RU2364360C1 - Hyaline cartilage repair method in treatment of intraarticular fractures - Google Patents

Abstract

FIELD: medicine; traumatology and orthopaedics.

SUBSTANCE: hyaline cartilage repair in treatment of intraarticular fractures is ensured by introduction into the osteochondral defect area of a collagen sponge containing collagen type I and bone powder in the ratio 1:1 w/w.

EFFECT: more rapid repair of bone tissue and the cartilage.

7 dwg

Description

The invention relates to medicine, in particular to transplantology, traumatology and orthopedics, and is intended for the restoration of bone-cartilage intraarticular defects.

In traumatological practice, intraarticular fractures account for approximately 1/3 of all skeleton injuries. Restoring the anatomical integrity and congruence of articular surfaces during intraarticular fractures is an important link in the treatment of these injuries and the prevention of post-traumatic complications, which are accompanied by the development of arthritis, the formation of ankylosis and disability of patients. In this regard, methods for treating osteochondral intraarticular defects are constantly being improved.

It is known that large osteochondral defects are not independently restored [Jackson DW, Labor PA., Aberman HM. et al. Spontaneous Repair of Full-Thickness Defects of Articular Cartilage in a Goat Model. J Bone Joint Surg. 2001; 83: 53]. The articular surface is covered with hyaline cartilage, ensuring the sliding of the articular surfaces relative to each other. Hyaline cartilage is formed by chondrocytes growing from a subchondral zone located at the border between the bone and cartilage and a massive collagen layer corresponding to the degree of load that falls on this portion of the cartilage [Muir H. The chondrocyte, architect of cartilage. Biomechanics, structure, function and molecular biology of cartilage matrix macromolecules. Bioessays 1995; 17 (12): 1039-48]. In case of articular cartilage injury, the intensity of the restoration processes depends on the depth of the lesion, the area of the damaged area, the degree of disorder of the intercellular matrix and the conditions for the existence of cartilage after the injury [V.N. Pavlova, T.N. Kopieva, L.I. Slutsky, G.G. Pavlov . Cartilage. - M.: Medicine. - 1988. - p. 479].

Despite the variety of techniques that stimulate restoration of the cartilage plate, significant defects of the cartilage surface are replaced by coarse connective tissue or fibrous cartilage, the mechanical properties of which do not correspond to articular loads, sliding conditions are violated, which is manifested by serious functional impairments.

Currently, the elimination of extensive traumatic defects of the cartilage of the joints is carried out mainly by the method of surgical correction. An alternative to surgical techniques can be the methods of cell and tissue engineering, which are associated with increasing the effectiveness of treatment [Repin B.C., Susha G.T. Medical cell biology. BEBiM, 1998 .-- v. 126. - No. 10. - S.459].

A known method of stimulating the reparative regeneration of hyaline cartilage of joints by implantation of embryonic chondrocytes or stem cells of germinal connective tissue (German patent No. 341063). The disadvantage of this method is the use of allogeneic tissues, which very often leads to their rejection.

A known method of repairing a defect in hyaline cartilage by introducing into the joint suspension autologous bone marrow stromal progenitor cells grown previously in vitro (patent RU No. 2142285). However, this method of repairing a hyaline defect carries a danger associated with the replication of cells in artificial conditions. A large number of researchers indicate a high probability of damage to the genome of mesenchymal cells during cultivation [Bochkov NP, Voronina ES, Kosyakova NV and others. Chromosomal variability of multipotent mesenchymal cells of human stromal cells. Cellular Technologies in Biology and Medicine, 2007, No. 1. - C.11-15].

A known method of restoring hyaline cartilage by introducing a high concentration of autologous bone marrow mesenchymal cells into the osteochondral defect and fixing them with fibrin glue [Oshima Y., Watanabe N., Matsuda K-i. et al. Behavior of Transplanted Bone Marrow-derived GFP Mesenchymal Cell in Osteochondral Defect as a Stimulation of Autologous Transplantation. J. Histochem. Cytochem. 2005; 53 (2): 207-216]. However, this method also has the disadvantages of the previous method. In addition, mesenchymal cells do not express integrin αvp3, which secures attachment to fibrin, therefore, fibrin glue prevents the proliferation and movement of mesenchymal cells and, probably, the histogenesis of hyaline cartilage, as the defect is filled mainly with fibrous cartilage [Clark RAF. Fibrin and Wound Healing. Ann.N.Y. Acad. Sci. 2001; 936: 355].

