Cellular and tissue preparations in regenerative medicine (treatment of burn disease)

Our experience with blood preparations has more than 30 years: this period includes experimental research in cryoconservation of all cell populations of peripheral blood, corpse and donor bone marrow, embryonic liver hemapoietic cells of humans and animals, and cord blood. Preparations received by us have passed clinical trial and found application not only in clinical practice but in cattle breeding as well. Application of cord blood in clinics was preceded by a long stage of experimental research in development of cryoconservation method. Special attention was paid to conditions of receipt, storage and fractionating of mononuclei and also to the development of freezing program. As result regiment parameters of cooling, long-term storage, and defrosting have been estimated that allowed saving 90-95% of cord blood cells in vital state. It is worth to emphasize that amount of hemapoietic stem cells in one sample of cord blood is 700 times less than is needed for transplantation to an adult patient. That is why it was necessary to exclude cell loss at all the stages of technological process.

Cord blood is the most preferable source of stem cells due to many parameters. First, there are no ethic problems, prohibiting its receipt, second - easiness of procedure of mononuclei isolation, third, it is competitive to bone marrow in stem cells and early cells-precursors content and exceeds it in proliferative potential. Besides this, while comparing content of lymphoid cells of peripheral and umbilical blood, significant differences in their population content were revealed: non-activated, immature cells and suppressor cells predominate in umbilical blood that excludes development of disease “transplant versus host” even in application of umbilical blood stem cells with incomplete HLA-compatibility.

World experience in umbilical blood stem cells application in scheme of hemoblastosis, hereditary pathologies and non-tumor diseases treatment is rather extensive, however, how it was shown in many works, spectrum of cells action is not limited by hemapoietic system restoration. Body reaction to preparation administration is unpredictable: nervous cells pathologies may be eliminated, liver parenchyma and pancreatic cells recover, homeostasis normalizes, etc.

Population of mesenchymal stem cells that in definite conditions in culture may transform into fibroblasts, cells of bone, fat and fibrous tissue are of special interest.

Source material for cells cultivation are mesenchymal stem cells (CD34-), that were isolated for the first time from bone marrow by Fridenstein (Fridenstein et al., 1973)

CD34-negative cells-precursors of bone marrow can leave niche and go out to bloodstream. Getting with blood to appropriate microenvironment they come into signal-regulatory interactions with mature cells and form stromal frame where their differentiation into glia tissue and fibroblasts is possible. Immature mesenchymal cells, circulating in blood, may return to stroma of bone marrow tissue and transform into CD34-positive hemapoietic cells /Schuldiner, 2000/.

Also it was shown that in local injures mesenchymal stem cells receive signals from destroyed tissues that trigger mechanisms of reparative regulation /Tavasolli et al., 1991/.

Study of application of allogenic fibroblast-like mesenchymal stem cells for treatment of deep burns was performed by Shumakov with co-authors (2003). They showed that with application of allogenic fibroblast-like mesenchymal stem cells the rate of wound healing was higher than in transplantation of embryonic fibroblasts.

Cultivated allogenic keratinocytes and fibroblasts, autokeratinocytes and autofibroblasts with following dermoplastic were applied over the last twenty years.

Mesenchymal and hemapoietic stem cells were found also in cord blood /Nieda M, Nicol A, Denning-Kendal P et. al. Endothelial cell precursors are normal component of human umbilical cord blood. Br J Haematol 1997; 98; 775-777/. In experimental research it was shown that in long-term cultivating of cord blood mononuclei endothelial precursor cells form fibroblasts colonies in culture under the influence of definite cytokines.

Repopulation potential of cord blood hemapoietic cells is confirmed by numerous transplantations in treatment of many diseases including aplastic anemias /Gluckman et al., 1997/.

Burn disease developing at the background of vast thermal trauma (more then 10% of body), includes complex of numerous clinical syndromes. These are burn shock, acute burn toxemia, septic toxic period and period of recovery. Every stage of disease is followed by appropriate tactics of treatment.

At present time biological preparations of complex action directed toward restoration of patient’s homeostasis, processes of reparation and regeneration of damaged skin zones and general flow of burn disease have been started to apply together with traditional methods.

In such a way the preparations containing erythropoietin / Patent №458887 A61M1/38/ are used for anemia treatment and prophylaxis at the period of burn shock. At the period of burn toxemia placenta tissue is used as immunomodulator in development of infection complications.

