Effect of acoustic pulses and EHF radiation on multipotent marrow stromal cells in tissue engineering constructs

R. K. Chailakhyan*¶, V. I. Yusupov, Yu. F. Gorskaya*, A. I. Kuralesova*, Yu. V. Gerasimov*, A. P. Sviridov, A. Kh. Tambiev, N. N. Vorobieva, A. G. Grosheva*, V. V. Shishkova*, I. L. Moskvina* and V. N. Bagratashvili‡ *N. F. Gamaleya Research Institute of Epidemiology and Microbiology Ministry of Health of the Russian Federation, 18 Gamaleyi, Moscow 123098, Russia V. I. Il'ichev Paci ̄c Oceanological Institute, Far-Eastern Division of Russian Academy of Sciences, 43 Baltiiskaya, Vladivostok 690041, Russia Institute on Laser and Information Technologies of Russian Academy of Sciences, 2 Pionerskaya, Troitsk, Moscow 142190, Russia Biological Faculty of Moscow State University 1-12 Leninskiye Gory, MSU, Moscow 119234, Russia ¶rubenchail@yandex.ru


Introduction
Nowadays, cell technologies constitute one of the most rapidly developing areas of biology and medicine. Regenerative medicine makes most widespread use of multipotent stromal cells (MSCs), 1-3 that were discovered and described, as a new population of marrow stromal precursor cells in the studies. 4,5 Multipotent stromal cells are already being utilized in clinics to repair tissue defects in cases of extensive deep thermal skin injuries, 6 to¯ll in facial soft tissue defects, 7,8 to regenerate myocardium, 9,10 to recover the integrity of bone tissues and hyaline cartilages of joints. 11,12 The creation of various organs is the subject of current scienti¯c research in many countries. [13][14][15] However, the quantity of stem cells in a marrow punctate taken from a patient is insu±cient for therapeutic purposes. The concentration of stromal precursor cells in the bone marrow is (1-5)Â10 À4 ; and in a suspension of hematopoietic cells they are strongly disunited by cells of di®erent kinds. For this reason, to increase the quantity of the stromal cells prior to their transplantation to the recipient, they are cultivated in vitro. E®ective methods were worked out to isolate these cells from the marrow and cultivate them in vitro, which provided for the increase in their numbers by more than 10 5 times. 16,17 Stromal progenitor cells, as well as their descendants have an enormous proliferation potential. 18 By the 10th passage the numbers of¯broblasts in some strains increase up to (1.2-7.2)Â10 9 cells. The cells undergoing up to 31-34 doublings in the course of their evolution.
Investigations 19 demonstrated that no abnormalities occurred in the chromosome set of MSCs preparations at¯rst passages, while at higher ones (7th-10th) certain abnormalities, such as cells of altered caryotype, polyploidy, and so on, revealed themselves in individual cultures. In this regard, the development of methods for increasing their content in the bone marrow and activation of proliferative potencies in vitro, is one of the key goals of tissue engineering.
One of the approaches that have been developed in recent years is the exposure of tissues and cell cultures to various physical factors capable of stimulating these processes. Such factors, applied in the case of stem cells, include low-intensity laser and extra-high frequency (EHF) electromagnetic radiations, acoustic waves and mechanical vibrations, and even ionizing radiation in small doses. 20 To illustrate, exposure of mesenchymal stem cells grown in culture to electromagnetic radiation in the red and near-infrared regions of the spectrum in a dose on the order of 1 J/cm 2 increases their proliferation. [21][22][23] A low-intensity mechanical stimulation of the cells at a frequency of 90 Hz 24 or 200 Hz 25 increases their proliferation and osteogenetic di®erentiation. Exposure to ultrasonic waves 1.5 MHz in frequency 26 and low-intensity electromagnetic millimeter waves 27 also proves e®ective.
In our previous in vivo studies, a short-term irradiation with a CW laser of moderate intensity 1.56 m in wavelength doubled the concentration of MSCs in the bone marrow. 28 Exposure of MSCs in vitro to a low-intensity He-Ne laser together with low-intensity EHF radiation resulted in a 1.5-2-fold increase of their proliferation activity. 29 Another approach used by us is that cells are exposed to acoustic pulses of laser-induced hydrodynamic (ALIH) processes, which were found in water and water-saturated biotissues near¯ber tip with laser radiation. [30][31][32] Interest in conducting such investigations stems from the fact, that according to recent information, the mechanism, responsible behind the high curative e®ect of moderate-power (1-10 W) laser radiation in the treatment of various diseases, including such serious ones as osteochondrosis and osteomyelitis, 32,33 is associated exactly with ALIH processes. We believe that the main role in the enhancement of the activity of cells is played by the wideband acoustic signal accompanying these processes. 34 As it was demonstrated in vitro studies 35,37 the action of the ALIH processes and EHF radiation reliably resulted in a 40-80% increase of the proliferation activity of MSCs.
