MODERN POSSIBILITIES OF THE USE OF STROMAL-VASCULAR FRACTION OF ADIPOSE TISSUE IN TRAUMATOLOGY AND ORTHOPEDICS Miromanov A.M., Miromanov M.M., Miromanova N.A. Chita State Medical Academy, Chita, Russia
Chita State Medical Academy, Chita, Russia
Over the last centuries of practice in traumatology, some various conservative and surgical techniques for treatment of locomotor system abnormalities have been developed, as well as techniques for stimulation of reparative regeneration of tissues. Despite of various medical technologies, the problem of fast and complete restoration of bones, cartilages and other tissues is still actual. During the last years, the important topic is research of influence of stem cells (SC) on the processes of tissue regeneration [43].
Objective – to reveal the possibilities of the mesenchymal multipotent adipose tissue cells, to compare their osteogenic and chondrogenic differentiation with the stem cells of the bone marrow, and also to outline the boundaries of their use in traumatology and orthopedics.
Human stem cells have become the attractive candidates for cellular therapy promoting the lost functions of cells and tissues. These unique cells can self-update endlessly and differentiate into other tissues [6, 13, 25]. The use of the potential of these pluripotent stem cells can offer other variants of therapeutic treatment of various diseases. From the moment of primary creation of SC lines in 1998, some great advances have been achieved in better understanding of stem cell biology and of requirements for pluripotency maintenance [42].
Confirmation of the first clinical tests of SC for treatment of spinal cord injuries and macular degeneration in 2010 has marked the new era in regenerative medicine [37].
When studying the fat tissue as one of the main sources of stems cells, some scientists gave their attention to stromal-vascular fraction used as the physiological regenerative substrate [24. 40].
This fraction promoted the provision of tissue homeostasis and influenced on regeneration of bone, cartilaginous and other tissues by means of an ability to self-update and differentiate in several lines. The main component is multipotent mesenchimal stromal cells (MSCs) of perivascular location [22, 29]. These cells can differentiate into various tissues by means of inductors and microenvironment of the cell – “the specific niche” [44, 45].
The bone marrow substance was considered as the source of multipotent cells over the long time. However in 2001, Zul et al. described the new adipose tissue-derived stem cells (ADSCs) after the procedure of liposuction [50]. The liposuction tissue is prepared with collagenase with subsequent centrifugation to get the packed cells on the bottom of the test tube. The packed cells are presented by so-called stromal vascular fraction. Actually, ADSCs present the heterogenous cellular population of red blood cells, fibroblasts, endothelial cells, smooth muscular cells, perithelial cells and adipose tissue stromal stem cells which show the plastic adhesive properties. After cultivation of ADSCs in vitro, the cell population becomes homogenous over time, and is mainly presented by ADSCs [20]. Multipotent mesenchymal stromal cells of adipose tissue stromal—vascular fraction also demonstrate the ability to differentiate into adipocytes, osteoblasts, chondrocytes and myocytes. Moreover, the liposuction procedure is simple, more comfortable and is associated with lower amount of complications [12].
ADSCs are easier to derive since they are located near the periendothelial region of vessels, and the adipose tissue with high amount of vessels is still considered as the most common and available source of these cells, whereas bone marrow stem cells (BMSCs) are located in deep bone structures. The amount of ADSCs is higher than cells derived from bone marrow since the bone marrow aspirates give 6 × 106 of nucleated cells per ml on average, and stem cells – only 0.001-0.01 %. Conversely, 2 × 106 of cells can be derived from 1 g of adipose tissue, and 10 % of cells are considered as stem cells. This feature of ADSCs means the good source of cells for clinical administration. For example, 10 ml of bone marrow aspirates of an adult patient with only 6 × 103 – 6 × 104 of stem cells mean the insufficient cell population for clinical use. However about 1,000-2,000 cm3 of lipoaspirate can contain about 2 × 108 – 4 × 108 of stem cells in a patient without discomfort or complications. Such amount of SCs is sufficient for restoration of a small bone defect. An extensive passage in vitro for receive of adequate amount of cells is usually required for BMSCs, but not for ADSCs. The disadvantages of long term passage in vitro are possible contamination, long term labor-dependent and possible gene mutations during passage. Therefore, stromal-vascular fraction of adipose tissue can be considered as the most appropriate source of SCs in comparison with bone marrow [21, 22, 27, 30].
