WO2023048552A1

WO2023048552A1 - Allogenic implants for the treatment of cartilage injuries

Info

Publication number: WO2023048552A1
Application number: PCT/MX2022/050017
Authority: WO
Inventors: Anell Olivos Meza
Original assignee: Anell Olivos Meza
Priority date: 2021-09-24
Filing date: 2022-03-11
Publication date: 2023-03-30
Also published as: MX2021011695A

Abstract

The present invention provides a graft or construct formed by isolated cartilage chondrocytes from young donors (<20 years old), seeded on a three-dimensional membrane, composed of one of the main chondrogenesis-promoting substances (hyaluronic acid), in high densities (1x106) with autologous serum, and sealed with a fibrin adhesive for treating a cartilage injury, with tissue formation that has greater durability, a lower cost and fewer risks than treatments currently available for said injuries. Furthermore, a method for the treatment of articular cartilage injuries is provided, by means of implanting an allogenic chondrocyte graft that has the capability to satisfactorily generate or repair the cartilage of the injury.

Description

IMPLANTS FOR THE TREATMENT OF INJURIES OF

CARTILAGE

TECHNICAL FIELD

The present invention is located in the field of medicine, particularly in cell therapy and tissue engineering, it refers to treatment methods for articular cartilage injuries, particularly knee cartilage injuries. Likewise, it refers to the development of an implant or construct of allogeneic chondrocytes for the treatment of said cartilage lesions.

BACKGROUND

Cartilage injuries in weight-bearing joints such as the knee, ankle and hip are difficult to heal without surgical treatment. The incidence of this type of injury is high in young patients (30-35 years), especially in the knee joint, found in up to 65% of arthroscopies performed on this joint to treat other pathologies (ligament injuries and /or menisci). ^1-4 The consequences of not adequately and promptly treating these injuries are pain, gradual loss of function, disability, and early osteoarthritis. ^{3 4} Obviously, this type of injury is a major health problem with significant epidemiological repercussions and a great impact on health and quality of life, generating significant costs for individuals, institutions, and the health system in general.

Currently there are various surgical techniques to treat cartilage injuries with variable results. However, despite this diversity of techniques, they present drawbacks that need to be overcome for the good of the patient, their quality of life, treatment and recovery times and, of course, economic costs. It has been documented that the treatment that has given the best results in the long term, in terms of pain relief, improvement in function and delay in the development of early osteoarthritis, is Autologous Chondrocyte Implantation (ACI, for its acronym in English; Autologous Chondrocyte Implantation) ⁴⁵ . ACI is currently the only cell therapy for cartilage repair in the UK. This technique was carried out and published by two Swedish orthopedics in 1987. Until 2010, 35,000 cases had been carried out worldwide, reporting that up to 87% of patients undergoing this procedure present clinical results that are classified as good to excellent. compared with other techniques (microfractures, autologous osteochondral transfer, augmented microfractures) that show less encouraging results.

The ACI technique is a procedure that comprises two surgical stages. In the first surgical procedure, cartilage biopsies are taken in a "non-load" area of the knee of the same patient (autologous). The cartilage biopsy is sent to the laboratory, where the cells that produce cartilage (chondrocytes) are extracted and cultured in vitro for a period of 6 to 8 weeks with the purpose of increasing the number of cells available, which will be used in the next surgical procedure. Once the appropriate number of cells is available, a second arthroscopy is performed to place the chondrocytes, previously cultured on a commercially available polymer of hyaluronic acid, in the lesion to promote the formation of a tissue very similar to cartilage with the aim of passage of time, repairing the defect, alleviating the symptoms of the injury; this allows the patient to resume activities of daily living, and even, in the long term, to perform sports activities.