In addition, the methods of isolation and cultivation of road cells are extremely laborious, and only very large specialized medical centers have laboratories that can replicate cells for each patient in need.

Today, no one doubts the wide potential of bone marrow mesenchymal cells capable of transformation into cells of various tissues, including chondrocytes, both in vitro and in vivo. The direction of differentiation of mesenchymal cells is determined by their microenvironment. It is known that with acute trauma, a unique microenvironment (growth factors, cyto- and chemokines) is created at the site of injury, activating bone marrow stem cells located in nearby intact tissues [Howes R., Bowness JM., Grotendorst GR. et al. PDGF enhances demineralized bone matrix-induced cartilage and bone formation. Calcif. Tissue Int .; 1988; 42 (1): 34-38]. Due to the fact that both chondrocytes and mesenchymal cells actively secrete collagen proteins, it can be assumed that in the presence of an ostechondral articular defect, when there is direct contact with mesenchymal cells of the bone marrow stroma, the latter should repopulate cartilage tissue with chondrocyte progenitors. However, in order to perform their professional functions in the regeneration of destroyed tissue, progenitor cells must advance into the damage zone using matrix intercellular space proteins (mainly collagen) that are absent in the early stages of the damage zone.

The objective of the invention is to develop a method to improve the restoration of bone tissue and hyaline cartilage in the treatment of intraarticular fractures.

The problem is solved in that a collagen sponge consisting of type I collagen with bone chips in a ratio of 1: 1 (w / w) is placed in an osteochondral defect.

The method of Abedin et al. Was taken as the basis for the production of type I collagen. [Abedin MZ., Riemschneider R. Heterogeneity collagens and them biological mean. Die Angewandte Makromolekulare Chemie 1983, 11 (N1701): p.107-122]. Type I collagen was isolated from rat tail tendons at 4 ° C. Grinded with scissors to a porridge-like state of the tendon was placed in a solution of 1M NaCl, buffered Tris-HCl, pH 7.2-7.5. With constant stirring in a solution of 1 M NaCl, non-collagenic tissue proteins and nucleic acids were washed out, and the insoluble collagen under these conditions was retained in the form of white aggregating fibers. The extraction solution was changed daily for 2-3 days. Then the collagen fibers were collected, washed thoroughly with distilled water to remove NaCl and dissolved in 0.5 M acetic acid with constant stirring for 2-3 days. The resulting collagen solution was filtered through nylon fabric to remove insoluble material.

For the implant, a collagen sponge with bone chips obtained from rat skeleton bones was prepared. For this, 50 ml of a 2% solution of rat collagen type I was mixed with 1 g of bone crumb and lyophilized (collagen: bone crumb ratio = 1: 1 (w / w).

In the experiment, 2 groups of rats were used: control (No. 1) and experimental (No. 2). In rats, a bone-cartilage defect was created on the patella surface of the femoral condyles. Under general anesthesia with ketamine, the knee joint was opened with a longitudinal, anteroposterior parapatellar incision (Fig. 1a), the patella was dislocated outward (Fig. 1b), a cutter with a diameter of 2.3 mm created a defect on the patella surface of the condyles with a depth of 2.5-3 mm ( figv).

In control group No. 1, the articular surface defect was not filled. In experimental group No. 2, the defect was filled with an implant from a collagen sponge with bone chips. The patella was straightened, the wound was sutured in layers. Animals were kept one at a time.

