Cultivation of three-dimensional cartilage-carrier-constructs under reduced oxygen tension

Three-dimensional cartilage-carrier-constructs were produced according to a standard protocol from chondrocytes of an adult mini-pig. Experiments with different oxygen concentrations (21, 10 and 5%, v/v O(2)) were performed and the constructs were compared qualitatively and quantitatively. The appearance of the cartilage obtained under reduced oxygen tension seemed to be closer to native cartilage with respect to shape of the cells, distribution of the cells within the matrix, smoothness of the surface, etc. The thickness of the cartilage formed by free swelling was always in the same range as for native cartilage (approximately 1mm). Qualitatively the most stable attachment of the cartilage on top of the carrier was found for 10% O(2) (v/v). Especially at 5% O(2) (v/v) the attachment between cartilage and carrier was not sufficient. The constructs generated at lower oxygen tensions had a significantly higher amount of glycosaminoglycan per DNA, but still significantly less when compared to native cartilage. Furthermore, the cultivated cartilage contained a large amount of collagen type II. The experiments proved the applied concept for generation of cartilage-carrier-constructs and the usefulness of cultivation under reduced oxygen tension.     

Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min(-1)) and gradually increasing (0.075-0.2 mL min(-1)) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0-4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min(-1). GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.     

Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development

A concentric cylinder bioreactor has been developed to culture tissue engineered cartilage constructs under hydrodynamic loading. This bioreactor operates in a low shear stress environment, has a large growth area for construct production, allows for dynamic seeding of constructs, and provides for a uniform loading environment. Porous poly-lactic acid constructs, seeded dynamically in the bioreactor using isolated bovine chondrocytes, were cultured for 4 weeks at three seeding densities (60, 80, 100 x 10(6) cells per bioreactor) and three different shear stresses (imposed at 19, 38, and 76 rpm) to characterize the effect of chondrocyte density and hydrodynamic loading on construct growth. Construct seeding efficiency with chondrocytes is greater than 95% within 24 h. Extensive chondrocyte proliferation and matrix deposition are achieved so that after 28 days in culture, constructs from bioreactors seeded at the highest cell densities contain up to 15 x 10(6) cells, 2 mg GAG, and 3.5 mg collagen per construct and exhibit morphology similar to that of native cartilage. Bioreactors seeded with 60 million chondrocytes do not exhibit robust proliferation or matrix deposition and do not achieve morphology similar to that of native cartilage. In cultures under different steady hydrodynamic loading, the data demonstrate that higher shear stress suppresses matrix GAG deposition and encourages collagen incorporation. In contrast, under dynamic hydrodynamic loading conditions, cartilage constructs exhibit robust matrix collagen and GAG deposition. The data demonstrate that the concentric cylinder bioreactor provides a favorable hydrodynamic environment for cartilage construct growth and differentiation. Notably, construct matrix accumulation can be manipulated by hydrodynamic loading. This bioreactor is useful for fundamental studies of construct growth and to assess the significance of cell density, nutrients, and hydrodynamic loading on cartilage development. In addition, studies of cartilage tissue engineering in the well-characterized, uniform environment of the concentric cylinder bioreactor will develop important knowledge of bioprocessing parameters critical for large-scale production of engineered tissues.     

Flow characterization of a wavy-walled bioreactor for cartilage tissue engineering

Cartilage tissue engineering requires the use of bioreactors in order to enhance nutrient transport and to provide sufficient mechanical stimuli to promote extracellular matrix (ECM) synthesis by chondrocytes. The amount and quality of ECM components is a large determinant of the biochemical and mechanical properties of engineered cartilage constructs. Mechanical forces created by the hydrodynamic environment within the bioreactors are known to influence ECM synthesis. The present study characterizes the hydrodynamic environment within a novel wavy-walled bioreactor (WWB) used for the development of tissue-engineered cartilage. The geometry of this bioreactor provides a unique hydrodynamic environment for mammalian cell and tissue culture, and investigation of hydrodynamic effects on tissue growth and function. The flow field within the WWB was characterized using two-dimensional particle-image velocimetry (PIV). The flow in the WWB differed significantly from that in the traditional spinner flask both qualitatively and quantitatively, and was influenced by the positioning of constructs within the bioreactor. Measurements of velocity fields were used to estimate the mean-shear stress, Reynolds stress, and turbulent kinetic energy components in the vicinity of the constructs within the WWB. The mean-shear stress experienced by the tissue-engineered constructs in the WWB calculated using PIV measurements was in the range of 0-0.6 dynes/cm2. Quantification of the shear stress experienced by cartilage constructs, in this case through PIV, is essential for the development of tissue-growth models relating hydrodynamic parameters to tissue properties.     