Over time, the ACI technique has undergone multiple modifications, which are usually referred to as "generations", currently 3 generations are recognized, with the aim of counteracting complications and improving long-term results. In the 3rd generation, an important modification is that the autologous chondrocytes are seeded in a collagen matrix to later be placed in the defect or lesion site, this reduces the leakage and loss of chondral cells outside the matrix as it used to happen. in the 1 ^§ and 2- generation. The third generation is also known as MACI (Matrix Associated Chondrocyte Implantation), and has shown better results than previous generations, so it has been observed that culturing the cells on the matrix is more effective than injecting them into the defect and then covering it. with a membrane sutured to the adjacent cartilage. Although the results with this technique MACI are better than those obtained with previous generations, this technique still has various drawbacks, among which are its high costs and even more limiting is the inability for any hospital or Health Institute to carry it out. practice due to the required infrastructure and trained personnel to carry out tissue engineering work.

The high economic cost of this treatment is due to the fact that two surgical procedures are required, in addition to the long time required for cell culture, including laboratory material, infrastructure, and human resources; which amounts to €17,000 in ⁶ European countries and approximately $250,000 in Mexico.

Another great limitation also occurs in the source of autologous cartilage, since this requires taking healthy cartilage from a knee of the same patient, which implies 2 problematic scenarios for the patient, on the one hand, there is the limitation of the amount of cartilage healthy that can be taken from the patient and, on the other hand, there is a great risk of developing premature joint wear in the donor area (early osteoarthritis).

With the aim of reducing costs and the two-step procedure, a one-step technique called STACI (Single treatment Autologous Chondrocyte Implantation) has been described in which the laboratory is taken to the operating room and during the cartilage biopsy surgery, the chondrocytes are isolated with an enzymatic procedure, they are stimulated with growth factors in one hour and mixed with bone marrow mononuclear cells, obtained from the same patient. This mixture of cells is seeded on a matrix of collagen or hyaluronic acid, to be placed in the lesion during the same procedure. The results of STACI (one surgery) at two years are similar to those reported in ACI (two surgeries), however, the reduction in costs makes this technique an attractive option for cartilage regeneration. ⁷

Allogeneic chondrocyte implantation has currently been explored to resolve the limitations and the multi-step technique that autologous chondrocyte implantation requires, however, what is reported in the literature is still limited. ⁸ Document EP2919794 was found in the patent literature, which refers to compositions for use in methods of treatment of a defect in cartilage, bone, ligament, tendon, meniscus, joints or muscle in a subject. In one modality, the method comprises administering a composition of fragmented or particulate cartilage (0.25 to 5 mm), derived from a cadaveric human donor, wherein said cartilage particles possess viable chondrocytes and also contains a biocompatible vehicle, comprising a solution of cryopreservation (DMSO+serum) or a culture medium (DMEM+5%SFB; high glucose DMEM).

Patent document EP2338441 refers to a product also composed of fragments or particles of cartilage derived from a cadaveric donor, but in this case from young donors (under 15 years of age), which enhances a greater capacity to form a hyaline cartilage matrix compared to the that can occur with chondrocytes from adult donors. The product is presented as a kit in a sterile container for the treatment of cartilage lesions composed of fragmented cartilage particles from a cadaveric donor containing viable chondrocytes and a storage solution.

A major drawback of the previously described patents is that cadaveric donor-derived cartilage retains the extracellular matrix, although it functions as a three-dimensional scaffold, like a chondroreactor for fragmented cartilage particles, it is also true that such matrix also poses a higher risk. of immunological response and rejection reactions by the recipient, since the cartilage contains donor proteins that can be identified by the recipient and produced from a simple rejection mechanism, which prevents the formation of the desired tissue and its integration, up to a greater immunological reaction both local and systemic.

Yet another drawback of the technology described in said patents results from the fact that the tissue is derived from rib cartilage, nasal cartilage, tracheal cartilage, sternal cartilage and other unspecified sources of cartilage, the nature of which, composition and structure is very different from that of the cartilage of the joints (knee, ankle, hip, shoulder), for which it is desirable that chondrocytes are derived from hyaline cartilage, mainly knee (as in the case of the present invention) to form articular cartilage.

