BioNanoSci. DOI 10.1007/s12668-016-0306-x
Application of Allogenic Adipose-Derived Multipotent Mesenchymal Stromal Cells from Cat for Tibial Bone Pseudoarthrosis Therapy (Case Report) Elena Yu Zakirova 1 & Anastasiya N. Valeeva 2 & Ruslan F. Masgutov 1,3 & Ekaterina A. Naumenko 1 & Albert A. Rizvanov 1
# Springer Science+Business Media New York 2016
Abstract A clinical case reported here demonstrates the possibility of using the multipotent mesenchymal stromal cells obtained from the donor animal to stimulate the process of bone formation in surgical treatment of tibia pseudoarthrosis in the cat. The cells were isolated from adipose tissue of the donor cat and express on their surface main markers of mesenchymal stromal cells—CD 44 and Thy-1. As a result of manipulation, the bone regenerate was formed and the support function of the injured limb was restored. Keywords Pseudoarthrosis . Mesenchymal stromal cells
1 Introduction Fracture of the pets’ limb bones is quite a common injury encountered in veterinary practice [1]. One of the complications in the treatment of this pathology is the formation of pseudoarthrosis, which is characterized by the appearance of bone mobility in the uncharacteristic location whereby the limb loses its supporting function. Still in veterinary as well is in human medicine, there is no optimal noninvasive technique for the treatment of pseudoarthrosis and surgical treatment is still used in most cases for this pathology [2]. * Elena Yu Zakirova
[email protected] * Albert A. Rizvanov
[email protected] 1
Institute of Fundamental Medicine and Biology, Kazan (Volga Region) Federal University, Kazan, Russia
2
Kazan State Academy of Veterinary Medicine, Kazan, Russia
3
Republic Clinical Hospital, Kazan, Russia
Stem cells have attracted attention of scientists because of simplicity of isolation from patients and their ability to differentiate into various cell types [3, 4]. In particular, there are a number of studies dedicated to the regeneration of bone tissue using adult stem cells [5–7]. At present, the prospects of using allogenic mesenchymal stromal cells (MSCs) in veterinary medicine are great. MSCs are widely used in the treatment of various pathologies in dogs and horses, but information about the treatment of cats is very rare. The properties of dogs’ and horses’ adipogenic MSCs are characterized enough, while the information about adipogenic MSCs of cats is insufficient [8]. Currently are known the studies on the application of allogenic MSCs for the treatment of chronic kidney disease in cats. By the results of these studies, it can be concluded that the intravenous administration there has no significant improvement of renal function immediately after injection [9, 10]. The use of MSCs for the treatment of traumatic lesions of the eye in cats has yielded positive results: subconjunctival administration of a suspension of allogenic adipose-derived MSCs stimulated the healing of posttraumatic corneal ulcers in cats [11]. Autologous adipose-derived MSCs were successfully used for the treatment of chronic gingivostomatitis in cats [12]. The introduction of allogenic adipose-derived MSCs has a positive impact on the treatment of chronic enteropathy in cats [13]. In the available literature, we have found no articles describing the use of allogenic adiposederived MSCs in the treatment of injuries and diseases of the musculoskeletal system in cats. However, there are some reports demonstrating cell therapy efficacy in the treatment of pseudoarthrosis. As we have shown previously, the autologous multipotent mesenchymal stromal cells (MMSCs) exert a stimulating effect on osteoregeneration processes during the surgical treatment of pseudoarthrosis of the dog’s tibia [14]. Furthermore, the possibility of stem cells using in the treatment of pseudoarthrosis
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Fig. 1 Radiograph of the tibia bone. (a) Full diaphyseal fracture of the left tibia. (b) Full diaphyseal comminuted fracture of the right tibia
was demonstrated in a rat model [15]. We hypothesized that allogenic MMSC derived from a donor will be as effective as autologous, during surgical treatment of bone pathologies. In this study, we described a clinical case of using of allogenic MMSC for the treatment of pseudoarthrosis of the cat’s tibial bone.