Effect of a mechanical stimulation bioreactor on tissue engineered, scaffold-free cartilage

Achieving sufficient functional properties prior to implantation remains a significant challenge for the development of tissue engineered cartilage. Many studies have shown chondrocytes respond well to various mechanical stimuli, resulting in the development of bioreactors capable of transmitting forces to articular cartilage in vitro. In this study, we describe the production of sizeable, tissue engineered cartilage using a novel scaffold-free approach, and determine the effect of perfusion and mechanical stimulation from a C9-x Cartigen bioreactor on the properties of the tissue engineered cartilage. We created sizable tissue engineered cartilage from porcine chondrocytes using a scaffold-free approach by centrifuging a high-density chondrocyte cell-suspension onto an agarose layer in a 50 mL tube. The gross and histological appearances, biochemical content, and mechanical properties of constructs cultured in the bioreactor for 4 weeks were compared to constructs cultured statically. Mechanical properties were determined from unconfined uniaxial compression tests. Constructs cultured in the bioreactor exhibited an increase in total GAG content, equilibrium compressive modulus, and dynamic modulus versus static constructs. Our study demonstrates the C9-x CartiGen bioreactor is able to enhance the biomechanical and biochemical properties of scaffold-free tissue engineered cartilage; however, no additional enhancement was seen between loaded and perfused groups.     

Computational fluid dynamics modeling of momentum transport in rotating wall perfused bioreactor for cartilage tissue engineering

In this study, computational fluid dynamics (CFD) analysis of a rotating-wall perfused-vessel (RWPV) bioreactor is performed to characterize the complex hydrodynamic environment for the simulation of cartilage development in RWPV bioreactor in the presence of tissue-engineered cartilage constructs, i.e., cell-chitosan scaffolds. Shear stress exerted on chitosan scaffolds in bioreactor was calculated for different rotational velocities in the range of 33-38 rpm. According to the calculations, the lateral and lower surfaces were exposed to 0.07926-0.11069 dyne/cm(2) and 0.05974-0.08345 dyne/cm(2), respectively, while upper surfaces of constructs were exposed to 0.09196-0.12847 dyne/cm(2). Results validate adequate hydrodynamic environment for scaffolds in RWPV bioreactor for cartilage tissue development which concludes the suitability of operational conditions of RWPV bioreactor.     

Computational fluid dynamics modeling of steady-state momentum and mass transport in a bioreactor for cartilage tissue engineering

Computational fluid dynamics (CFD) models to quantify momentum and mass transport under conditions of tissue growth will aid bioreactor design for development of tissue-engineered cartilage constructs. Fluent CFD models are used to calculate flow fields, shear stresses, and oxygen profiles around nonporous constructs simulating cartilage development in our concentric cylinder bioreactor. The shear stress distribution ranges from 1.5 to 12 dyn/cm(2) across the construct surfaces exposed to fluid flow and varies little with the relative number or placement of constructs in the bioreactor. Approximately 80% of the construct surface exposed to flow experiences shear stresses between 1.5 and 4 dyn/cm(2), validating the assumption that the concentric cylinder bioreactor provides a relatively homogeneous hydrodynamic environment for construct growth. Species mass transport modeling for oxygen demonstrates that fluid-phase oxygen transport to constructs is uniform. Some O(2) depletion near the down stream edge of constructs is noted with minimum pO(2) values near the constructs of 35 mmHg (23% O(2) saturation). These values are above oxygen concentrations in cartilage in vivo, suggesting that bioreactor oxygen concentrations likely do not affect chondrocyte growth. Scale-up studies demonstrate the utility and flexibility of CFD models to design and characterize bioreactors for growth of tissue-engineered cartilage.     