The patent document WO 2019/1 13558 A1 discloses the treatment of chondral and osteochondral lesions, using a composition of allogeneic cells, chopreserved in a cell bank, cultured in a resorbable membrane of porcine collagen (type I and/or II), at a density of 250,000 cells per cm ² . In this document it is indicated that chondrocytes can be obtained from "various tissues", which implies the possibility that the cells obtained are not chondrocytes specific for hyaline cartilage. On the other hand, before cryopreserving the chondrocytes, they are expanded and cultured for at least two passages (in one section they describe less than 5), which conditions a dedifferentiation of the primary chondrocytes and, therefore, there is a elevated risk of fibrocartilage formation instead of hyaline cartilage. In addition, another event that can condition the formation of fibrocartilage instead of hyaline cartilage is the low density of cells seeded in the collagen membrane, another factor that favors cell dedifferentiation and the formation of fibrous tissue.

On the other hand, there is also the drawback that the chondrocyte cultures are supplemented with fetal bovine serum, a serum of animal origin that can clearly trigger immunogenic reactions in a human recipient.

OBJECT OF THE INVENTION

The present invention refers to an orthopedic implant made up of cadaveric allogeneic chondrocytes on a hyaluronic acid support or membrane, sealed with a fibrin adhesive, for the treatment of a cartilage lesion, with tissue formation that has greater durability, at a lower cost. cost and with fewer risks than currently available treatments for the management of such injuries.

Another object of the present invention refers to a kit for the treatment of cartilage that comprises an orthopedic implant, made up of allogeneic chondrocytes, derived from cadaveric donor, seeded on a hyaluronic acid membrane and covered with a biocompatible adhesive, where said chondrocytes are adhered to the membrane and present the formation of extracellular matrix; wherein said container also comprises a culture medium, supplemented with autologous serum and antibiotics-antimycotics.

Another object of the present invention refers to a procedure for the treatment of articular cartilage lesions, through the implantation of an orthopedic construct made up of cadaveric allogeneic chondrocytes on a hyaluronic acid support or membrane, sealed with a fibrin adhesive, where said chondrocytes They are adhered to the membrane and present the formation of extracellular matrix.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 . Flowchart of the method for obtaining the implant or construct with allogeneic chondrocytes of the present invention, where a) is the biopsy; b) cell culture; c) cell expansion; and d) implant formation.

Figures 2A-2F. Photographic sequence of the procedure for obtaining cartilage biopsies from cadaveric donors.

Once a tissue donor is identified, arthrotomy of both knees is performed and cartilage fragments are taken (see figure 2A to 2D), which are placed in sterile containers (figure 2E to 2F). containing physiological solution or culture medium (DMEM-F12) supplemented with 10% antibiotic-antifungal.

In one embodiment of the present invention, cartilage samples are taken from the femoral condyles and patella (Fig. 2A and 2B), with the help of a scalpel, with #20 blades. The tissue is taken from the most superficial part (Fig. 2C and 2D), with the purpose of not deepening the cut to the subchondral bone, to avoid the exit of bone marrow stem cells and contact with intramedullary fluids or tissue contamination. with cells of other origin. At the biopsy procurement site, the donor's data is documented to standardize characteristics that may influence the viability and chondrogenic capacity of the cells (age, gender, associated pathologies, and cause of death). Likewise, the associated variables such as time of death and time between death and taking the cartilage are verified.

Figures 3A-3E. Photographic sequence of the isolation method and viability determination of chondrocytes derived from cadaveric donor cartilage.

Briefly, the tissue is washed several times with PBS and 10% antibiotic-antifungal, until fluids and donor debris are completely removed (A). Subsequently, mechanical digestion is performed with a scalpel to obtain tissue fragments smaller than 1 mm (B), followed by enzymatic digestion using type 2 collagenase (0.1 to 0.3% w/v), for at least For 1 hour at 37°C, with continuous agitation, the supernatant containing the cells released by the digestions previously described (C) is recovered, and the undigested fragments are subjected to a second digestion stage with a fresh enzyme solution, for at least minus another hour at 37°C.

