultured for 2 weeks, after 6 weeks most of the degradation of the body, but can still see the outline of residual stand (dark red stain), stent pore filling into a group of classes within the cartilage, no significant inflammatory response (Figure 5 ). Toluidine blue staining metachromatic material within a large number of tissues, cells and extracellular matrix were significantly coloring (Figure 6). Type ? collagen immunohistochemical staining
showed positive reaction to new organization can be seen (Figure 7).
Tissue engineering scaffold material is an important issue, the ideal scaffold for cartilage tissue engineering should possess the following characteristics: (1) good biocompatibility, degradation products of its own, or the body of seed cells and non-
toxic, does not cause inflammation and immune rejection; (2) the appropriate bio-
degradability, degradation rate and the need to match the speed of tissue regeneration may eventually completely absorbed; (3) Good compatibility with the structure, with a certain degree of strength and plasticity, can maintain a stable three-dimensional
structure, the porous three-dimensional stent should have interconnected pores to
facilitate the nutrients and metabolites from spreading. Porosity of 85%, and pore size appropriate for the uniform distribution of seed cells and growth to provide sufficient space; (4) a good surface compatibility and a certain degree of biological activity, surface conducive to cell adhesion and growth of seed 〔 4〕. Authors applied bone
tissue engineering scaffold material for cartilage cells, a good compatibility of type ?
collagen, chitosan as the main body, through a specially designed freeze-dried cross-
linking technology that enables the integration of stent material under the premise of the nano-TCP powder evenly mixed in the lower material in order to play its role in the promotion of osteogenesis. Experimental proof of scaffold material with good pore structure and porosity, non-cytotoxic and are conducive to the seed attached to cell proliferation, for the three-dimensional culture in vitro basis. Growth curve (Figure 4) Section 6 ~ 9 d cell proliferation was not obvious, analyze the reasons may be increased as the number of cells, which also increased the demand for nutrition, 1.5 ml/2d exchange of fluid volume far from being able to meet a large number of the need for cell proliferation, resulting in cell proliferation due to lack of nutrient supply is limited;
later changed to a daily exchange of medium, to the first 12 d, when the value of a rapid increase in MTT. Material also has good degradation in vitro cultured for 2 weeks after the materials, the general shape and pore structure remained basically unchanged,
while the in vivo degradation after 6 weeks, and the majority, but the basic outline of the material is still preserved the basic right to meet the tissue engineering material degradation performance requirements.
Figure 5 cells were implanted in muscle pouches of scaffold complexes 6 weeks (HE
stain × 100) Figure 6-cell stent implanted in muscle pouches of compound 6 weeks
(toluidine blue stain × 200) Figure 7-cell stent implanted in muscle pouches of
compound 6 weeks , ? collagen immunohistochemical staining (× 400) as the source of
chondrocytes for the area is limited and there is a series of negative factors such as injury, so the present study, BMSC as seed cells. BMSC derived from the body easily, there is no immune rejection response, both ex vivo expansion ability, and self-renewal
fast, multi-differentiation potential of characteristics such as bone cartilage tissue engineering the most ideal seed cells 〔5〕. BMSC not only in vitro for cartilage cells
or bone cells, but also the growth in the body are subject to different environmental
stimuli to differentiate into cartilage or bone 〔6〕. BMSC to the cartilage or bone
differentiation require specific culture conditions, although the plane and three-
dimensional culture conditions, culture can be successfully induced BMSC
differentiation to cartilage cells, but the right three-dimensional culture conditions
more conducive to the cartilage or bone phenotype maintenance 〔7 〕. In this study,
amplification of the adult rabbit isolated BMSC, with the scaffold materials in its three-
dimensional culture in vitro, confirming the material has good biocompatibility, suitable for the growth of BMSC proliferation; and then the cell material into a complex three-dimensional cartilage induction, confirmed the induction of appropriate
conditions, the material of the cells can be differentiated into chondrocytes. As the nano-TCP has good biodegradability and bone conductivity, nano-TCP / collagen
composite is considered to best meet the needs of bone tissue engineering scaffold, one
of 〔8〕. It was found that scaffold material in vitro cultured for 2 weeks after the lower the apparent mineralization of TCP powder, which in this study into the role of bone remains to be further experiments confirmed that, but the literature has shown
that nano-tricalcium phosphate not only as a BMSC the cell skeleton, but also can
accelerate bone formation 〔9〕 〔10〕.
Conclusion: The collagen / chitosan / nano-
layer gradient prosthesis has good physical properties and biocompatibility, suitable for three-dimensional induced BMSC culture, is expected to become a new type of tissue engineering scaffold material for bone repair cartilage damage.
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