Of TiSSue enGineerinG TechnOlOGY fOr fOrMaTiOn Of huMan arTicular carTilaGe in PerfuSiOn BiOreacTOr

В специально разработанном биореакторе проведены эксперименты по формированию тканеинженерной конструкции хрящевой ткани. На первом этапе была сформирована клеточно-инженерная конструкция, состоящая из биополимерного микроструктурированного коллагенсодержащего гидрогеля, мезенхимальных стромальных клеток жировой ткани человека (МСК ЖТч) и индукционной хондрогенной культуральной среды, которая была помещена в проточный биореактор. В ходе эксперимента установлено, что на 16-е сутки МСК ЖТч приобретают характерную для хондробластов уплощенную форму и проявляют высокую пролиферативную активность с формированием собственного внеклеточного матрикса. Анализ гистологической картины показал, что на данном этапе можно говорить о начале формирования тканеинженерной конструкции хрящевой ткани человека.


inTrODucTiOn
Recently, the development of new approaches to the treatment of cartilage tissue damage based on the use of tissue-engineering technologies and regenerative medicine attracted major attention [1,2].The existing technologies can be divided in two basic groups [3]: -in situ implantation of cell-engineered construct of human articular tissue (CEC ATh) for the stimulation of regenerative processes of damaged articular cartilage; -creation of a tissue-engineered construct of human articular tissue (TEC ATh) using a special devicea bioreactor where TEC ATh is formed from the placed in it CEC ATh with a subsequent implantation for full or partial replacement of damaged cartilage.
In both cases CEC ATh consisting of resorbable matrix, autologous chondrocytes (ACs) and/or stem cells, as well as induction chondrogenic culture medium, containing the necessary signal biomolecules, is preliminarily cultured in vitro.
When using CEC ATh for the stimulation of the regeneration of damaged cartilage, the main matrix function is the provision of functional cellular activity at the site of implantation during a time period sufficient for the launch of regenerative processes of cartilage tissue.In a bioreactor the matrix plays a role of a temporary artificial extracellular matrix which for a long period of time provides the conditions necessary for cell proliferation/differentiation and their synthesis of their own extracellular matrix (ECM).
The goal of this work was a study of the effectiveness of CEC ATh consisting of biopolymer microstructu-red collagen-containing hydrogel (BMCH), mesenchymal stromal cells of human adipose tissue (hADMSCs), and induction chondrogenic culture as for the regeneration of damaged articular cartilage and the formation of TEC ATh in a dynamic environment of a perfusion bioreactor.

MaTerialS anD MeThODS Biopolymer heterogeneous hydrogel matrix
As a biodegradable matrix to develop cell-engineered construct of cartilage, the heterogeneous composition of the implantable gel Sphero®GEL (ZAO «Biomir service», Krasnoznamensk, Moscow Region) was selected.Sphero®GEL, an injectable form of biopolymer heterogeneous hydrogel registered in Russia for clinical use, is produced on the basis of tissue extracellular matrix components of farm animals, possesses high biocompatible and biostimulating properties, and is designed for soft tissue defects replacement, including application in cell technology [3][4][5][6].

Preparation of mesenchymal stromal cells and composition of cec aTh
The study used hADMSCs obtained from a healthy donor at the Academician V.I.Shumakov Federal Research Center of Transplantology and Artificial Organs of the Ministry of Health of the Russian Federation with patient's informed consent.The cells were separated and cultured using the standard methods [12] on a complete growth medium (CGM) for mesenchymal human stem cells MesenPRO RS™ without glutamine (Gibco®byLifeTechnologies™, USA) using MesenPRO RS™ GrowthSupplement (Gibco®byLifeTechnologies™, USA) and L-Glutamine 2 mM (PanEko, Russia).In order to create CEC hATh the second passage cells were used.All procedures were conducted in aseptic conditions.Cells were removed from culture plastic using a reagent TrypLe (Invitrogen, USA) after rinsing the monolayer with Versene solution (PanEko, Russia).Then the cell suspension in CGM was centrifuged to the needed concentration of 6 × 10 6 cells/ml.In order to obtain CEC ATh, 500 μl of the suspension obtained were mixed in layers with 1 ml of BMCH biomatrix.The obtained sample was applied to a nylon polymer membrane and placed in a bioreactor culture chamber.