The German surgeon Gustav Neuber (1850-1932) used the fat tissue for grafting in surgery in 1893. He used the adipose autograft for correction of the lower boundary of the orbit [34]. Simultaneously, the German surgeon Eugene Hollaender (1867-1932) offered the mixture of human and mutton fat to prevent the reabsorption and, as result, complications after grafting [28]. However the highest amount of such operations were inefficient since the adipose tissue did not survive to the full degree, and oil cysts appeared in the region of its extinction, with subsequent transition into the necrosis zone under influence of microcirculation disorders [36].
Subsequently, Erich Lexer (1867-1937) published a study of clinical use of fat tissue for correction of posttraumatic deformation of the face, asymmetry of glandula mammaria and Dupuytren's contracture. He became one of the first authors who had shown the accuracy of collection of the allograft for successful survival [19].
After the detailed study of adipose tissue, M. Rodbell separated it into two fractions: mature adipocytes and stromal-vascular fraction including fibroblasts, perithelial cells, endothelial cells and pre-adipocytes [39].
Currently, separation and grafting of the fat autograft is both possible and safe [46]. Moreover, the valuable experience has become the estimation of the inductors influencing on differentiation of the stem cell into other tissues (the table) [2, 4, 5, 7, 17, 21, 48, 50].
Table
The inducing factors influencing on differentiation of multipotent mesenchimal stromal cells of stromal-vascular fraction of adipose tissue in other tissues
|
Author |
Phenotype |
Differentiation |
Differentiation-inducing factors |
|
Gronthos S. åt al. (2001) |
Positive: HLAABC, CD9, CD10, CD13, CD29, CD34, CD44, CD59, CD105, CD49e, CD54, CD55, CD166 Negative: HLADR, CD11a, CD11b, CD11c, CD14, CD18, CD31, CD45, CD50, CD56 |
In vitro: osteogenic, adipogenic |
Osteogenic: vitamin D3, dexamethasone Adipogenic: insulin, dexamethasone 1-methyl-3-isobutylxanthine BRL49653
|
|
Zuk P.A. åt al. (2001) |
Positive: CD13, CD29, CD44, CD49d, CD71, CD90, CD105, SH3, STRO1 Negative: CD31, CD34, CD45, CD14, CD16, CD56, CD61, CD62E, CD104, CD106 |
In vitro: osteogenic, chondrogenic, myogenic, neurogenic |
Osteogenic: vitamin D3, ascorbate, β-glycerophosphate
Chondrogenic: insulin, TGFβ1, ascorbate Myogenic: bovine and human serum, hydrocortisone
Neurogenic: β-mercaptoethanol
|
Considering the osteogenic differentiation of ADSCs and BMSCs, one should note the much better characteristics of BMSCs in relation to development of the bone matrix for future clinical administration. The determinate drug resistance factor is based on the issue: do ADSCs demonstrate much better osteogenic potential than BMSCs? If the answer is yes, ADSCs can be undoubtedly used instead of BMSCs for formation of the bone matrix [3].
In 2001, Zuk P.A. et al. firstly described the derivation of ADSCs from fat tissue and conducted some experiments for estimation of the phenotype and multiple potency. In their study, they found that the activity of alkaline phosphatase was higher in the human osteoinduced ADSCs than in BMSCs within three weeks of induction, whereas six weeks of induction caused 35 times higher matrix calcification in ADSCs and 68 times higher in BMSCs. Moreover, the authors realized the gene expression (specific osteogenic gene osteocalcin, alfa-1 subunit, Runt-associated transcription factor 2, osteonectin, osteopontin, bone morphogenic protein-2) of osteoinduced ADSCs and BMSCs. They showed the efficiency of ADSCs for recovery of both bone (filling of intraosseous cysts or for acceleration of bone tissue consolidation after surgery) and cartilaginous tissue [14, 16, 31, 50].
The positive results in treatment of cartilaginous defects of surfaces of big joints were noted by other researchers. After introduction of ADSCs into the joint cavity, the examination with magnetic resonance imaging showed the complete closure of defect by homogenous tissue with structure similar with cartilaginous tissue after one month. Moreover, the homeostasis of intraarticular system was noted with fast decrease in the inflammation factor with subsequent disappearance of pain syndrome [32, 38].
The efficiency of conservative therapy was identified in a study by Startseva O.I. et al. (2016) who investigated the combined intraarticular introduction of ADSCs and platelet-enriched fraction of the blood [39].