The number of cells and their viability are determined by counting in the Neubauer chamber and trypan blue (D). The isolated chondrocytes are cryopreserved (E) without expanding, with the purpose of conserving and maintaining both their viability, their morphology and functionality; various methodologies are known in the state of the art for this purpose. The freezing medium comprises culture medium, commercial human serum or autologous serum, and a cryoprotective substance. In one embodiment of the present invention, the obtained chondrocytes were deposited in freezing medium [DMEM/F12 (80%), autologous serum (10%) and DMSO (10%)], at a density of a maximum of 500,000 cells per cell. ml of medium, and cryopreserved in liquid nitrogen or at -192°C.

Figures 4A-4D. Photographs of a Grafting Kit or three-dimensional construct. The figure shows a kit for the treatment of cartilage that includes an orthopedic implant, made up of allogeneic chondrocytes, derived from cadaveric donors, seeded on a hyaluronic acid membrane and covered with a biocompatible adhesive, where said chondrocytes are adhered to the membrane and present the formation of extracellular matrix; wherein said container also comprises a culture medium, supplemented with autologous serum and antibiotics-antimycotics.

Figures 5A-5D. Presence of proteoglycans in the formed cartilage.

Shown are histological sections of tissue formed from allogeneic cadaveric chondrocytes of the present invention (5B), chondrocytes derived from living donor (P0), neither expanded nor cryopreserved (5C), chondrocytes derived from living donor (P2) expanded for 2 passages , without cryopreservation (5D) and hyaline cartilage control tissue (5A), stained with alcian blue that allows identification of the characteristic extracellular matrix of hyaline cartilage.

Figures 6A-6D. Quantitative evaluation of the quality of the cartilage formed.

Quantitative analysis of histological staining was carried out by three independent and blinded observers, using the modified O'Driscoll histological scale. In this, the amount of cartilage formed is evaluated, which is evidenced by red staining, giving it the following score of 80-100% (8 points), 60-80% (6 points), 40-60% (4 points), 20-40% (2 points) and 0-20% (0 points); integrity of the structure of the tissue formed, assigning the following score: Normal (2), slight disruption (1) and severe lack of integration (0); Cellularity, which evaluates how similar the shape of the cells is to normal cartilage and how many cells there are, assigning them the following score: normal cellularity (3), hypocellularity <25% (2), moderate hypocellularity >25% (1), severe hypocellularity (0).

At least three fields of each histological section were evaluated. A maximum score of 14 was given to native cartilage (Figure 6A); the closer the score is to 14, which is assigned to histological samples, it means that the tissue formed presents a higher quality and that it is more similar to healthy cartilage. Figure 7. Diagram of the implant or construct placement procedure in the area of the lesion.

Figures 8A-8F. Preparation and measurement of the size of the lesion by arthroscopy.

The figures show an image of the lesion to be treated (8A), and the debridement of the edges of the lesion (8B, 8C), to obtain a square or rectangular area (8F) and the measurement at the top and bottom. width by using an arthroscopic hook palpator (8D and 8E).

Figures 9A-9D. Implantation and fixation of the construct at the lesion site.

The image shows how the implant or construct is inserted into the area of the lesion (9A), as well as its extension on the surface of the lesion (9B), ensuring contact of the graft with all the edges of the adjacent native cartilage (9C ) and coating with fibrin glue (9D).

Figures 10A-10B. Process of evaluation of the integration of the construct by NMR.

A Nuclear Magnetic Resonance study of a knee treated with the implant of the present invention is shown, showing the tissue generated at 12 months with adequate contact with the native cartilage.

Figures 1 1 A-11 F. Cartilage lesion repair at 12 months: ACI vs ALCI.

Arthroscopic evaluation, 12 months after treatment of cartilage lesion in the lateral trochlea of both knees in the same patient. In the figures of sections A to C, the images of the right knee are shown, with a 20x15 mm lesion, treated with autologous chondrocyte implantation (ACI); On the other hand, the left knee ( Fig . 1 1 D, 1 1 E and 1 1 F), with a lesion of 18x15 mm, was treated with the allogeneic chondrocyte implant of the present invention (ALCI).

