Sevastianov V.I., Perova N.V., Nemets E.A., Egorova V.A., Onishchenko N.A., Bryukhovetskiy A.S., Shumakov V.I.
(National Research Institute of Transplantology and Artificial Organs, Moscow, Russia; e-mail: viksev@yandex.ru)
Stem and pre-differentiated cell transplantation is the most promising line of research in the modern transplantology and it can become a powerful tool in treatment of different diseases. However, it is still important to develop technologies that allow for target delivery of viable cells to the site of injury, their retainment and regular functions there. A number of transplantation protocols prescribe cell suspension introduction directly to the blood stream or to the site of injury. These techniques have a number of limitations and disadvantages, particularly the difficulty of controlling cell localization at the site of injury and their low viability. Cell transplantation with support scaffold (matrix) may appreciably improve the effectiveness of this technique for function recovery of essential organs and tissues [1].
One of the main problems during cell culture and tissue formation in vitro following transplantation is the development and design of temporary polymer biodegradable scaffolds possessing essential physicochemical and biological properties [2].
Natural polymers and derivatives such as alginate, chitosan, gelatine, collagen, hyaluronic acid, polyoxyalkanoates have the biggest potential. Biopolymers, besides being biocompatible, are additionally high-performance biostimulants. The products of biodegradation are water and carbon dioxide.
Two biopolymers: high-molecular animal protein - collagen, - and a bacterial polymer - poly(polyhydroxybutyrate-co-hydroxyvalerate) were chosen to develop the scaffolds.
The goal of this study is the development and pre-clinical investigation of biodegradable scaffolds on the basis of collagen and a bacterial polymer for cell transplantation.
Collagen SpheroOGel matrix
Due to hydrolytic destruction, collagen implants are resorbed during 3-4 weeks. This leads to scar tissue formation as in the case of biostable implants. We supposed that the formation of heterogeneous structure in collagen gel would result in collagen bioresorption rate decrease. The optimal composition obtained as a result of technological stage was of heterogeneous implantable gel SpheroOGel (Fig. 1) [3].
Figure 1. Injectable form of SpheroOGel.
At room temperature the SpheroOGel matrix represents a clear slightly opalescent viscous рН- balanced long-term storage stable and non-splitting gel. Water absorbtion is 86.6 w.%.
The SpheroOGel contains a crosslinking collagen in the form of microspheres (~10-15 mm) and a homogeneous viscous collagen. Matrix diffusion pore size ranges from 100 to 300 mm (Fig. 2).
The time of SpheroOGel degradation depends on the degree of crosslinking in the volume of homogeneous viscous collagen and can vary from several weeks to 6 months.
Figure 2. Scanning electron micrograph of bulk of SpheroOGel. SEM JSM Т-330 (JEOL, Japan)
In vivo experiments demonstrated that the heterogeneity of collagen did result in the decrease of biodegradation rate of matrix.
After 1 month of implantation into musculus rectus femoria of a rat the destruction of SpheroOGel was not observed (Fig. 3). After the 3rd month of implantation partial gel resorption appears. The fibrous capsule consists of 2-3 fibroblast layers (Fig. 4).
Figure 3. Histologic section of musculus rectus femoria of a rat after 1 month of SpheroOGel implantation with Hematoxilyn-eosin staining. Magnificationх400.
Figure 4. Histologic section of musculus rectus femoria of a rat after 3 months of SpheroOGel implantation with Hematoxilyn-eosin staining. Magnificationх200.
B. Film matrix ElastoPOBO on the basis of bacterial copolymer
The bacterial polyethers - homopolymer polyhydroxybutyrate and copolymer poly(polyhydroxybutyrate-co-hydroxyvalerate) - are thermoplastic and biodegradable and are the promising medical polymers [4]. However their hydrophobic and inadequate flexible properties constrain their application.
We supposed that the addition of high-molecular hydrophilic agent to bacterial copolymer would improve its mechanical strength and hydrophylic properties without lack of biocompatibility.
To develop biopolymer film matrix we chose high-purity copolymer poly(polyhydroxybutyrate-co-hydroxyvalerate) (Insitute of Biophysics, Siberia Division of RAS, Krasnoyarsk). According to preliminary experiments polyethylene glycol was chosen as a hydrophilic agent.
