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DOI: http://dx.doi.org/10.20534/ESR-17-1.2-126-129
Khadjibaev Abdukhakim Muminovich, Republic Research Centre of Emergency Medicine Chief of Emergency Surgery Department Professor, MD, PhD Tilyakov Akbar Buriyevich, Republic Research Centre of Emergency Medicine MD, PhD, senior researcher of the traumatology department
E-mail: [email protected] Magrupov Bokhodir Asadullaevich, Republic Research Centre of Emergency Medicine Chief of pathologic anatomy department Professor, MD, PhD Urazmetova Maisa Dmitriyevna, Republic Research Centre of Emergency Medicine Chief of experimental medicine department Professor, MD, PhD Ubaydullaev Bobur Sabirovich, Republic Research Centre of Emergency Medicine Department of traumatology, MD Republican Research Centre of Emergency Medicine 2, Tashkent, Republic of Uzbekistan
The role of allofibroblasts transplantation in cartilaginous tissue regeneration process
Abstract:
Aim of investigation. Ground of embryonal allofibroblasts in the process of cartilaginous tissue regeneration. Material and methods. Investigation is based on the study the results of stimulation cartilaginous tissue regeneration process in the conditions of embryonal allofibroblasts application in 24 experimental sexually mature rabbits in which the model of symphysis pubis rupture with its following recovery have been used. Pieces of cartilaginous tissue have been fixed in 10% neutral formalin and embedded in paraffin. Histological sections were colored with hematoxylin, eosin and by Van-Gieson. Experiments duration — 7, 14, 21and 30 days.
Results. After pelvic bones symphysis pubis rupture, cartilaginous tissue is undergone alternative changes with following formation of lobulose chondromatous nodules of different sizes and sclerosis of surrounding muscular and osseous tissue. In the second series of experiment after pelvis symphysis pubis rupture and symphysis recovery with the help of cerclage, morphologic investigation results testify formation of mixed initial callus consisting of connective and chondromatous tissue elements. In the third series of experiment embryonal allofibroblasts were introduced into symphysis pubis rupture defect after its recovery by cerclage. It has been shown that within 4 weeks of experiment connective tissue having intensive basophil stain is formed in the area of symphysis pubis rupture. Connective tissue formation wit following formation of cartilaginous cells have been also noted in surrounding sift tissue structures.
Conclusion. Cartilaginous tissue formation process in the area of symphysis pubis rupture in experiment testifies stimulating influence of allogenic fibroblasts on cartilaginous tissue regeneration process in the experiment and it creates premises for applying this method in clinical practice.
Keywords: embryonal allofibroblasts; cartilaginous tissue regeneration.
Background
Cultivated allofibriblasts are considered as a perspective way of connective tissue regeneration [1; 2; 3; 4; 5; 6]. There are reports of good clinical results after allofibroblasts transplantation [2; 3; 4; 7; 8; 6; 9]. The stimulation of reparative osteo- and chondrogenesis to be considered as one of the possible way of their applying [10; 5; 8; 6; 9]. But a serious analysis is required subject to many issues which can occur at implantation of received culture of cells.
Fibroblasts have mesenchymal origin and are morphologically characterized as cells ofround fusiform flat form with processes and flat oval nucleus. Fibroblasts synthesize tropocollagen, collagen's precursor, intercellular matrix and basic substance of connective tissue, amorphic jelly-like substance filling space between cells and fibers of connective tissue and taking part in wound healing. Due to differentiation fibroblasts transform to less active mature cells — fibrocytes [2; 7; 8; 11].
Quantity of offered methods testifies about the necessity of going on search of more perfect, available, less cost-based ways of reparative osteogenesis stimulation which would allow to save advantages of traditional methods and to reduce their disadvantages [11; 12].
Goal — the experimental study of influence of embryonal allo-fibriblasts to the cartilaginous tissue regeneration processes.