Figures 12A-12D. Fibrocartilage formation in the donor area in the ICA Arthroscopic evaluation of the osteochondral biopsy in autologous chondrocyte implantation (Fig. 12A and 12B). Subsequently, by means of a second arthroscopic view 12 months after autologous chondrocyte implantation, filling of the donor area with abundant presence of fibrous, irregular and fibrillar tissue is clearly observed (Fig . 12C and 12D).

Figures 13A-13F. Hip construct placement.

Left hip arthroscopy. In images 13A and 13B, the cartilage lesion is identified in the anterior-superior region of the acetabulum (25 x 10 mm), while in images 13C and 13D, a lesion of the same size is indicated, but in the posterior area. - superior. Both lesions involve 40% of the total articular surface. Images 13E and 13F show the placement of the allogeneic chondrocyte implant and its fixation with fibrin glue.

Figures 14A-14B. Quality of repaired cartilage in the hip.

Evaluation of the quality of the repair tissue by measuring the relaxation time of the water 3 months after the hip implant. The red arrow indicates the value of the control cartilage (ROI-1) versus the measurement of the repair tissue (ROI-2) (47.5 ms vs 50.5 ms), the values are very similar.

Figures 15A-15F. Knee construct placement.

The figure shows the placement of the graft with cadaveric chondrocytes in the patella. 15A) Resection of a bony prominence (osteophyte) to prevent erosion of the trochlear cartilage. 15B) Osteophyte at the base of the anterior cruciate ligament (ACL). 15C) ACL reconstructed with synthetic graft. 15D) Cartilage lesion on the lateral facet of the patella (30 x 30 mm). 15E) Placement of the graft or hyaluronic acid construct seeded with allogeneic chondrocytes in the area of the lesion. 15F) Application of fibrin glue to promote adherence of the implant in the lesion and in the adjacent cartilage.

Figures 16A-16B. Quality of cartilage repaired in the knee. The figure shows the evaluation of the quality of the repair tissue by measuring the relaxation time of the water 3 months after the knee implant. The red arrow indicates the control cartilage value (ROI-1) versus the repair tissue measurement (ROI-2) (47.7 ms vs 56.9 ms). The repair tissue value still reflects the presence of immature cartilage at 3 months.

Figures 17A-17B. Hip functional assessment scales before and after surgery.

The functional evaluation of the hip was carried out using widely recognized scales, for example, the Harris hip scale is the most widely used instrument to evaluate the results obtained after hip arthroplasty. The evaluation was carried out before and after surgery to assess the movements of the transverse, anteroposterior and longitudinal axes or mobility arcs.

Figures 18A-18E. Functional evaluation scales of the knee before and after surgery.

The functional evaluation of the knee was carried out using widely recognized scales, for example, the TEGNER, LYSHOLM, International Knee Documentation Committee (IKDC) and KUJALA scales. The evaluation was carried out before and after surgery to assess the range of motion, pain and function of the knee.

DESCRIPTION OF THE INVENTION

The present invention relates to an implant or construct for the treatment of cartilage lesions in an individual's joints, wherein said implant is made up of allogeneic chondrocytes, derived from a cadaveric donor, cultured on a hyaluronic acid scaffold, sealed with a biocompatible glue. Likewise, it refers to a kit for the treatment of cartilage that comprises primary culture of allogeneic chondrocytes, derived from cadaveric donor, seeded on a hyaluronic acid membrane, and covered with a biocompatible glue, arranged in an appropriate container or container for such purpose, wherein said container also comprises a culture medium, supplemented with autologous serum and antibiotics-antimycotics.

The invention of the present application clearly contributes to solving at least one of the many latent drawbacks in the technical area. One of the great advantages of the present invention is that it favors the formation of a better quality of hyaline cartilage, which favors the integration of the implant to the adjacent native tissue and we have also observed in all treated patients that the risk of immunogenic reactions is reduced, every time we have observed that all patients subjected to this technique have not presented local or systemic rejection data, such as: increase in joint volume, increase in local temperature, absence of rejection or detachment of the implant when evaluated by magnetic resonance. Another of the great advantages of the present invention lies in the fact that enough material is obtained from the donor tissue to treat large lesions, that is, there is no limitation in the number of cells to carry out the medical treatment. Another great advantage of the present invention is that the procedure for obtaining the graft or construct requires short times (approximately 1 week) compared to the different methodologies (approximately 8 weeks) described in the state of the art. In the present invention, chondrocytes isolated from articular cartilage (knee and/or patella) from young donors (<20 years old) are used, seeded on a three-dimensional membrane, composed of one of the main chondrogenesis-promoting substances (hyaluronic acid), in high densities (1x10 ⁶ ) with autologous serum and sealed with a fibrin adhesive.

