Structure and Morphogenetic Properties of Collagen Matrixes Obtained from Connective Tissue Sheaths of Paravertebral Tendons

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Abstract

The morphogenetic properties of a collagen gel prepared by acetic acid extraction from the tendon sheaths (peritenons) of the paravertebral tendons of Wistar rats were studied. The gel was used as a substrate during in vitro cultivation together with mesenchymal stromal cells for 14 days in the growth and osteogenic incubation media. It has been established that the collagen framework of the peritenon substrate is strengthened by increasing the connectivity of fibrillar nodes and is structured with the formation of lamellar and tangle formations. Sesamoid globules, penetrating into the substrate from the initial peritenon gel, during cultivation remain inert in the growth medium, but exhibit an increased ability to structure calcium phosphates in the osteogenic medium. The formation of cell-mediated structures occurs by directions of fibro-, tendo-, ligament- and osteogenic differentiation. The fibrogenic direction provides a structuring framework; the tenogenic direction – the formation of embryonic tendons according to the mechanism of lateral assembly of collagen subfibrils on cell surfaces and their autonomization in the form of tendon filament primordia; the ligamentogenic direction – structuring of collagen ribbons associated with tangles and elastic fibers; the osteogenic direction – the formation of lamellar, trabecular and nodular osteoid structures through intramembranous ossification, accompanied by activation of alkaline phosphatase and mineralization. The formation of enthesis predictors is the organization of commissures between mechanically different-phase components of osteoid structures and frame. A classification of taxonomic forms has been developed and a hypothesis has been proposed about the role of evolutionary tools in the structuring of the collagen framework in tissue cultures in vitro. The classification of taxonomic forms has been developed and a hypothesis has been proposed about the role of evolutionary tools in the structuring of the collagen framework in tissue cultures in vitro.

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А. А. Gaidash

Institute of General and Inorganic Chemistry, National Academy of Sciences of Belarus

Author for correspondence.
Email: algaidashspb@gmail.com
Belarus, Minsk

A. I. Kulak

Institute of General and Inorganic Chemistry, National Academy of Sciences of Belarus

Email: algaidashspb@gmail.com
Belarus, Minsk

V. K. Krut’ko

Institute of General and Inorganic Chemistry, National Academy of Sciences of Belarus

Email: suber@igic.bas-net.by
Belarus, Minsk

M. I. Blinova

Institute of Cytology, Russian Academy of Sciences

Email: algaidashspb@gmail.com
Russian Federation, St. Petersburg

O. N. Musskaya

Institute of General and Inorganic Chemistry, National Academy of Sciences of Belarus

Email: algaidashspb@gmail.com
Belarus, Minsk

S. A. Aleksandrova

Institute of Cytology, Russian Academy of Sciences

Email: algaidashspb@gmail.com
Russian Federation, St. Petersburg

K. V. Skrotskaya

Research Institute for Physical Chemical Problems, Belarusian State University

Email: algaidashspb@gmail.com
Belarus, Minsk

V. A. Kulchitsky

Institute of Physiology, National Academy of Sciences of Belarus

Email: algaidashspb@gmail.com
Belarus, Minsk

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2. Fig. 1. SEM images of the structure of the collagen network of the gel and the framework of the peritenone gel substrate in growth and osteogenic media: (a) the structure of the initial peritenone gel; (b) deformation of the network by thickened collagen fibers; (c) arrows – nascent fibrillar protons; (d) ellipse – mature fibrillar node; (e) calcium phosphates in the area of forming fibrillar nodes; (f) ellipse – a mature fibrillar node in a peritenone substrate (osteogenic medium).

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3. Fig. 2. SEM images of lamellar formations of the peritenone collagen framework: (a) arrows – converged plates (growth medium); (b) arrow – converged plate (osteogenic medium; (c) polymorphism of converged plates: arrow 1 – compactified fibers with clear boundaries, arrow 2 – fused collagen fibers with blurred contours (growth medium); (d) arrows – compact plates (growth medium); (e) ellipses – concentric plates with asymmetric walls in the fenestered hole of the MSC (growth medium); (e) Arrow 1 is a concentric plate with a thickened asymmetric wall, arrow 2 is a lamellar formation in the collagen framework (growth medium).

