Deformational loading represents a primary component of the chondrocyte physical environment in vivo. This review summarizes our experience with physiologic deformational loading of chondrocyte-seeded agarose hydrogels to promote development of cartilage constructs having mechanical properties matching that of the parent calf tissue, which has a Young's modulus EY = 277 kPa and unconfined dynamic modulus at 1 Hz G*=7 MPa. Over an 8-week culture period, cartilage-like properties have been achieved for 60 × 106 cells/ml seeding density agarose constructs, with EY = 186 kPa, G*=1.64 MPa. For these constructs, the GAG content reached 1.74% ww and collagen content 2.64% ww compared to 2.4% ww and 21.5% ww for the parent tissue, respectively. Issues regarding the deformational loading protocol, cell-seeding density, nutrient supply, growth factor addition, and construct mechanical characterization are discussed. In anticipation of cartilage repair studies, we also describe early efforts to engineer cylindrical and anatomically shaped bilayered constructs of agarose hydrogel and bone (i.e., osteochondral constructs). The presence of a bony substrate may facilitate integration upon implantation. These efforts will provide an underlying framework from which a functional tissue-engineering approach, as described by Butler and coworkers (2000), may be applied to general cell-scaffold systems adopted for cartilage tissue engineering.

/pdf/a-paradigm-for-functional-tissue-engineering-of-articular-ma6qqd1lcj.pdf

A Paradigm for Functional Tissue Engineering of Articular Cartilage

We performed culturing of a cell-engineered construct of human cartilage tissue consisting of biopolymer microstructured collagen-containing hydrogel, human adipose tissue mesenchymal stromal cells, and induction chondrogenic culture medium in a specially designed flow-through bioreactor. On day 16 of the experiment, human adipose tissue mesenchymal stromal cells acquired flattened shape typical for chondroblasts, demonstrated high proliferative activity, and formed extracellular matrix. The observed histological changes in the cultured system attested to the beginning of the formation of a tissue-engineered construct of human cartilage tissue.

Formation of Tissue-Engineered Construct of Human Cartilage Tissue in a Flow-Through Bioreactor.

A method for obtaining a microdispersed tissue-specific matrix from decellularized porcine articular cartilage while maintaining morphological and functional properties of the extracellular matrix with no signs of cytotoxicity was developed. The cartilage particle size distribution in suspension after cryogenic grinding was determined using laser diffraction analysis. The range of sizes of the obtained cartilage microparticles suggests the possibility of their administration by injection (<220 μm). The combination of stages, including three freeze/thaw cycles (–196/37°C) followed by treatment with solutions of surface-active substances (surfactants), sodium dodecyl sulfate and Triton X-100, and DNase resulted in the complete removal of non-decellularized microparticles. The residual DNA content was 9.11 ± 1.13 ng/mg of tissue. The effectiveness of surfactant washing was assessed by the cytotoxicity of the matrix on the culture of human adipose derived mesenchymal stromal cells (hADSCs). To assess the hemocompatibility of the obtained samples, their hemolytic activity was studied in vitro. The adhesion and proliferation of hADSCs on the matrix surface were studied on day 21 of cultivation. The matrix did not exhibit hemolytic activity and cytotoxicity with respect to hADSCs. hADSCs in the samples were actively proliferating on the matrix surface. The biocompatibility and hemocompatibility of the obtained matrix in vitro indicate its potential for application in cartilage regenerative medicine.

Fabrication of Microdispersed Tissue-Specific Decellularized Matrix from Porcine Articular Cartilage

Despite great progress in the field of biomaterials for tissue engineering and regenerative medicine, the high requirements placed on artificial matrices (matrices, carriers, scaffolds) are the reason for the ongoing search for natural or synthetic extracellular matrix mimetics. Among such materials, decellularized umbilical cord (UC) stroma appears to be very attractive – it has a high content of hyaluronic acid, cytokines, and growth factors, and there are no ethical restrictions for its production. Decellularized UC stroma has been found to promote cartilage, liver tissue and nerve tissue repair, as well as wound healing. The review critically analyzes and summarizes published data on the ability of decellularized UC stroma to maintain the necessary conditions for adhesion, migration, differentiation and functional activity of adherent cells, thus stimulating the internal (physiological) regenerative potential of tissues. Literature was searched for in the following electronic databases: Medline/PubMed (www/ncbi. nlm.nih.gov/pubmed), Cochrane library (https://www.cochrane.org), and eLIBRARY/Russian Science Citation Index (https://www.elibrary.ru). Inclusion criteria were the presence of biomaterials obtained from decellularized human UC stroma. Exclusion criteria for papers included research objects as decellularized umbilical cord vessels (veins and arteries) and umbilical cord cell cultures. Twenty-five original articles in English and Russian were selected for analysis of the products obtained, their applications, decellularization methods and research results. The review also discusses the prospects for decellularized umbilical cord in medicine.