Placental tissue is applied in ointment composition and chorion cells are used in a form of cell suspension that is applied to wound surface (Preparation “chorion cell suspension” for treatment of burns, method of receipt and method of treatment with application of this preparation 20040806939/К19.08.2004 A61K35/50) in septic toxic period in traumatic bandage change and staged nectotomias.

Fibroblasts and keratinocytes culture is applied to the wound surface in vast wound surfaces, in trophic ulcers, long-term persisting wounds at the phases of regeneration and epithelization. Cell culture transplantation is performed independently, in combination with collagen-containing preparations or in combination with aytodermoplastics /Patent №2002105467/.

Own results

Above mentioned results and also own experience in application of stem hemapoietic cells in clinical practice /G.S.Lobyntseva, Yu.V.Gladkih and others. Stem embryonic hemapoietic human cells (theory and clinical practices), 2004/ allowed to supply a common scheme of patient with burn trauma treatment with complex of biopreparations, application of which in connection with particular heaviness of case could bring positive results (fig.1).

Fig.1

Patient N., 51 years old, entered the clinic with vast burn trauma of 36% of body. Deep thermal burn induced development of burn disease of 3-4 degree in patient that was characterized with severe burn shock, burn of respiratory tract, sepsis and development of septic pneumonia (fig.2)

Fig.2

Cryoconserved cord blood stem cells in amount of 0,2x109 nucleated cells were administered intravenously by droplets for elimination of the development of anemia state in patient during the 2-nd day on the background of sharp decrease of hemoglobin, erythrocytes and leukocytes figures (fig. 3)

Fig.3

Tactics of patients with vast burn skin lesions of IIIВ- IV degree treatment for intoxication reduction and elimination of complications envisages removal of damaged areas.

Fig. 4

Decompression sections were performed (fig. 4) during the 2-nd day and surgical excision of necrotic tissues (8% of all body surfaces) on abdomen and chest during the 4-th day (Fig.5).

Fig. 5

Suspension of epithelial cells in amount of 1-2 x 106/ml was applied to wound regions on abdomen and chest and it was closed by chorion tissue (fig. 6).

Fig. 6

As a result of performed manipulations the period till autodermoplastics provision was reduced by 6-7 days. Autoimmune reactions against applied biological preparations were not observed. Due to this full transplant engraftment occurred without tissue rejection and its lysis. This facilitated preservation of limited own skin resources.

Granulation and epithelization were accelerated and they ran without apparent inflammation process that led to decrease of formation of scars and contractures.

Fig. 7

The wound treated with biopreparation remained clean, without apparent plasmorrhea. It was revealed that at the sections that were treated by embryonic epithelial cells and that were closed by cryoconserved chorion tissue wound healing proceeded significantly faster then without it. Skin surface at the places of deep skin injure was closed by even layer of granular tissue inside of which sections with spreading of terminal loops of vascular system could be seen (fig. 7).

The wound was ready for autodermoplastic performance during 17-th day (fig. 8, 9).

Fig. 8

During bandaging the smears were made where cells with long phylopodias, big nucleuses and 1-2 nucleoli can be seen.

Region of the left hand after skin removal was closed by chorion tissue, after its lysis fibroblasts that were grown in culture were applied to the wound section.

Results of patient’s peripheral blood examination during a month showed stable dynamics and absence of anemic state (indices of hemoglobin and erythrocytes see on fig. 12).

Fig. 9

Significant changes of biochemical indices were not revealed (fig.10).

Short-term increase of total protein was observed in response to skin autotransplantation.

Fig. 10

Creatinine and liver tests remained stable during all the process of treatment.

Application of biological preparations includes mechanisms of physiologic regeneration and inflammatory proliferation in response at burn wound area. Stem cells presence in the chorion does not exclude also the possibility of their migration and proliferation in wound region.

As result of these processes we observed generation of specialized granular tissue and connective-tissue elements. Growth factors containing in chorion tissue stimulate proliferation of granular cells and fibroblasts.

Further observation of the process of autoskin engrafting flow has shown that there were no complications connected with transplant rejection, skin surface was even and bright (Fig.11).

Fig. 11

To summarize, practicability of cord blood stem cells and preparations of fetoplacenta complex application for treatment of burn wounds was confirmed one more time by our research. High proliferative potential of stem cells, growth factors content in the preparations that stimulate cells division, decreased antigenic properties and bactericidal action make biopreparations unique and irreplaceable for combustology.