Materials and Methods
Investigation is based on studying results of stimulation ofpel-vic bone's joint cartilaginous tissue regeneration process stimulating due to applying embryonal allofibriblasts (AF) in 26 sexually mature rabbits of "Chinchilla" race with the weight of 2,3-3,5 kg.
For approximation of experiment to those pathologic conditions which are observed at pelvis injuries in human, there was created an experimental model of pubic joint rupture. Care and experimental animals keeping were standard in accordance with requirements and principles of European Convention (Strasbourg, 1986) and Helsinki Declaration ofWorld Medical Association about humane treatment of animals (1996). Rabbits were bought from vivarium of Tashkent Medical Academy and kept in the vivarium of experimental department of Republican Research Centre of Emergency Medicine at 12 hours lighting, 20±2 °C room temperature, 50-70% humidity. Animals feeding was fulfilled according to prescribed ration with the use of mixed fodder. Animals have been divided into the following groups:
There were not underwent reconstructive-recovery surgeries in the first control group (8 animals) after pubic joint rupture.
There was underwent a symphysis restoration after pubic joint rupture with the help of cerclage from tantalum wire, 1,2 mm in diameter in the second group (8 animals).
ef Pic. 1. Cartilaginous tissue of symphysis pubis regeneration at different time points
Note: a - week 2. Necrosis and necrobiosis of cartilaginous tissue on the rupture border. Stained with hematoxylin and eosin (X10,20.) b - week 4. Chaotic order of fibrous structures in cartilage and dense interlayers in fibromatous tissue. Van Gieson's stain (X10, 40). c, d - week 1. Local intracellular edema of cartilaginous tissue. Stained with hematoxylin, eosin (X10,40). e, f - week 3. Adhesion of defectbetween cartilaginous tissue stumps with formation of vessels and connective tissue in it. Stained with hematoxylin, eosin (X10,40).
In the third group (8 animals) there was also underwent a symphysis restoration after pubic joint rupture with the help of cerclage from tantalum wire, 1,2 mm in diameter, then in the area of injury a solution of grown embryonal allofibroblasts in the quantity of 30 ml with the help of 5 ml syringe without needle has been injected.
X-ray investigation has been performed before slaughter of animals.
After decapitation of experimental animals an intake of investigated part of pelvis has been carried out. Material has been fixed in 10% solution of neutral formalin (ph 7.2-7.4).
An experimental animal in sterile conditions has been fixed the back to desk with parted extremities. After intramuscular introduction of thiopental sodium 60 mg/kg, droperidol 2 mg/kg+fentanyl 0,04 mg/kg longitudinal incision by pubic joint view has been made. After dissection of skin, subcutaneous fat, symphysis was bareB in the wound (it is necessary to mention that symphysis's height in rabbits reaches up to 2,5-3,0 sm. and width is 0,4-0,5 sm.). With the use of sharp straight raspatory a disruption of pubic joint has been carried out, with the help of straight surgical scissors and Mikulicz's clamp pelvis's half have been pulled apart and so instability of anterior semicircle ofpelvis has been made. After d-bridement by betadin solution its layered closure has been carried out (Pict. 1).
Embryo intake has been made at the 3ld week of pregnancy, embryonic allofibroblasts were prepared by professor M. D. Uraz-
metova's method [13]. Slaughter of animals has been carried out by introduction thiopental sodium (90 mg/kg) with posterior de-hematizing by decapitation.
Histologic sections received on sledge microtome after dewax-ing were colored by hematoxylin and eosin, collagenous fibers revealed by picrofuchsin by Van Gison (morphologic investigations were carried out at pathologic anatomy department of Tashkent Medical Academy).
Results
In the first series of experiments a tentative model of pubic joint rupture has been created as a control and the process of cartilaginous tissue regeneration in the dynamics has been investigated during four weeks.