Decellularized umbilical cord stroma in tissue engineering and regenerative medicine: a systematic review

Objective: to study the effect of trypsin pretreatment in the porcine articular cartilage decellularization protocol on the ability to restore the biochemical composition and functional properties of the resulting finely dispersed tissue-specific scaffold when co-cultured with human adipose-derived stem cells (hADSCs).Materials and methods. Porcine articular cartilage was micronized to a maximum size of 250 μm. The resulting porcine articular cartilage microparticles (CMps) were treated with trypsin (0.05, 0.25, 0.50%) / EDTA solution at +37 °C for 24 hours. Then, the CMps were successively incubated for 24 hours in three surfactant solutions containing 0.1% sodium dodecyl sulfate and increasing concentration of Triton X-100 (1, 2, 3%) at room temperature and in DNase I solution at +37 °C for 48 hours. The degree of change in the biochemical composition and the ability of decellularized CMps (DCMps) scaffolds within cell-engineered constructs (CECs) to support hADSC adhesion and proliferation, as well as their potential ability to exert a stimulatory regenerative effect, were then assessed. DNA, glycosaminoglycans (GAGs) and collagen content in the DCMps and CECs were examined. The morphology of the samples was examined using histological and immunohistochemistry staining.Results. Histological analysis showed that there were no cells and detritus in the DCMp samples. Pretreatment of CMps samples гыштп a solution with the lowest content of trypsin (0.05%) / EDTA in the samples retained 5.14 ± 0.87 ng/mg DNA in the samples, while GAG content decreased to 5.34 ± 0.9 μg/mg and collagen to 154 ± 34 μg/mg. By day 28 of CEC cultivation, adherent cells had produced their own extracellular matrix (ECM) containing GAGs and collagen. The amount of DNA in it was 6.30 ± 0.11 μg/CEC and that of GAGs was 19.36 ± 0.73 μg/CEC.Conclusion. Pretreatment with trypsin allows achieving uniformly complete decellularized CMps. At the same time, onset of changes in the ECM composition indicates a decrease in the ability of hADSCs to synthesize GAGs and type II collagen during co-culturing with DCMps. The increased proliferative activity of adherent hADSCs, as well as the tissue specificity of the DCMp scaffold will allow further research towards a hydrogel matrix capable of enhancing the specific and stimulating regenerative potential when co-cultured with cells of the same phenotype.

Effect of trypsin on biochemical and functional properties of decellularized porcine articular cartilage

Introduction. Extracellular matrix play an essential role in providing structural integrity and physiological support to Langerhans islets in pancreas. Imitation of the native microenvironment can be useful for viability of isolated pancreatic islets in vitro and in vivo. Aim. The purpose of this study was to characterize and compare the effect of biopolymer microstructured collagen-containing hydrogel (BMCH) on isolated rat islets survival. Materials and methods. Islets were isolated by classic collagenase techniques with some modifications. There were used hystological, immunofluorescence and immunohystochemistry methods. Results. Rat islets cultured with collagen-based gel don’t revealed destructive changes of structure and remained viabile 7 days incubation. Conclusion. Positive effect of BMCH to rat islet survival was revealed.

/pdf/sravnitelnyi-morfologicheskii-analiz-izolirovannykh-1x6hde2btq.pdf

Сравнительный морфологический анализ изолированных островков поджелудочной железы крысы, культивируемых в стандартных условиях и с биополимерным микрогетерогенным коллагенсодержащим гелем

Some of the most pressing health problems of the industrial society are the damage and degeneration of articular cartilage associated with the limited capacity of tissues to regenerate. The review describes the existing and developing technologies for the recovery and replacement of damaged joint cartilage tissue. The results obtained are analyzed covering two major areas: the stimulation of regeneration of damaged cartilage tissue and the growing of cartilage tissue elements in bioreactors.

/pdf/tekhnologii-tkanevoi-inzhenerii-i-regenerativnoi-meditsiny-v-4otght9q4p.pdf

Технологии тканевой инженерии и регенеративной медицины в лечении дефектов хрящевой ткани суставов

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Технологии тканевой инженерии и регенеративной медицины в лечении дефектов хрящевой ткани суставов