The process of restoration of bone-cartilage defects was evaluated morphologically. The terms of the morphological study were: 1 and 2 weeks, 1 and 3 months after surgery. We studied 4 animals at each time point (32 animals in total). After the operation, the animals were withdrawn from the experiment at the indicated time by introducing an intraperitoneal lethal dose of hexenal, the knee joints were removed and fixed in a solution of 10% neutral formalin. The operated site of the femoral condyles was subjected to morphological study. Samples were decalcified with the Biodek preparation and embedded in paraffin. Using a microtome, sections 4–5 μm thick were obtained, which were stained with hematoxylin and eosin, van Gieson picrofuxin and toluidine blue.

In a morphological study of the region of the patella condyle of the femur of the rat femur in the control group (No. 1) 1 week after the operation, most animals revealed that the bone defect was filled with organized fibrin, red blood cells, and in deeper areas there are signs of the formation of islets of immature loose connective tissue (figure 2, uv.kh400) After 2 weeks, the severity of inflammatory changes decreases, granulation tissue increases in volume and matures. In the depth of the defect, bone tissue regeneration is just beginning, immature osteoid beams are formed. 1 month after surgery, inflammatory infiltration disappears, granulation tissue turns into mature connective tissue, and osteogenesis intensifies in the depth of the defect. 3 months after surgery, the defect decreases in volume and fills closer to the surface with dense fibrous connective tissue without signs of cartilage tissue regeneration, and inside the defect - bone regenerate, which matures by this time and occupies about 4/5 of the volume of the former defect (Fig. 3, UV.x400).

In experimental group No. 2, a matrix was used to seal the defect, which consisted of type I collagen and bone chips. After 1 week, the newly formed maturing bone tissue, consisting of osteoid beams, occupies 4/5 of the defect volume, and the outer layer is formed by mature connective tissue, but still without signs of cartilage regeneration. Visible areas of resorption by macrophages of fragments of bone crumbs (figure 4, uv.kh400). After 2 weeks, there is no collagen filling due to resorption of the implanted collagen. Bone tissue matures. During this period, numerous foci of cartilage regeneration are formed. Around the remaining fragments of the implanted collagen and bone chips, an environment of differentiated chondrocytes typical of hyaline cartilage is observed from the columns (Fig. 5). One month after the operation, the formation of large foci of fibrous and hyaline cartilage is noted. After 3 months, the defect zone is filled with bone tissue, and outside the areas of connective tissue and regenerated hyaline cartilage (Fig. 6, u.v200) When a visual inspection of the sliding surface of the femoral condyle is made, the place of the defect is not detected (Fig. 7).

The introduction of collagen type I, a universal intercellular matrix into osteochondral wound defect, to which all cells participating in the wound process have specialized integrin receptors that determine cellular movement, is a prerequisite for the directed movement of cells into the lesion zone [Lynch MP., Stein JL., Stein GS., Lian JB. The influence of type I collagen on the development and maintenance of the osteopath phenotype in hoary and passage rat calvarias osteoblasts: modification of expression of genes supporting cell growth, adgesion, and extracellular matrix mineralization. Exp Cell Res; 1995; 216 (1): 35-45]. In addition, the collagen sponge densely seals the bone-cartilage wound, preventing the outflow of blood into the joint bag, which also favorably affects the course of the early postoperative period. The role of bone crumbs is probably that it is a source of bone morphogenic protein (MBP). The KMB family is known to be involved in various development programs, and its members are powerful inducers of osteo- and chondrogenesis [Canalis E., Economides AN., Gazzerro E. Bone Morphogenetic Proteins, Their Antagonists, and the Skeleton. Endocr. Reviews, 2003; 24 (2): 218-235; Cook SD., Patron LP., Salkeld SL. et al. Repair Articular Cartilage Defects with Osteogenic Protein-1 (BMP-7) in Dog. J Bone Joint Surg. 2003; 85: 116-123]. Thus, the combination of the biological properties of type I collagen and bone chips in the implant actively contributes to the restoration of bone and hyaline cartilage in an intraarticular defect.

Claims (1)

A method of restoring hyaline cartilage in the treatment of intraarticular fractures by introducing a collagen sponge into the osteochondral defect region, characterized in that a collagen sponge consisting of type I collagen and bone chips is introduced in a ratio of 1: 1w / w.

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