In the present invention we have observed that in the development of the described implant it has been observed that the seeding of the chondrocytes on the membrane or scaffold has made it possible to maintain a three-dimensional environment, which in turn is necessary to maintain the chondral cells in appropriate conditions and, in this way, prevent them from dedifferentiating into “fibrochondrocytes”. It is highly desirable to avoid obtaining fibrochondrocytes, since the repair cartilage formed from these cells is of poor quality and durability, since these cells produce very different structural components from native hyaline cartilage. On the other hand, we have observed that the structural nature of the membrane of hyaluronic acid allowed us to obtain an implant with the flexibility and desirable resistance to be manipulated during arthroscopy. Likewise, during the development of the present invention and with the methods described, we found that more than 80% of the chondrocytes obtained from cartilage, coming from the knees of cadaveric donors, up to 48 hours after death, maintain their viability and preserve the capacity. to form cartilage in in-vitro cultures (figures 4 and 5).

For the present invention, the terms or expressions "construct", "graft", "chondrocyte graft", "implant", "chondrocyte implant" or "orthopedic implant" are used interchangeably, with the same meaning and scope of protection. ”. In these we refer to a cartilage substitute with the ability to functionally and structurally repair, regenerate or replace cartilage in a patient who has lost the function of said tissue, or when said tissue has suffered some mechanical or physiological damage.

For the present invention, the expression "cadaveric donor" refers that the cartilage tissue is derived from a deceased donor, where said donor is approximately 20 years old or less, preferably less than 20 years old at the time of donation. Normally, chondrocytes derived from a young individual have a greater capacity to synthesize/organize the extracellular matrix of hyaline cartilage compared to those derived from an adult individual.

For the present invention, the expression "allogeneic chondrocytes" takes the conventional meaning in the technical area, which refers to chondrocytes from different individuals of the same species.

For the present invention, the terms or expressions "membrane", "scaffold" or "support" are used interchangeably, with the same meaning and scope of protection. In these we refer to a support structure, made up of one or more materials of a different nature, preferably biocompatible, resorbable and resistant materials. This membrane provides a mechanical support on which the chondrocytes are deposited, which facilitates their placement at the site of the defect to be treated, and also allows said cells to remain at the site of the defect. In one embodiment the membrane is composed by a layer of a synthetic biopolymer of hyaluronic acid or its derivatives, particularly esterified derivatives, also does not contain animal or human by-products, which prevents allergic reactions from other products. In another modality, the membrane is composed of a layer of a synthetic biopolymer of hyaluronic acid esterified with benzyl alcohol and some of its characteristics are: it is biodegradable, mechanical resistance, biocompatible, non-cytotoxic (does not destroy cells), non-antigenic (does not causes an immune response), promotes cell proliferation and differentiation, is flexible and elastic, and allows the passage of nutrients and metabolic waste, and its metabolization mechanism is known and safe. This membrane degrades through hydrolysis (decomposition by action of water) of the ester, which generates the release of HA and benzyl alcohol.

For the present invention, the term or expression "fibrin tissue adhesive" or "fibrin glue" refers to cell adhesive compositions based on human fibrinogen and thrombin, which allows the generation of a fibrin clot for hemostasis, sealing and healing of tissues and for improve tissue adhesion. In one embodiment, it is desirable that said adhesive contain only components of animal or synthetic origin, in order to avoid adverse reactions thereto by the receiving organism. In another embodiment, the adhesive may contain other components than those previously mentioned to strengthen its adhesive property. In another modality, the fibrin glue can be obtained from autologous serum during surgery, or one of synthetic origin and for commercial use. Some of the fibrin-based adhesive products that are marketed are, for example: Beriplast® Behering, Tisseel® from Baxter, Evicel ® from J&J, etc.