2 Case Report 2.1 Veterinary Anamnesis
Fig. 3 Radiograph of the tibia bone. (a) A false joint in the right shin. (b) Resorption of excessive regenerate in the left shin
Homeless cat, age about 3 years, received an appointment with dysfunction of both pelvic limbs. Animal defense community BZoozabota^ took care of the animal. During examination, the asymmetry in the area of left and right shins, pain, swelling, breach of skin integrity, and bone mobility outside the joint were found. It has been assigned the additional radiographic examination. X-ray images were taken using X-ray machine Maxivet HF (Germany). Radiographs cover all the damaged bone with two adjacent joints for the correct anatomical and topographical orientation. Based on clinical data and radiographs, they were diagnosed with an open full diaphyseal fracture of the left tibia with displacement along the length with shortening and open full diaphyseal comminuted fracture of the right tibia with an offset angle (Fig. 1 (a), (b)).
All manipulation with the animal was in accordance with a resolution of a local Ethical Committee of Kazan (Volga Region) Federal University. General potentiated anesthesias were used for the operation, including premedication with 0.3 ml of 0.1 % solution of atropine sulfate (BDalkhimpharm,^ Russia) subcutaneously, 1 % diphenhydramine solution (BBiosynthesis,^ Russia) was applied by intramuscular injection of 0.3 ml, ketofen (BMerial,^ France) subcutaneously 0.6 ml, and 2 % solution of xylazine (BInterchemie,^ the Netherlands) by intramuscular injection of 0.6 ml. For the general anesthesia, Zoletil (BVirbak,^ France) was used at a dose of 4 mg by intravenous bolus injection. As a method of treatment, the submersible osteosynthesis was chosen by the uncoated steel plate 12H18H9T, which was mounted on the tibial bone with cortical
Fig. 2 Osteosynthesis of left and right tibial bones with plates
Fig. 4 A false joint in the right shin. The limb twisted at an angle of 180°
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Fig. 5 X-ray of the right shin after repeated repositioning of bone fragments with MMSC in between-fragment gap
screws (Fig. 2). All procedures were made under the conditions of veterinary operating room in compliance with the rules of aseptic and antiseptic. Fibulas left unfixed. Operating access was made from the medial surface of the tibia using wide incision. An operative wound was sutured by broken knotted sutures. After 30 days, plates were removed under anesthesia. Wherein in the region of the right shin, a Bfalse^ joint (pseudoarthrosis) was formed, which was accompanied by pathological mobility in the fracture zone and absence of bone callus formation evidence. In the zone of fracture, the osteoporosis of bone fragments ends, blunting their sharp teeth, the lack of intermediate and endosteal callus, and the presence of between-fragment gap were visualized (Fig. 3 (a)). In the left shin, fuzzy boundary ends of the fragment and thickening of regenerate shadow with nonuniform density were noted. In the former site of the damage newly formed cortical layer with adjacent to it, periosteum on the outer surface and endosteal layer on the inside were detected. At the same time, resorption of excess regenerate bone occurred (Fig. 3 (b)). The support function of the left shin was restored. The right tibia was twisted at an angle of 180°. As a result, its support function was completely disturbed (Fig. 4).
Fig. 6 The results of flow cytometry of cat’s MMSC
It was decided to carry out a second operation to eliminate the pseudoarthrosis and repositioning of bone fragments. To stimulate the process of osteoregeneration, we have proposed the single injection of a suspension of allogenic MMSC in the composition of fibrin glue Tissucol® Kit (BAXTER AG, Austria) in the place of bone fragment matching during the surgery process. For this purpose under the general potentiated anesthesia, surgical approach from the medial surface of the right lower leg was performed. The osteoclasis of the proximal and distal fragments and their reposition were performed, and fragments were fixed using the technique of submersible osteosynthesis with steel plate. The edges of the surgical wound were sewn with broken knotted sutures. Finishing radiograph (Fig. 5) showed a stable position of the tibia fragments and the presence of cloudy substance with fuzzy boundaries (arrow) which we determinate as Tissucol® Kit with MMSC in the bone diastase field. Isolation of MMSC and their subsequent cultivation was performed according to the previously described protocol [8]. In vivo observation of cells’ growth was performed using inverted microscope AxioObserver Z1 (Carl Zeiss, Germany). Cells isolated from cat’s adipose tissue had the fibroblastlike morphology. To determine the expression of surface markers in the MMSC of cat, fluorochrome-conjugated antibody to the antigen CD44—APC/Cy7 (no. 103028), Thy-1—PE/Cy5 (no. 328112), and CD 45—PerCp (no. 304026) (BioLegend, USA) were used. Staining of cells was performed according to the protocol of the manufacturer. The measurements were carried out using flow cytometer FACS Aria III (BD Biosciences). The high expression of MMSC markers (Thy-1—98 %, CD 44—95 %) was demonstrated using flow cytometry method while the marker of hematopoietic cells (CD 45) was not expressed (Fig. 6). Cell preparation containing 2,000,000 cells was made immediately prior to transplantation. Cells on the sixth passage were trypsinized, counted, and washed by centrifugation from the remainders of the growth medium and trypsin. As a matrix for the MMSC, the fibrin glue Tissucol® Kit was used locally at the fracture site in the amount of 0.3 ml.