Rotating versus perfusion bioreactor for the culture of engineered vascular constructs based on hyaluronic acid

It is generally accepted that dynamic culture conditions are required for vascular tissue engineering. We compared the effects of two dynamic culture systems, a perfusion and a rotating bioreactor, using tubular constructs based on hyaluronic acid seeded with porcine aortic smooth muscle cells (SMC), that we recently showed to be adequate for the generation of vascular tissue. In perfused constructs mechanical stimulation importantly affected cell morphology, increased the incidence of cell proliferation and reduced apoptosis. However, extracellular matrix deposition, cytoskeletal organization and mechanical properties were poor. In rotated constructs cell proliferation was also higher and apoptosis lower than in static controls. Rotated constructs showed the highest ultimate stress and the lowest elastic modulus. Our data indicate that the rotating bioreactor is more efficient than the perfusion bioreactor and we then suggest that this method can be considered a valid alternative to complex bioreactor systems described in the literature.     

Effect of streptomycin on the active force of bioengineered heart muscle in response to controlled stretch

In this study, we describe a bioreactor system to deliver controlled stretch protocols to bioengineered heart muscle (BEHMs) and test the system when streptomycin (an aminoglycoside antibiotic, which blocks stretch-activated channels) is either added to or excluded from the culture medium. Streptomycin is a very commonly used component of cell culture antibiotic-antimycotic media additives, so its effects on muscle development and functional response to mechanical signals in vitro is worthy of investigation. Our hypothesis is that BEHMs will not adapt to the applied mechanical stretch protocol when streptomycin is present in the culture medium, but will do so when streptomycin is excluded. Bioengineered heart muscles were formed by culturing primary neonatal cardiac myocytes in a fibrin gel using a method previously developed in our laboratory. A custom bioreactor system was designed using SolidWorks and structural components manufactured using fusion deposition modeling. We utilized a stretch protocol of 1 Hz, 10% strain for 7 d. BEHMs were stretched in the presence and absence of streptomycin. As controls, BEHMs were maintained in a cell culture incubator with and without streptomycin. The contractile properties of all BEHMs were evaluated to determine the active force. We were able to demonstrate compatibility of the bioreactor system with BEHMs and were able to stretch 58 constructs with zero incidence of failure. When the BEHMs were stretched in the absence of streptomycin, the active force increased from a mean value of 51.7 +/- 5.6 (N = 10) to 102.4 +/- 16.3 muN (N = 10), with p < 0.05. However, BEHMs that were stretched in the presence of streptomycin did not show any significant increase in active force generation. The average active force of BEHMs increased from a mean value of 57.6 +/- 10.2 (N = 10) to 91.4 +/- 19.8 muN (N = 10) when stretched in the presence of streptomycin. In this study, we demonstrate compatibility of the a bioreactor system with BEHMs, stability of the BEHMs in response to stretch protocols, and significant functional improvement in response to controlled stretch only when streptomycin is excluded from the culture medium, supporting our hypothesis.     

Modeling evaluation of the fluid-dynamic microenvironment in tissue-engineered constructs: a micro-CT based model

Natural cartilage remodels both in vivo and in vitro in response to mechanical stresses, hence mechanical stimulation is believed to be a potential tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis in engineered cartilage constructs. The quantification of the hydrodynamic environment is a condition required to study the biochemical response to shear of 3D engineered cell systems. We developed a computational model of culture medium flow through the microstructure of a porous scaffold, during direct- perfused culture. The 3D solid model of the scaffold micro-geometry was reconstructed from 250 micro-computed tomography (micro-CT) images. The results of the fluid dynamic simulations were analyzed at the central portions of the fluid domain, to avoid boundary effects. The average, median and mode shear stress values calculated at the scaffold walls were 3.48, 2.90, and 2.45 mPa respectively, at a flow rate of 0.5 cm(3)/min, perfused through a 15 mm diameter scaffold, at an inlet fluid velocity of 53 microm/s. These results were compared to results estimated using a simplified micro-scale model and to results estimated using an analytical macro-scale porous model. The predictions given by the CT-based model are being used in conjunction with an experimental bioreactor model, in order to quantify the effects of fluid-dynamic shear on the growth modulation of tissue-engineered cartilage constructs, to potentially enhance tissue growth in vitro.     