This work is aimed at investigating the e®ect of ALIH pulses and EHF radiation on the in vivo formation and¯nal size of a bone marrow organ comprising a bony and a hematopoietic tissue.

Materials and Methods
Experiments were carried out on CBA mice con- A suspension of precipitated bone marrow cells from CBA mice were exposed to ALIH pulses and EHF radiation separately and also in combination in di®erent sequences ALIHþEHF, EHFþALIH. Tissue engineering constructs (scaffolds in the form of gelatin sponges 2 by 2 by 2 mm in size containing 10 7 nucleated bone marrow cells) were then prepared. To grow bone marrow organs, all the tissue-engineering constructs were implanted under the renal capsules of syngeneic mice. All the experimental mice were divided into the following six groups (of seven animals each) in accordance with the kind of the physical factor their transplanted cells were exposed to: (1) EHF, (2) EHFþALIH, (3) ALIH, (4) ALIHþEHF, (5) ALIH in sponge, (6) control-unexposed. The cells in group 5 (ALIH-sponge) were¯rst embedded in a sponge and then exposed to ALIH pulses. The transplants were examined within three and¯ve months of treatment. Subject to investigation in the newly formed hematopoietic organs were the total number of hematopoietic cells, e±ciency of colony formation of MSCs (ECF-MSCs), the number of MSCs, and the weight of the bone capsule of the transplant.

Physical factors used to treat bone marrow cells.
(1) MSCs, either precipitated in a test tube or embedded in a sponge, were exposed to ALIH pulses in an original apparatus-ALIH simulator. 35 Prior to experiment, the memory block of the simulator was loaded with an acoustic signal obtained when forming laser channels in the nucleus pulposus of intervertebral disc in vitro. 32 As can be seen from Fig. 1, this wideband acoustic signal is a quasiperiodic sequence of short pulses 2 kPa in pressure amplitude, which are known [29][30][31]33 to be associated with the generation and cavitation collapse of steam-gas bubbles near the hot tip of the laser¯ber. During experiment by means of a piezoceramic transducer the recorded signal initiates the acoustic pulses, similar to those shown in Fig. 1, in the water-¯lled chamber of the ALIH apparatus. The MSCs under test, either precipitated or embedded in a sponge, were placed, together with the culture solution, in a test tube that was then immersed in the operating cell of the ALIH apparatus and exposed to ALIH pulses for 50 s.
(2) Exposure of precipitated MSCs to a lowintensity EHF radiation 7.1 mm in wavelength and 5 mW/cm 2 in power density was carried out with a EHF source (Akvastin IRE-Polyus, Russia). A test tube, containing the precipitated MSCs of interest, was placed at the output of the EHF emitter. The exposure time came to 30 s, which corresponded to an exposure dose of 150 mJ/cm 2 .
(3) The combined e®ect of ALIH pulses and EHF radiation on bone marrow cells was studied using MSCs precipitated in test tubes were treated using one of the following four exposure regimens: ALIH, EHF, ALIHþEHF, and EHFþALIH. The cells embedded in sponges were exposed to ALIH pulses only. The controls were sponge transplants containing unexposed cells.
Preparation of Bone Marrow Suspension. The shin and thigh bones of ether-killed mice were excised. The epiphyses of the bones were cut o® and the bone marrow was then washed out into a nutritional medium with a syringe. A homogeneous marrow cell suspension was prepared. The suspension obtained was¯ltered through a 4-ply kapron lter and the number of cells was then counted. The cells were centrifuged (10 min, 1000 rpm) at 4 C.
Transplantation under the Renal Capsule. A longitudinal incision was made on the left side of the anterior abdominal wall of mice under Nembutal anesthesia (40 mg/kg), through which the left kidney was exteriorized. With the kidney held fast, a small pouch was formed with a pair of eye forceps under its capsule, into which a sponge containing 10 7 marrow cells was transplanted.
Extraction of Hematopoietic Cells from Transplants and Their Explantation to Tissue Cultures. The formed hemopoietic organ was isolated from the renal capsule and placed in a hole with 1 ml of medium. Hematopoietic tissue was separated from the bone capsule using of an ophthalmic scalpel; a suspension of bone marrow cells was prepared and is ltered through a 4-ply nylon¯lter; the number of isolated cells was counted. Cells were explanted in 25 cm 2 culture°asks (NUNC) (on the basis of 1.0-2.0 h 105 cells per vial) with 5 ml of complete medium consisting of 80% of -MEM (Sigma), 20% serum cow embryos (Ny clone) and antibiotics.