experimental model (in vivo study)
An analysis in vivo of the influence of intra-articular introduction of CEC ATh on the regeneration of hyaline cartilage of the rabbit tibia was performed on the experimental model of the adjuvant arthritis (female Soviet Chinchilla rabbits) with the further development into osteoarthrosis (OA) confirmed by the clinical, biochemical, and radiological trials [7].The right joint served as a negative control; the left one was injected with the BMCH scaffold only for one group of animals (n = 5), while the other group (n = 5) received CEC hAC.

Description of a bioreactor
In order to conduct a study of formation of TEC ATh from CEC ATh, a bioreactor was developed as seen in Fig. 1.A BMCH matrix with cells was placed on a nylon membrane with pore size 10 μm (Merck Millipore Ltd., Ireland) which was located on the porous substrate of a bioreactor culture chamber.
During the preliminary trials on cell line NIH 3T3 mouse fibroblasts it had been shown that nylon memb-ranes are not cytotoxic to cells nor influence their morphology.It has to be noted that only singular cells were attaching to the surface of nylon membranes without further proliferation.
Bioreactor contained two independent circulation systems.In each of them two culture chambers (height 5 mm, base diameter 22 mm) were simultaneously attached to the reservoir with culture medium.Constant medium circulation with a given speed was provided by four peristaltic pumps (MasterFlex, USA) located outside the СО 2 incubator.The flow control was performed via the controller connected to a computer (Harvard Apparatus, USA) using ORCA Controller Software.Culture chambers and reservoirs with medium were placed in an incubator where 37 °С temperature, 90-100% relative humidity, and composition of gas mixture CO 2 -5% and О 2 -20% were maintained.

Vital cell liVe/DeaD staining
After a defined time period a sample of CEC ATh was taken out of a bioreactor culture chamber, placed in a Petri dish and stained with fluorescent dyes Live/ Dead.(Invitrogen, USA).This set consists of two components: calcein AM produces green fluorescence of live cells registered at 515 nm wave length, while ethidium homodimer-1 penetrating the damaged cell membrane and linking with DNA, results in red fluorescence at 635 nm.The dyes are distinguished by low flash values and good hydrogel diffusion which allows to use them for evaluation of cell condition in a sample.The needed amount of dyes was mixed with sterile phosphate buffer DPBS and then added to the Petri dish with a TEC fragment.After 30 minutes of incubation at room temperature the picture of the sample was taken with a fluorescent microscope Nikon Eclipse TS100.

histological study of samples
The samples were fixated in 10% buffered formalin solution (no less than 4 h), rinsed during 15 minutes in running water and dehydrated in alcohols of rising concentrations (in two portions of 70, 80, 96% ethanol; 5 minutes in each replacement of alcohol solution), let stand 5-7 minutes in a mixture of ethanol and chloroform, in chloroform, and poured into paraffin.Sections with a thickness of 4-5 μm obtained using microtome Leica RM3255 were deparaffinized, rehydrated and stained per the standard methods with hematoxylin and eosin, alcian blue to detect mucopolysaccharides and connective tissue using Masson's method.The analysis and imaging of the samples obtained were conducted using Nikon Eclipse microscope with a digital camera.

Staining of paraffin sections of samples with hematoxylin and eosin
Sections were stained with Mayer's hematoxylin during 10 min, washed in running water for 3-5 min, rinsed with distilled water and stained with 1% eosin solution for 30-50 sec.Then they were rinsed in distilled water, dehydrated in ethanol with rising concentrations (70, 80, 96%), clarified in carbolxylol, xylene and placed in balm.