Growth factors for sequential cellular de- and re-differentiation in tissue engineering

A model system for the in vitro generation of cartilaginous constructs was used to study a tissue engineering paradigm whereby sequentially applied growth factors promoted chondrocytes to first de-differentiate into a proliferative state and then re-differentiate and undergo chondrogenesis. Early cultivation in medium with supplemental TGF-β1/FGF-2 doubled cell fractions in 2-week constructs compared to unsupplemented controls. Subsequent culture with supplemental IGF-I yielded large 4-week constructs with high fractions of cartilaginous extracellular matrix (ECM) and high compressive moduli, whereas prolonged culture with supplemental FGF-2 yielded small 4-week constructs with low ECM fractions and moduli. Sequential supplementation with TGF-β1/FGF-2 and then IGF-I yielded 4-week constructs with type-specific mRNA expression and protein levels that were high for type II and negligible for type I collagen, in contrast to other growth factor regimens studied. The data demonstrate that structural, functional, and molecular properties of engineered cartilage can be modulated by sequential application of growth factors.     

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.     

Bi-zonal cartilaginous tissues engineered in a rotary cell culture system

In this study, we aimed at validating a rotary cell culture system (RCCS) bioreactor with medium recirculation and external oxygenation, for cartilage tissue engineering. Primary bovine and human culture-expanded chondrocytes were seeded into non-woven meshes of esterified hyaluronan (HYAFF-11), and the resulting constructs were cultured statically or in the RCCS, in the presence of insulin and TGFbeta3, for up to 4 weeks. Culture in the RCCS did not induce significant differences in the contents of glycosaminoglycans (GAG) and collagen deposited, but markedly affected their distribution. In contrast to statically grown tissues, engineered cartilage cultured in the RCCS had a bi-zonal structure, consisting of an outgrowing fibrous capsule deficient in GAG and rich in collagen, and an inner region more positively stained for GAG. Structurally, trends were similar using primary bovine or expanded human chondrocytes, although the human cells deposited inferior amounts of matrix. The use of the presented RCCS, in conjunction with the described medium composition, has the potential to generate bi-zonal tissues with features qualitatively resembling the native meniscus.     

The role of bioreactors in cartilage tissue engineering

Cartilage tissue engineering is concerned with developing in vitro cartilage implants that closely match the properties of native cartilage, for eventual implantation to replace damaged cartilage. The three components to cartilage tissue engineering are cell source, such as in vitro expanded autologous chondrocytes or mesenchymal progenitor cells, a scaffold onto which the cells are seeded and a bioreactor which attempts to recreate the in vivo physicochemical conditions in which cartilage develops. Although much progress has been made towards the goal of developing clinically useful cartilage constructs, current constructs have inferior physicochemical properties than native cartilage. One of the reasons for this is the neglect of mechanical forces in cartilage culture. Bioreactors have been defined as devices in which biological or biochemical processes can be re-enacted under controlled conditions e.g. pH, temperature, nutrient supply, O2 tension and waste removal. The purpose of this review is to detail the role of bioreactors in the engineering of cartilage, including a discussion of bioreactor designs, current state of the art and future perspectives.     

Development of large engineered cartilage constructs from a small population of cells

Confronted with articular cartilage's limited capacity for self‐repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large‐sized engineered constructs (≥3 cm²) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm² tissue construct). Using rabbit donor tissue, the bioreactor‐cultivated constructs were hyaline‐like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid‐prototyped defect tissue culture molds. Similar hyaline‐like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large‐sized osteochondral constructs. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

A novel rotating-shaft bioreactor for two-phase cultivation of tissue-engineered cartilage