Cultivation was conducted at 37 C under 5% CO 2 . Discrete colonies clones, consisting of several thousand¯broblast, were formed in monolayer cultures of bone marrow cells at 12-14 days (Fig. 2). The clonal nature of the colonies permits to determine the number of MSCs in the test suspension. 4 To calculate the number of colonies, cultures were washed twice with saline solution,¯xed 70 ethanol and stained with azure-eosin. The e±ciency colony formation (ECF) of MSCs (number of grown colonies produced after explantation of 10 5 bone marrow cells) was estimated by the number of grown colonies of stromal¯broblasts.
Determination of the Dry weight of the Bone Capsule. The bone capsules, left to stay overnight in 70 alcohol, were removed and transferred for 30 min to an ether/alcohol (50/50) mixture and thereafter to pure ether for an hour. Within 5-10 min after the removal of the capsules from ether, their dry weight was determined.
Statistical Processing of the Results. The data of interest were presented in the form of averages for no less than three experiments, together with standard deviations, M AE m. The results were compared using the Student t-test, the di®erences being considered statistically valid at p 0:05.

Results and Discussion
This work is a continuation of research of separate or combined e®ect of various physical factors on MSCs. It was shown that the impact of the certain physical factor, in particular, on the precipitated cells of di®erent origins gives a various results. Exposure to EHF radiation and acoustic pulses of ALIG on MSCs of rabbit did not lead to increasing proliferative activity of cells in vitro. The trend towards inhibition of proliferative activity was observed for the combined impact in sequence EHFþALIG. However, a statistically signi¯cant (p < 0:05) increase in cell proliferation (80%) relative to the control results were observed in all cases at the action of acoustic pulses on human MSCs. At the same time, the proliferative activity of the human and rabbit MSCs, which were adhered on plastic culture plates, increased in 2.5-3 times under EHF radiation. Studies such as the perosseous heating of bone marrow of a rat shin using the helium-neon laser radiation in vivo, as well as the impact of EHF radiation by means of a submersible antenna on the suspension of bone marrow cells in vitro, which led to similar results-a doubling of MSCs in the bone marrow, deserve attention. Previously, we have studied the e®ect of physical factors either on a change of the proliferative activity of MSCs in vitro, or on MSCs content in the bone marrow. In this study, the impact of physical impacts on MSCs that form the heterotopic hematopoietic organs under the kidney capsule was studied along¯ve parameters.
Investigations conducted within three and¯ve months after transplantation showed that in all cases hematopoietic organs successfully formed in place of the transplants (Fig. 3). As one can see from this¯gure, distinct under the renal capsule is a bone capsule; and beneath it and over the renal tissue there are hematopoietic cells.
During the period between three and¯ve months after transplantation, the transplants grew much larger. This agrees with the data, 36,38,39 obtained earlier, and is supported by the results of the hematopoietic cell counts (Tables 1 and 2). Within ve months the number of these cells in the control group came to (5:9 AE 1:2Þ Â 10 6 ; i.e., they multiplied by more than three times as compared with their quantity within three months of transplantation. The number of hematopoietic cells in the rest of the groups increased during this period by a factor from 3 to 10, the increase being as high as 20-fold in  One can see from Tables 1 and 2 that the maximum MSCs concentration was observed in the transplants of the EHFþALIH group, both within three and¯ve months after treatment, and was more than two times as high as the corresponding control values. It is interesting to note that treatment in the opposite order, namely, ALIHþEHF group, reliably led to a gradual decrease of the MSCs concentration in the transplants to 50% within three months and to 26% of the corresponding control values within¯ve months of treatment. During the period between three and¯ve months after transplantation the increase in the MSCs concentration in all the other groups is caused mainly from the increase in the number of hematopoietic cells in a transplants. The extent of this increase in the EHFþALIH and ALIH-sponge groups (in 3.9-fold) did not di®er signi¯cantly from the control group (4-fold). The increase in the EHF group was higher (5.6-fold), while in the ALIHþEHF group it was reliably lowered (2.1-fold during three months).
The minimum MSCs concentration per transplant was observed after three months in the ALIH group, amounting to 20% of the control values. However, it increased to 50% of control values after ve months. In the ALIH-sponge group the MSCs concentration was substantially higher (by six times) than in the ALIH group and exceeded the control level by a factor of 1.3, but it was lower by a factor of 1.7 than in the EHFþALIH group. After ve months exceeding the MSCs concentration in the ALIH-sponge group over the ALIH group was only by a factor of 2.5 times. At EHF treatment, the MSCs concentration was higher by 1.2 times than in the control group after three months and 1.7 times after¯ve months.