The optimal composition obtained in the set of in vitro and in vivo experiments was biodegradable biopolymer membrane ElastoPOBO (Fig. 5) [5] which allows to develop the film matrix possessing essential physicochemical and biological properties with the facility of surgical manipulations during the implantation of ElastoPOBO and cell culture adhesion, spreading, and proliferation. The presence of high-molecular hydrophilic agent results in elasticity and flexibility improvement as well as a substantial decrease of matrix surface roughness.
Figure 5. Biodegradable biopolymer membrane ElastoPOBO.
C. Biological safety of SpheroOGel and ElastoPOBO matrices
The analysis of biological safety of SpheroOGel and ElastoPOBO in vitro and in vivo according to GOST ISO 10993-99 standards showed that the matrices can be used as implantable materials [3, 5].
D. Functional properties of SpheroOGel and ElastoPOBO scaffolds
The ability to maintain cell growth in vitro was tested for the SpheroOGel and ElastoPOBO scaffolds with the use of rat bone marrow stem cells (Fig. 6, 7).
Figure 6. SpheroOGel matrix with rat bone marrow stem cells (7 days, magnificationx400).
Figure 7. The surface of ElastoPOB® with rat bone marrow stem cells (7 days, magnificationx200).
Biofunctionality of SpheroOGel matrix in vivo was investigated on the experimental complete spinal injury animal model [6]. A positive dynamics in regeneration of the motor function was observed in all cases of SpheroOGel implantation.
The effectiveness of ElastoPOBO application for reconstructive cell therapy was demonstrated on skeletal muscle injury and deep burns animal models [7, 8].
The experimental data received suggests that developed scaffolds SpheroOGel and ElastoPOBO are a suitable cell support for different cell types (stem cells, cardiomyocytes, nerve cells, hepatocytes, chondrocytes, b-cells, fibroblasts etc.) and may be applied as independent implantable system.
The clinical trials of the SpheroOGel and ElastoPOBO matrices were authorized under Department of Drug and Medical Devices Registration of Russian Ministry of Health, decision of September 29, 2005, № 03-228s/0559 and № 03-229с/0559. Several cases of clinical results will be presented.
Literature
1. Skaletskiy N.N., Onishchenko N.A. Cell transplantation: progress and aspects. Vestnik transplantologii. 2001, № 3-4, pp. 94-102 (Russ.).
2. Shumakov V.I., Sevastianov V.I. Biopolymer matrices for artificial organs and tissues. Zdravoohranenie i meditsinskaya tehnika. 2003, №4, pp. 30-32 (Russ.).
3. Perova N.V., Porunova Yu.V., Uriash V.F., Faminskaya L.A., Krasheninnikov М.Е., Rasulov М.F., Onishchenko N.A, Sevastianov V.I., Shumakov V.I. Biodegradable collagen matrix Spherogel for cell transplantation. Perspektivnye materially, 2004, № 2, pp. 52-59 (Russ.).
4. Volova T.G., Sevastianov V.I., Shishatskaya E.I. Polyoxyalkanoates - biodegradable polymers for medicine (Ed. V.I. Shumakov),- Krasnoyarsk, Publ. “Platina Group” -2006, p. 206 (Russ.).
5. Sevastianov V.I., Egorova V.A., Nemets Е.А., Perova N.V., Onishchenko N.A. Biodegradable biopolymer material ElastoPOB for cell transplantation. Perspektivnye materially, 2004, № 3, pp. 35-41 (Russ.).
6. Brukhovetski A., Yaryghin V., Chekhonin V., Savchenko E., Sevastianov V., Perova N., Abrosimov S. Implantation of the biodegradable polymer matrix Spherogel and embryonic cells under experimental complete spinal injury. Abstract of the 7th International Congress of the Cell Transplant Society, USA, Boston, November 17-20, 2004, p.58.
7. Rasulov М.F., Sevastianov V.I., Egorova V.A., Bogatyrev S.R., Zaidenov V.А., Potapov I.V., Onishchenko N.A., A comparative study of the dynamics of deep burn healing in using medullary allogenic fibroblast-like mesenchymal stem cells immobilized on biodegrading membrane or taken from cultural plastic. Patofiziologiya i eksperimentalnaya terapiya, 2005, №2, pp. 20-23 (Russ.).
Potapov I.V., Ilinskiy I.M., Sevastianov V.I., Egorova V.A., Zaidenov V.А., Rasulov М.F., Onishchenko N.A., Film systems ElastoPOBO with immobilized bone marrow stromal cells optimize regeneration conditions of injured tissues. Kletochnye tehnologii v biologii i meditsine, 2005, №3, pp. 151-157 (Russ.).
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