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4. Fig. 3. SEM images of tangle formations in a peritenone substrate (growth medium): (a) arrow – a tangle in an ex tempore gel; (b) arrows – the beginnings of tangles in the form of tangled collagen subfibrils; (c) arrows – protoclubs with homogenized and calcified centers; (d) Strelka is an emerging resident tangle; (e) mature resident tangles; (f) arrow – an involutive resident tangle.

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5. Fig. 4. SEM images of sesamoid globules in peritenone in vivo: (a) arrows – sesamoid globules; (b) arrow – lamellar compactification of collagen fibrils.

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6. Fig. 5. SEM images of solidified spheroids in the ex tempore gel and the substrate (growth medium): (a) arrow - sesamoid globule in the gel; (b) arrow – deformed spheroid; (c) solidified spheroid in the depth of the collagen framework; (d) The arrow is a solidified spheroid covered with collagen fibers and cytoplasmic appendages.

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7. Fig. 6. SEM images of growing spheroids in a peritenone substrate (growth and osteogenic media: (a) a spheroid on a substrate (growth medium); (b) a composite growing spheroid; (c) a growing spheroid with a rough surface and open pores (growth medium); (d) a growing spheroid covered with fenestrated cytoplasmic membrane (growth medium); (e) arrow – focal growths on the surface of a growing spheroid (growth medium); (f) arrow – a growing spheroid with microcrystallites of calcium phosphates (osteogenic medium).

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8. 7. SEM images of MSCs and matrix vesicles: (a) attachment via cytoplasmic processes releasing a multilayer network of collagen fibers (growth medium); (b) arrows – attachment via filopodia in the form of actin “whiskers” (growth medium); (c) matrix vesicles contouring under the MSCS membrane (growth medium); (d) arrow – matrix vesicles at the site of extrusion of collagen material (growth medium); (e) arrow – cluster-shaped clusters of matrix bubbles and granules of calcium phosphates on the surfaces of collagen fibers (osteogenic medium).

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9. 8. SEM images of teno- and ligamentous structures in the collagen framework: (a) arrow - spindle–shaped cell (growth medium); (b) arrow – spindle–shaped cell (osteogenic medium); (c) arrow - collagen subfibrils assembled on the lateral surface of the process cell (growth medium); (d) arrow – a process cell with a split mineralized process (growth medium); (e) arrows – ligamentous structures “thrown” through a conglomerate of calcium phosphates (osteogenic medium); (e) the arrow is a ligamentous strip associated with a tangle and elastic fiber (osteogenic medium).

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10. Fig. 9. SEM images of osteoid structures: (a) arrow is a lamellar structure pushed out of the MSCs (growth medium); (b) arrow is a lamella with mineralized marginal sections (growth medium); (c) mineralized bone trabecula with haversoid holes (growth medium); (d) arrow is a spicule embedded in a collagen framework (osteogenic medium); (e) arrow–spicules connecting a mineralized osteocyte–like cell to a trabecula (osteogenic medium); (f) arrow - spicule connected by digitalization to a cell (osteogenic medium).

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11. 10. Light microscopic images of the structure of osteoid nodules and diformazane deposits: (a) loose network of cytoplasmic processes in the nodule (growth medium); (b) dense network of cytoplasmic processes in the nodule (osteogenic medium); (c) average activity of alkaline phosphatase in nodule cells (growth medium); (d) high activity of alkaline phosphatase in nodular cells (osteogenic environment).

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12. Fig. 11. SEM images of enthesis-like structures (osteogenic medium): (a) arrows – lamella attached to the collagen framework by infiltration of compact processes deep into the peritenone substrate; (b) arrow – ridge-shaped structure at the junction of the trabecula with the collagen framework.

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13. Fig. 12. Graphical representation of the Gompertz inverse sigmoid, reflecting the relationship between the specific surface area (Sv) and the diameters (given in ranks) of resident tangles during cultivation of a peritenone substrate together with MSC for 14 days in a growth medium.

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