ADSCs also are used for recovery of biceps function by means of remodeling of brachial plexus. This fraction was put onto the nerve suture. It accelerated the regeneration process and increased the hyperexpression of neurotrophic factors in the site of the suture [18].
One should note that the great potential of differentiation into various tissues makes the risk of oncologic predisposition of this type of cells. According to the authors’ opinion, it was always the stumbling block for wide use of SCs in medicine. One of few studies of influence of ADSCs on breast cancer cells (in vitro and in vivo model) showed that ADSCs really increased the growth of active, but not resting cells of breast cancer cells. The authors state that extrapolation of these results can suppose the ability of ADSCs to stimulate breast tissue regeneration, but without influence on condition of dormant residual cancer cells [49]. A decrease in apoptosis rate in presence of ADSCs supposes the increasing growth of a tumor in the medium with ADSCs, despite of absence of increasing formation of timorous vessels [47].
In the individual mice model, the combined transplantation of ADSCs and active cells of prostate cancer cause more than three times increase in the tumor volume in comparison with mice without administration of ADSCs [33].
Other studies showed that human ADSCs, which were cultivated with triple negative cellular lines of breast cancer, did not influence on growth in the culture, but stimulated the metastases in other organs of mice in vivo. Such effects were not observed in the control group without ADSCs. One case showed the increase in vascular endothelial growth factor and density of microvessels. It means the increase in tissue angiogenesis, which can cause the disorders in the tumor bed [15, 35].
The short review of studies estimating the influence of MSCs (including human ADSCs) on growth of tumors and metastases indentified some difficulties in estimation of safety already at the preclinical stage. Having the data indicating the influence of MSCs on stimulation or alternative inhibition of tumor growth, the authors concluded that our modern knowledge on the mechanisms of MSCs influence is still poor, i.e. behavior of the cells is impossible to predict reliably. The authors note the absence of any signs of formation and growth of a tumor directly relating to the use of MSCs in all treated patients [26].
It is evident that subsequent reproducible studies are required. These studies should minimize the discrepancies in donor tissues, recipient cells, time of MSCs administration and parameters of monitoring. However the available findings are probably sufficient to exclude the use of grafts with ADSCs since there are few data on possible recurrence of tumor and metastases. Also the disadvantage of ADSCs is their difficult derivation. The first way is manual derivation with washing in saline with phosphate buffer to remove the blood cells, with collagenase (for simplification of subsequent derivation of various types of cells) and centrifugation for production of sediments including the vascular stroma and stem cells [8, 9, 23], or use of the special column with unwoven viscose and polyethylene fibers for derivation of cells of stromal vascular fraction from solutions. In contrast to centrifugation, this technique precludes the extensive process of hemolysis, resulting in provision of quality and purity of the derived material [10]. Another technique includes the use of automated equipment which is combined into the single system to prevent the influence of the human factor on the process. It decreases the risk of negative influence of external factors and precludes the microbial contamination [11, 41]. Active mitotic division of the fraction is initiated after three days. Moreover, the acceleration of this process requires for condition of physiological hypoxia of the cell, when intracellular level of oxygen is 5 % [1].
All above-mentioned facts suppose the availability of big laboratories, but it is impossible for many facilities. Unfortunately, the use of automated blocks for derivation and selection of SCs will be possible in the Russian Federation only in 2020. Certainly, this technique is the perspective direction in traumatology and orthopedics, but it requires for further extensive researching to decrease the various risks and to successfully use these findings for increase in efficiency of treatment.
CONCLUSION
Therefore, over the last years, the multiple experimental models of SCs in regeneration of organs and tissues have been developed. SCs show their restorative potential both through direct way of differentiation and through indirect way of influence on the “cellular niche”. The special interest is associated with adipose tissue-derived cells, i.e. stromal-vascular fraction including both mature and multipotent cells. The improvements in the modern technologies and tools have allowed to find and characterize the molecular mechanisms of regeneration of injured tissues. However due to great differential potential it is impossible to make the final conclusion on their clinical efficiency. Moreover, the studies of the differentiation of ADSCs in natural conditions did not find any evident results mainly due to absence of standards for use of this material. Certainly, the main task is creation of standard protocols for derivation, selection and differentiation of this cellular culture that will allow using this technology in traumatology and orthopedics in treatment of abnormal processes.
Information on financing and conflict of interest
The study was conducted without sponsorship. The authors declare the absence of any clear or potential conflicts of interest relating to this article.