Effect of Cryopreserved Cord Blood Serum and Placental Extract on Cold-Wound Healing

The dynamics of planimetric and microbiological indices of cold wounds during therapy with cryopreserved cord blood serum (CCBS) and placenta extract (PE) has been studied. Cold wounds were modelled in Sphinx using the cryodevice with 8.0 mm applicator, temperature of –195°C and 60 sec exposure. Injections were started on day 3 following cryodestruction intramuscularly each other day by 0.1 ml/kg of body mass (5 injections in general). On day 7 after CCBC or PE introduction the wound area decreased 2.8 or 1.9 times, correspondingly. Wound area in non-trated animals decreased only to day 9. On day 21 the wound area in animals with PE treatment was 3.3 times smaller, than in non-treated ones, and CCBC application resulted in full epithelization of wounds. Application of CCBC and PE contributed to elimination of microbes in wounds. Number of germs in wounds of rats which were treated with CCBC or PE decreased starting from 7th day of observation (3.4 or 1.9 fold, correspondingly). On day 21 the number of germs in wounds of rats treated with PE was 8.4 times less than in control, and in case of CCBC no patogenic microflora was present. Thus, an expressed stimulation effect of CCBC and PE on wound healing was shown. The presented results offer the prospect to include the CCMA into the wound treatment protocols.

Processes of sanogenesis resulting from the injury and targeted to recover the anatomical and functional integrity of damaged tissues, i. e. wound healing, are affected by various factors of both general and local characters, in particular the degree of its contamination and microbial species [1,14,17]. The healing rate is a total index of wound healing efficiency, determining the time of patients disability. Searching the ways to reduce a wound healing time is an important task in current surgery. One of these ways may be the supplementation of treatment protocols with therapeutic agents of natural origin, containing a combination of biologically active substances with systemic regulatory impact on sanogenesis processes: natural regulators of metabolic processes. As we believe such agents include the cryopreserved cord blood serum (CCBS), the effect of which on metabolic processes was emphasised in papers of B.P. Vvedensky et al. [19], Ya.A. Popovich et al. [13], as well as placenta extract (PE), a medical product from pharmacotherapeutic group of agents affecting metabolic processes.
The research was aimed to study the dynamics of planimetric and microbiological indices of cold wounds in the rats treated with CCBS and PE.
Wound planimetric indices were studied to days 3, 7, 9, 14 and 21. To provide impartial evaluation of cold wound healing dynamics during experiment we calculated the absolute and relative planimetric indices. The wound area was measured in digital images using the Adobe Photoshop CS3 software (Adobe Systems, USA). The percentage of area reduction (PAR) of wound from initial one was calculated by the formula: PAR = (S 0 -S)/S 0 ×100%; S 0 was initial average wound area at the beginning of therapy, S was average wound area at the time of measurement [9]. The healing rate (HR) of wounds was calculated by the following formula: HR = (PAR 1 -PAR 0 )/T; where PAR 1 was the percentage of area reduction of wounds from initial one at the time of measurement; PAR 0 was the percentage of area reduction of wounds during previous measurement; T was a number of days between measurements [4].