Staining of paraffin sections with alcian blue
In order to prepare an alcian blue solution (Sigma, USA) 1 g of dye was diluted in 100 ml of 3% acetic acid.The solution was filtered and applied to deparaffinized and rehydrated sections for 30 minutes after which they were rinsed in distilled water and additionally stained during 10 minutes with hematoxylin.Section slides were placed in running water for 3-5 minutes, dehydrated in alcohols of rising concentrations, xylene, and placed in balm.

Masson's staining of paraffin sections
Deparaffinized rehydrated sections were stained with Weigert's iron hematoxylin for 2 minutes, rinsed with running water for 15 minutes and stained with acid fuchsine for 2 minutes.Afterwards the sections were quickly rinsed with distilled water and placed into 1% solution of phosphatotungstic acid for 10 minutes.The acid solution was drained without rinsing the sections and the slides were placed in aniline blue for 1-2 minutes.Then the sections were rinsed with running water and differentiated in 1% solution of acetic acid.Dehydration was performed in alcohols of rising concentrations, the sections were clarified in xylene and placed in balm.During this process the connective tissue fibers are stained blue, cell nuclei -black or dark-blue, cell cytoplasm -raspberry pink or red.

reSulTS anD DiScuSSiOn 1. intra-articular introduction of cec aTh for the regeneration of damaged articular cartilage
On Day 90 after OA modeling the control joints demonstrated erosion and exfoliation of the superficial lamina, deep layer cellular rarefaction, columnar structure disappearance, chaotic location of cartilage cells and local separation of cartilage matrix (Fig. 2, a).On Day 60 after the injection of the BMCH scaffold alone into the affected joint the morphological difference in the tissue specimens compared to the negative control is not pronounced.The beginning of columnar structure formation and the relative superficial layer integrity were observed.Meanwhile, over the same timeframe following the implantation of CEC hAC into the injured joint the specimens demonstrate the signs of partial cartilage structure regeneration such as chondrocyte columnar formation in the middle layer, rejuvenation of superficial layer (the cell number increase), the emergence of isogenic cartilage cell groups in the extracellular matrix (Fig. 2, b).Radiological studies of the dynamics of the knee joint OA development did not allow to monitor the process of hyaline cartilage regeneration upon the injection of CEC hAC.The obtained results of the functional effectiveness of CEC hAC on the experimental model which is xenogenic by the cellular component to the recipient animal can be explained by the stimulating action of CEC hAC on the regeneration processes of articular cartilage.It can be assumed that the regeneration potential of CEC hAC is due to its activating effect on migration of stem cells from the surrounding tissue into the damaged area with the subsequent differentiation into chondrocytes.