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Fig. 7 Zone of fracture after removal of the plate on the right shin
On the first day after surgery, the overall status of the animal was oppressed, the food excitability lacked, and the body temperature was within 39.7 °C. In terms of subsequent observation, general state of cat remained satisfactory. The body temperature on a third day after the operation returned to normal. Locally, the development of the inflammatory response was observed, which was manifested by edema, exudation, and violation of the support function of the operated limb. Wound process took place against a background of aseptic exudative inflammation. Operating wounds were healed by primary intention. On the first day, the process was more pronounced with wound exudate amount ≈1.5–2.5 ml, and on the next 5 days, it did not exceed ≈1–1.5 ml. The support function of the operated limb started to recover from the 10th day. The support function was maintained; when moving, the cat used the operated limb more confident. Moreover, medium degree of the leaning type lameness remained. The fracture was determined by palpation as a dense thickening area with considerable size, and there was a slight soreness. The mobility of bone fragments in the fracture zone was absent. Analysis of stepwise radiographs of leg bones in the lateral projection on the 10th day of the experiment has shown transverse fracture of the middle third of the tibial shaft fixed extramedullary by plate. The fracture line was clear and bone fragment consolidation signs were not observed. At the 20th day, the cat confidently relied on the operated limb. At the same time, the low degree lameness of leaning type was observed; pain was completely absent in the zone of fracture. During the X-ray on the 20th day, there was a trend of the boundary periosteal consolidation. Reposition of bone fragments without displacement, with initial signs of consolidation, was observed. On the radiograph, fuzzy fracture line was visualized. On the 60th day on the radiograph, the formation of callus limited by the periosteum with a clear cortical layer was determined, the fracture line was slightly visible, and the presence of endosteal callus was not expressed (Fig. 7). The integrity of the bone and the support function of the limb fully recovered. Bone fragments with adequate stress were fixed and painless. Palpation was determined dense, clearly limited thickening in the fracture zone.
In the described case, the nonunion formation in the cat was accompanied by the development of osteoporosis (Fig. 3 (a)). This disease significantly hinders the formation of a complete callus. Therefore, as a means of stimulating the processes of osteoregeneration, we have applied the MSCs. According to the literature, MSCs stimulate the regeneration processes in the experiments on the treatment of pseudoarthrosis of the femur of rats [15]. As a result, the treatment of a bone spur in cats was founded in physiological terms. Turning the limb in support function occurred on day 10, whereas dogs treated for experimental pseudoarthrosis of femur via extramedullary osteosynthesis without MSCs included the injured limb in supporting function only after 15 days [16]. Therefore, the introduction of MSCs did not have a negative impact on osteoregeneration and possibly stimulated it. We propose that the described protocol here can be used for the efficient treatment of pseudoarthrosis in veterinary practice. Acknowledgments The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University and subsidy was allocated to Kazan Federal University for the state assignment in the sphere of scientific activities. Some of the experiments were conducted using the equipment of Interdisciplinary Center for Collective Use of Kazan Federal University supported by Ministry of Education of Russia (ID RFMEFI59414X0003), Interdisciplinary Center for Analytical Microscopy and Pharmaceutical Research and Education Center, Kazan (Volga Region) Federal University, Kazan, Russia. Compliance with Ethical Standards All manipulation with the animal was in accordance with a resolution of a local Ethical Committee of Kazan (Volga Region) Federal University. Conflict of Interest The authors declare that they have no conflict of interest.
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