Cold wounds were microbiologically studied to day 7, 14 and 21 of the experiment. The material was prepared for inoculation and then seeded according to the requirements stated in the practical guidelines for handling with microorganisms [2,3,15]. Tissue fragments from fibrin-leukocyte wound crust were taken under aseptic conditions, the weight for each bioptic specimen was determined. The obtained bioptic specimens were disintegrated in homogenizers and placed into glass vials, supplemented with 5 ml of sterile physiological saline. The vials were shaken with mechanical shaker (350 rpm) for 30 min. Serial dilutions in physiological saline were prepared from obtained tissue suspensions, thereafter 0.2 ml were transfered for seeding to Petri dishes filled with following growth media: meat-peptone, 5% blood, chocolate, and vitelline-salt agars, anaerobic hem agar, MacConkey agar, and agarized Sabouraud medium [8,10,12]. Platings in anaerobic hem agar and a part of those in 5% blood agar were cultured in anaerobic jar for 4 days at 37°C in oxygen-free gas medium. The 'GasPak EZ' system (Dickinson and Co, USA) was used to create anaerobic conditions. Inoculations in chocolate agar were cultured within a day at 37°C in atmosphere with 6-8% CO 2 . Those in Sabouraud medium were cultured for 48 hours at 30°C. All other ones were cultured at 37°C for a day. Following the incubation of the samples the macrocolonies of microorganisms grown on agarized growth media were assessed and a number of microbial cells per 1 g of tissue taken from wound and adjacent tissues was calculated [2,3]. Colonies were counted in those dishes, where the visible clusters of growing bacteria were found. The grown colonies of microorganisms were identified in accordance with the formal recommendations [2,3,8].
Statistical processing was performed using the Excel 2003 (Microsoft, USA), and SPSS v.10.0 (SPSS Inc., USA) software package. The data were presented as mean ± standard deviation. The significance of the differences between samplings was estimated by non-parametric Mann-Whitney U-test, the differences were assumed as significant at p < 0.05.

Results and discussion
Reparative processes, occurring in skin injuries are universal and independent on a damaging agent type. Healing of any wound has a similar dynamics [5], providing the possibility to apply the objective criteria for assessing wound process irrespective of a wound type. The wound detersion, reduction and elimination of edema, hyperemia, and infiltration in wound area, the appearance and development of granulation tissue, contraction and epithelization of wound surface are the most obvious clinical signs, characterising the wound process course, which directly depend on the amount and type of microorganisms in a wound.
It is known that a site of skin-located cryonecrosis transforms in a few days into a dry crust rejected in 2-3 weeks, depending on a size of destroyed site [11]. All this time the cryonecrosis accomplishes the role of protecting biological dressing, therefore neither surgical treatment nor other manipulations with wounds were done.
The results of wound area measurement after skin cryoinjury in experimental animals are shown in Table 1. Proceeding from the features of cold wound reparative dynamics in different groups, the values of PAR and HR of wounds were calculated between such observation terms, when the statistically significant differences in a wound area were recorded. The wounds area in CG animals to day 7 was not significantly different from those to day 3 after cryodestruction of investing tissues. At the same time the CCBS and PE application resulted in a significant decrease of the studied index.
Результаты измерения площади ран после криоповреждения кожи у экспериментальных жи-testified to the severity of low temperature injury in tissues. Within this observation period the areas of wounds in experimental groups were significantly lower than in the control group: by 2.8 and 1.4 times after CCBS and PE administration, respectively. The wound PAR in EG-1 was 78.0, and in the rats received either physiological saline or PE no significant changes in wound area from days 7 to 9 was recorded.
To day 14 of the experiment the wound area in EG-1 was 6.1 times lower than in CG, and the wound PAR was equal to 91.5. In the EG-2 the wound area reduced by 2.1 times, the PAR was 74.8. The wound area in CG animals remained unchanged from days 9 to 14, the significant differences were found only between the indices of days 7 and 14 of observation, the wound PAR within this time period was 48.2.
To day 21 of the experiment the wounds in the rats received CCBS were completely epithelialized. If comparing with the results in CG the PE application resulted in a reduction of wound area in 3.3 times, the PAR was equal to 91.7. To the mentioned observation term the wound area in non-treated animals was reduced by 72.8%.
In non-treated rats the wound HR was also reduced with the course of time. Within the period from day 3 to 9 of the experiment it was 6.3%/day, from day 7 to 14 and from 14 to 21 it was 4.3%/day and 3.5%/day respectively.
Due to the fact that in this research we used the coat-deprived animals, the wound epithelialization had a marginal character. It is known that the creep of epithelium on the side of healthy skin is only possible if granulation tissue surface is present [17]. Proceeding from this fact the results obtained in the group of CCBS treated animals confirm an adequate development of granulation tissue and indirectly testify to the activation of local immune responses.