The formation of Tec aTh from cec aTh in a flow-through bioreactor
In a multi-day trial a sample of CEC ATh was applied to each nylon membrane (Millipore, Ireland) as 1.5 ml of hADMSC suspension in MesenPRO RS™ growth medium mixed by layers with 1 ml of matrix BMCH.At that, the final cell concentration in the mix was composed of 2 × 10 6 cells per 1.0 ml of CEC ATh.Four culture chambers of a bioreactor with CEC ATh were incubated for one day in MesenPRO RS™ growth medium (Gib-co® by Life Technologies™, USA) in static conditions.Afterwards all four culture chambers were placed in a bioreactor system previously filled with growth medium incubated in flow conditions at speed and volume of circulating medium of 0.5 ml/min and 110 ml respectively.After 24 h of growth medium circulation one culture chamber with a sample (№ 1) was removed from a bioreactor, while the growth medium in the bioreactor was replaced with a STEMPRO® (Gibco® by Life Technologies™, USA) medium inducing chondrogenic differentiation.On the next day of study (48 h of culturing in flow conditions, including 24 h in differential medium) the second culture chamber with Sample № 2 was removed from a bioreactor.On Day 14 of culturing in differential medium the last culture chamber (Sample № 3) was removed from the bioreactor.The differential medium was changed weekly.Samples № 1 and № 3 were additionally stained with fluorescent vital stain Live/Dead (Invitrogen, USA) to control cell viability according to manufacturer's instructions.After one day of the beginning of the study large amounts of cells were attaching to hydrogel particles while flattening.The cell state evaluation after two weeks of medium replacement with the differential one has shown that on the surface of BMCH microparticles there was a proliferation of the attached cells and, correspondingly, the growth of cellular mass (Fig. 3) with the formation on some hydrogel areas of uniform cellular layers with high cellular thickness.
Histological study of Sample № 1 demonstrated that after 2 days of culturing in MesenPRO RS™ growth medium (24 h in static and 24 h in flow conditions) the cell mass on the hydrogel surface was insignificant.Cells found on some BMCH particles were characterized by flattened elongated shape and oval nucleus.The attached cells were located strictly on the surface of hydrogel particles without penetration into biomatrix (Fig. 4).24 h after the growth medium was replaced with STEMPRO® differential medium (Sample № 2), a noticeable growth of cellular mass took place as well as the distribution of cells on the surface of practically all hydrogel fragments.Compared to a previous sample, the amount of multilayered cellular groups has increased where two morphologically different cellular types could be detected: cells of flattened shape and cigar-shaped cells with rounded nucleus (Fig. 5).Additionally, a penetration of cells into biomatrix was observed accompanied by hydrogel resorption.Cells obtained a flattened shape and formed layers.
After 14 days of flow incubation in STEMPRO® differential medium, on Day 16 of the study in Sample № 3 a significant increase in cellular mass was observed with progressive increase in the share of extracellular matrix (ECM) which proved not only proliferative but also high functional cellular activity.
The proportion of cells and matrix was changing in favor of the latter.The majority of cells in those three-dimensional structures had fibroblast-like shape.Masson's staining for connective tissue allowed not only to determine the presence of collagen in the extracellular matrix but also to reveal the matrix structure: thin blue fibers formed a net surrounding the fibroblastlike cells.At that, the densest packaging of collagen fibrils was in the immediate surrounding of producer cells (Fig. 6).
The use of alcian blue in order to detect mucopolysaccharides in extracellular matrix resulted in a weak blue-green staining which we evaluated as a positive reaction to glycosaminoglycans appearing in the cellsynthesized ECM (Fig. 7).The results obtained possib- It is important to note a significant difference in histologic pattern of CEC ATh samples in static and flow culturing.While culturing hADMSCs with BMCH matrix in chondrogenic medium in static conditions [8], after 14 days a formation of three-dimensional structures (microspheres) size ~ 30-300 μm was observed which was not noted while culturing CEC ATh in a perfusion bioreactor.Both at Day 14 and 28 in static conditions, as well as at Day 14 in flow differentiation conditions we were unable to detect the presence of type II collagen in the constructs obtained which may be caused by the short differentiation time.Let it be noted that during static culturing of CEC ATh, the cytoplasmic cell staining with human collagen II antibodies was detected only on Day 42 of chondrogenic differentiation [8].
Thus, the test results have shown that in a bioreactor at a flow rate of 0.5 ml/min after 14 days of hADMSC culturing with BMCH biomatrix in chondrogenic medium, an active cellular proliferation with a progressive increase of ECM produced by cells takes place.A histochemical analysis of samples at that time revealed the presence of collagen and glycosaminoglycans.The observed morphological changes in a bioreactor-cultured CEC ATh confirm that the formation of TEC ATh started on Day 16 of the study.

cOncluSiOnS
The obtained results demonstrate the effectiveness of CEC ATh consisting of biopolymer microstructured collagen-containing hydrogel (BMCH), mesenchymal stromal cells of human adipose tissue (hADMSCs), and induction chondrogenic culture as for the regeneration of damaged articular cartilage in experimental model of osteoarthrosis and the formation of TEC ATh in a dynamic environment of a perfusion bioreactor.

Fig. 2 .
Fig. 2. The histological structure of the cartilage in the joints of the tibia in the rabbit experimental model of OA 120 days from the start of the experiment: a -the right knee joint after 90 days after modeling OA (negative control); b -left knee joint after 90 days after modeling OA and 60 days after intra-articular introduction of CEC hATh.Colouring with hematoxylin and eosin (×200)