Note that the results of these in vivo studies into the proliferation of MSCs con¯rmed the results obtained by us in the preceding in vitro investigations, 35,37 where the enhancement of the proliferation activity of MSCs reached 40-80% in the cases of their exposure to ALIH pulses and to combined EHFþALIH e®ects.
Thus, the relationships between the MSCs concentrations in the corresponding three and¯ve months old transplants were similar; i.e., the differences in quality between the transplants due to their being exposed to di®erent physical factors were conserved and sustained over the entire lifetime of the transplants. Bearing in mind the fact that the organization of the transplants is realized by the cells from the MSCs population that are responsible for transplantability, 26 one can conclude that the physical factors used in this work were aimed, among others, at this category of cells.
As for the e±ciency of colony formation, during the period between three and¯ve months after transplantation the number of colonies in each group varied comparatively little: changes almost did not happen in the EHFþALIH, ALIH in sponge groups, and control; for EHF group it decreased by a factor of 1.7 after¯ve months. The exception is again the ALIHþEHF group, where the number of colonies dropped by a factor of 9.5.
The plots in Fig. 4 present the numbers of MSCs and dry weights of bone capsules, normalized to the corresponding average control values, within three and¯ve months after exposure to the physical factors. With such representation, the control values in both plots correspond to unity. As can be seen from Fig. 4(b) and Tables 1 and 2, the dry weight of the three months old bone capsules in the EHFþALIH and ALIH-sponge was greater by a factor of 1.7 and 2.4, respectively, than in the control groups. The bone capsule weights in the EHF groups and ALIHþEHF groups within three months after treatment did not di®er from the control groups.
The corresponding results for the¯ve months old bone capsules in the EHFþALIH and ALIH-sponge groups did not di®er reliably from the controls, whereas those for the bone capsule weights in the EHF and ALIHþEHF groups showed them to be more than half as high as in the controls.
The ability to osteogenesis is one of the fundamental properties of MSCs, which identi¯ed areas of initial use of MSCs for orthopedics and traumatology. Due to this, the determination of the number of colonies, positive for alkaline phosphatase, which is the main indicator of osteogenesis cells, is very relevant. In the three months old transplants, the number of phosphatase-positive colonies in the EHFþALIH and ALIHþEHF groups exceeded over the \control" groups by a factor of 1.3-1.4. The most high exceeding (by 1.6 times) of the number of phosphatase-positive colonies was in the EHF group. In the¯ve months old transplants such exceeding was kept in the groups mentioned above.
It could be assumed that in the newly formed blood-forming organ the number of nucleated cells can play a role of a determining parameter of physical factors in°uence. However, our studies have shown that there is no correlation between such parameters as the number of nucleated cells, the bone capsule mass, number of MSCs and other. This indicates that the formation of the transplants has its own characteristics after each physical e®ects in di®erent experimental groups. A correlation was observed only in separate groups, even for such parameters, close in their e®ects, as mass of the bone capsule and the percentage of phosphatasepositive colonies.
Our investigations showed that in transplants with a comparatively small number of hematopoietic cells the quantity of MSCs was, more often than not, lowered as well, there being no correlation between the weight of the bone capsule on the one hand and the numbers of hematopoietic cells and MSCs on the other hand. Also, no correlation was observed to occur between the number of hematopoietic cells in the transplants and the e±ciency of colony formation of MSCs. Thus, the abovementioned groups of transplants are observed to di®er by the relations between the number of hematopoietic cells, the e±ciency of colony formation of MSCs, their quantity, and bone capsule weight. From this, it apparently follows that the process of formation of the transplants following their exposure to each individual physical factor featured speci¯cs of its own.
On the whole, it follows from the results obtained that the combined e®ect of EHF radiation and ALIH pulses proved most favorable for the formation of bone marrow organs, providing for the maximum MSCs concentration in the transplants (2.2 times as high as in the control) and a bone capsule weight comparable with the control value. What is very important is that the bone capsule weight accumulation rate in the transplant of EHFþALIH and ALIH-sponge groups during thē rst three months after transplantation exceeded that in the control groups by a factor of 1.7 and 2.4, respectively. This suggests that these physical factors can be used to accelerate rehabilitation processes.
This work was supported by the Russian Foundation grant 14-25-00055 (in the part of LIH and EHF treatment methods) and by Russian Foundation for Basic Research, grants 13-02-00438 and 13-04-12032 (in the part of the cell and transplant experiments).