Any wound is the 'open gates' for pathogenic microorganisms entering. Presence of non-viable tissues in the wound, vascular and other trauma-induced disorders, contribute to microflora propagation, which, in its turn, leads to development of inflammation and accompanying cascade of morphofunctional disorders at a local and systemic levels. Disorder in local immune processes slows down the wound eradication from pathogenic microorganisms. Consequently the wound cleansing is slowed down, thereby preventing its epithelialization.
Скорость заживления, %/сутки Wound healing rate, %/day Срок наблюдения, сутки Observation term, days number in the wounds of CCBS animals decreased by almost two orders (91 times). Similar dynamics of the studied indices remained to day 21 of the experiment as well. Bacterial contamination of wounds in the control group rats was (1.95×10 5 ) CFUs/g, the PE administration was accompanied by a decrease in microorganism number down to (2.32×10 4 ) CFUs/g (in 8.4 times). The CCBS therapy resulted in wound eradication from pathogenic organisms.
the cold wound tissues of the control group animals yielded the following: Staphylococcus aureus, E. faecalis, Proteus vulgaris, S. pyogenes, Fusobacterium necrophorum, Peptostreptococcus spp. The tissues of PE treated animals had S. aureus, P. vulgaris, S. pyogenes, F. necrophorum, Peptostreptococcus spp. As mentioned above, there was no microflora growth observed in the inoculations from bioptic specimens of animals received CCBS.
The analysis of bacteriological parameters of cold wounds in rats of different groups demonstrated a significant effect of systemic administration of CCBS and PE on microbial contamination of wounds and the species composition of their microflora. If no therapy was performed the bacterial contamination of wounds decreased slowly, and the species composition of microflora remained unchanged. We believe this fact is one of the main reasons of slow cold wound healing in the control group animals. Delayed wound healing in non-treated animals was confirmed by absence of significant differences between the wound area between days 3 and 7; 7 and 9; 9 and 14 after cryoinjury.
The CCBS and PE introduction to animals resulted in a significant reduction in a number of germs in wounds, and it was accompanied by the narrowing of their species composition range as well. Of note is the fact, that we observed a wound eradication from the pathogens, significantly slowing down the reparative processes in it. The intensity of CCBS therapeutic effect was higher than the PE one.
One-direction effect of CCBS and PE we explain by their composition of biologically active substances, being physiological for animal organism and a balanced effect on immune, endocrine, nervous and hematopoietic systems, which are in a close functional interaction. Proceeding from the statements of the concept about multilevel regulated homeostasis maintenance [10], the active effectors in both studied regulators of metabolic processes may act at any level of regulation: central, systemic, cellular, and molecular ones. Immune system is one of the most sensitive integrative parts of the body [7]. Normal immune response is provided by a coordinated participation of cellular, humoral mechanisms and a specific immune reactivity. Since the CCBS and PE have no direct antibacterial effect, it is most likely, that they activate the immune system responses (possibly through cellular mechanisms) and other associated systems mentioned above. More pronounced CCBS effect on wound reparative processes is most likely due to differences in CCBS and PE composition.
Представленные результаты свидетельствуют о возможности включения КСКК в протоколы лечения ран. Перспективным направлением дальнейших исследований может стать сравнительное изучение влияния регуляторов метаболических процессов ксеногенного происхождения на динамику заживления ран. a narrowed range of species composition of wound microorganisms, and their accelerated epithelialization. The CCBS and PE effect was found to be one-direction, and was manifested in an improved healing mostly in the first phase of wound process, wherein the CCBS therapeutic efficiency was higher than the PE one.
The results presented offer the prospects for the possibility to include CCBS into the wound management protocols. A promising direction for further research could be a comparative study of the effect of regulators of metabolic processes of xenogenic origin on the wound process dynamics.