Morphological investigation results in the dynamics after creating pubic joint rupture showed that in early period of experiment cartilage defect is filled up with necrotic mass and blood elements, circumflex cartilaginous tissue primarily is underwent disorganizational, dystrophic and destructive changes, further inflammatory, reparative-regenerative process with following changes of both cartilaginous and circumflex soft tissues which is followed by appearance of proliferative infiltrate in in-terstitia and around vessels with forming lobulose chondroma-tous nodules of different sizes and fibromatosis of surrounding muscular, connective and bone tissue (Pic 1. a, b).
e f
Pic. 2. Cartilaginous tissue of symphysis pubis regeneration during different periods after allotransplantation of fibroblasts (3rdseries of experiment)
Note: a - week 1. Allofibroblasts replanting. Allofibroblasts adoption in cartilaginous tissue depth. Stain with hematoxylin, eosin (X10, 40). b - week 2. Allofibroblasts replanting. Formation of initial granulation callus from allofibroblasts and local tissue elements.Stain with hematoxylin, eosin (X10, 40). c, d - week 3. Neogenic chondromatous tissue with big quantity of fibrous structures. Van Giesonstaining (X10,40). e, f - Detection of fibrous structures in neogenic chondromatous tissue surrounding each chondrocyte. Van Giesonstaining (X10, 40).
In the second series of experiments after pubic joint rupture, recovery of symphysis was underwent with the use of cerclage from tantalum wire, 1,2 mm in diameter. Morphologic investigation results showed that during the 1st and the 2nd weeks of experiment connective tissue and vascular elements were formed in the rupture's zone with following adhesion of cartilaginous tissue. Development of active regenerative processes both in cartilaginous and surrounding connective tissues as proliferation of young cellular elements of fibro- and chondrocyte origin and also an increasing of basic intercellular substance testifies about forming of mixed primary callus consisting of fibrocyte and chondrocyte tissue elements (Pic 1. c, d).
In the third series of experiments the mass of stem fibroblasts has been put into defect of the rupture of pubic joint cartilaginous part and the process of cartilaginous tissue regeneration has been investigated. Morphologic investigation results during four weeks showed that at initial period necrobiosis development of allofobro-blasts some part has been noted and another part of them being activated move into the depth of cartilaginous tissue. From the second week of experiment transformation of allofibroblasts into fibroblasts and chondroblasts with forming neogenic bands of fibro- and chon-dromatous tissue has been noted. Further the forming of lobulose structures from neogenic cartilaginous tissue has been observed and its two layers are formed by the fourth week. There is a neogenic tissue in the defect zone, it is more intensively colored by hematoxylin into basophilic color and in circumference is embodied with old cartilaginous tissue in which cells hypertrophy and hyperplasia have
been observed. It is necessary to mention the fact that in dynamics of experiment proliferation, hypertrophy and hyperchromatism of fibroblasts and osteoblasts nucleus with thickening of bone beam have been observed from endoost side. Regeneration of fibroma-tous tissue with the following transformation into cartilaginous tissue has been noted from surrounding soft tissue structures (Pic 2.).
Conclusion
Hereby, study of forming new cartilaginous tissue in pubic joint rupture area in the experiment under allofibroblasts influence has shown that concerning changes taken place in the control group, cartilaginous tissue formation occur earlier mainly due to transplanted fibroblasts and formation of cartilaginous and partially bone tissues on 21-30 days of experiment prove that. Therefore, we have made a conclusion about stimulating influence of allogenic fibroblasts on cartilaginous tissue regeneration process course in the experiment and it creates prerequisites for applying this method in clinical practice.
Resume
1. Healing of cartilaginous defect in the conditions of stimulation with replanting allogenic fibroblasts, cartilaginous tissue is formed in the same sequence as in the control group.
2. Regeneration process starts earlier under allofibroblasts influence and passes more active and ends by 21-28 days of cartilaginous tissue forming.
3. Allogenic fibroblasts can be recommended for application as chondrogenesis stimulations.
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