A novel rotating-shaft bioreactor (RSB) was developed for two-phase cultivation of tissue-engineered cartilage. The reactor consisted of a rotating shaft on which the chondrocyte/scaffold constructs (7.5 mm diameter x 3.5 mm thickness) were fixed and a reactor vessel half-filled with medium. The horizontal rotation of the shaft resulted in alternating exposure of the constructs to gas and liquid phases, thus leading to efficient oxygen and nutrient transfer, as well as periodically changing, mild shear stress exerting on the construct surfaces (0-0.32 dyn/cm2 at 10 rpm), as revealed by computer simulation. Strategic operation of the RSB (maintaining rotating speed at 10 rpm for 3 weeks and lowering the speed to 2 rpm in week 4) in combination with higher seeding density (6 x 10(6) chondrocytes/scaffold) and medium perfusion resulted in uniform cell distribution and increased glycosaminoglycan (3.1 mg/scaffold) and collagen (7.0 mg/scaffold) deposition. The 4-week constructs resembled native cartilages in terms of not only gross appearance and cell morphology but also distributions of glycosaminoglycan, total collagen, and type II collagen, confirming the maintenance of chondrocyte phenotype and formation of cartilage-like constructs in the RSB cultures. In summary, the novel RSB may be implicated for in vitro study of chondrogenesis and de novo cartilage development under periodic mechanical loading. With proper optimization of the culture conditions, a RSB may be employed for the production of cartilage-like constructs.     

Nutrient gradients in engineered cartilage: metabolic kinetics measurement and mass transfer modeling

Since tissue-engineered cartilage is avascular, both nutrient supply and metabolic waste removal rely on diffusion. As a result, gradients of nutrients and wastes exist through the construct. Previous models usually calculate gradients of oxygen, glucose, and lactic acid separately, without taking into account the complex interdependence between concentrations of these substrates and rates of metabolism. In this study, these interactions were experimentally examined and incorporated into diffusion models. One-dimensional diffusion-reaction models were developed for three typical culture conditions, that is, static culture, perfusion culture, and suspended culture. The profiles of oxygen, glucose, lactic acid, and pH in the cultured constructs were calculated simultaneously using measured metabolic rates. The maximum construct size and cell density which could be supported before nutrients were depleted in the construct center was identified; a function predicting the relationship between construct dimension and the maximum viable cell density was developed. For constructs incubated under static culture the model demonstrated that the gradients which developed through the medium could not be neglected. Perfusion cultures could support a considerably higher cell density than static cultures, while for batch cultures in a rotating bioreactor, the volume of medium also influences the maximum cell density that could be supported. This study provides useful guidance for design of engineered cartilage constructs.     

Investigating ADAMTS-mediated aggrecanolysis in mouse cartilage

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.     

Differentiation of multipotent mesenchymal stromal cells of bone marrow into cells of cartilage tissue by culturing in three-demential OPLA scaffolds

Bone marrow multipotent mesenchymal stromal cells represent a perspective material for engineering of human three-dimensional transplants of cartilage tissue. We are demonstrated the opportunity of the directed differentiation of BM MMSC in cells of cartilage tissue by culturing them in three-dimensional scaffolds, presented by polymer OPLA in medium with inductors of chondrogenesis. For loading cells in porous scaffolds used method which essence consist in saturation of polymeric blocks by cellular suspension with the subsequent centrifugal force of cells in scaffolds and culturing of engineering constructs for 28 days in chondrogenic medium. Histological analysis derived in vitro of three-dimensional transplants showed uniform distribution of cells in the matrix with morphologically distinct chondrocytes-like cells of hyaline cartilage. Immunohistochemical analysis detected aggrecan and collagen type II within the extracellular matrix. Preclinical the researches lead on a livestock of immunodeficient mice have shown not toxicity of the engineering constructs.     

Differentiation of multipotent mesenchymal stromal cells of human bone marrow into cells of cartilage tissue by culturing in three-dimensional OPLA scaffolds

Bone marrow multipotent mesenchymal stromal cells show considerable promise for the engineering of human three-dimensional transplants of cartilage tissue. We demonstrated the directed differentiation of BM MMSC in cells of cartilage tissue by culturing them in OPLA polymer three-dimensional scaffolds in a medium with chondrogenesis inducers. Cells were loaded into porous scaffolds by saturating polymer blocks with a cellular suspension. This was followed by high-speed centrifugation of the matrix-embedded cells and cultivation of the engineering constructs in a condrogenic medium for 28 days. Histological analysis of the three-dimensional transplants derived in vitro showed a uniform distribution of cells in the matrix. Morphologically, the cells were similar to the chondrocyte-like cells of articular cartilage. Immunohistochemical analysis revealed aggrecan and collagen type 2, which are the major markers of chondrogenesis, in the constructs. Preclinical research on immunodeficient mice showed that the engineering transplants were not toxic.