For the present invention, the term or expression "culture medium" or "medium" refers to compositions suitable for the culture of chondrocytes, as long as they support or favor the maintenance and/or growth of cells in in vitro culture. In some preferred embodiments, it also refers to the culture medium supplemented with one or more additional components. In some embodiments, additional components can include, for example, serum, antibiotics, antifungals, growth factors, buffers, pH indicators, and the like. In other embodiments, the medium can be used in the process of isolating cells (eg, chondrocytes and/or chondrocyte precursors) from a tissue sample (eg, a cartilage sample). In some embodiments, the tissue is mechanically digested and then subjected to enzymatic digestion, combined with the medium which may comprise enzymes (eg, collagenase or protease) to digest tissue and release cells.

For the present invention the term or expression "cadaveric allogeneic chondrocytes" or "cadaveric chondrocytes" refers to chondrocytes that are isolated from cartilage derived from young cadaveric donors, up to 20 years of age. In some embodiments, the isolated cells can be used immediately, or they can be cryopreserved, without expanding (P0), until use.

EXAMPLES

These examples are illustrative and not limiting in nature, since a person skilled in the art will understand that there are variants that fall within the scope of protection of the present invention.

Example 1: Obtaining cadaveric chondrocytes.

Selection of cadaveric donors

For the present invention, biopsies or tissue samples are obtained from healthy young donors. In one modality, the donors are selected from the male gender, to avoid variables of a hormonal nature in the biology of the chondrocytes obtained. Young donors are selected from an age of up to 20 years, in one modality they are selected with an age between 14 and 20 years. The cartilage samples are obtained from the knees of the donors (see figure 2), taking as an important factor that they are macroscopically healthy joints (without the evident presence of traumatic cartilage lesions, crystal deposits, degenerative chondral lesions, previous or active infection). and/or rheumatic diseases). Therefore, the staff that procures the tissue corroborates that the tissue is smooth and whitish. In one embodiment, the biopsies or tissue samples that were used in some of the examples of the present invention were provided by the Biograft Skin and Musculoskeletal Tissue Bank of Mexico.

Donor serological panel

Once a cadaveric donor is selected and the biopsy or tissue sample is taken, this tissue is submitted to a panel of studies to rule out the presence of infectious diseases that could be transmitted through the donated tissue. The panel includes the study of antibodies against hepatitis B (Core and Surface), hepatitis C, HTLV, syphilis, HIV and detection of nucleic acids (NAT test) against hepatitis B, C and HIV. Once the biopsies or tissue samples present negative serology reports, they are considered appropriate for use in the formation of the implant or construct of the present invention.

In one embodiment of the present invention, some of the biopsies or tissue samples used in the present invention were sent to the Viromed/Labcorp laboratory, in Minnesota, USA, who performed said panel studies.

Obtaining the biopsy

Once a tissue donor is identified with the inclusion criteria described, the tissue procurement staff plus an expert orthopedist in cartilage collection go to the hospital where the death occurred, under aseptic, sterile conditions, the procedure is performed. arthrotomy of both knees and to take cartilage fragments (see figure 2), which are placed in sterile containers (figure 2E-2F), containing culture medium (DMEM-F12) supplemented with 10% antibiotics-antimycotics , later they are transferred to -4°C to the laboratory of the Tissue Bank (Biograft) to be processed.

In one embodiment of the present invention, cartilage samples are taken from the femoral condyles and patella (Fig. 2A and 2B), with the help of a scalpel, with #20 blades. The tissue is taken from the most superficial part (Fig. 2C and 2D), with the purpose of Do not deepen the cut as far as the subchondral bone, to avoid leakage of bone marrow stem cells and contact with intramedullary fluids or contamination of the tissue with cells of other origin.

At the biopsy procurement site, the donor's data is documented to standardize characteristics that may influence the viability and chondrogenic capacity of the cells (age, gender, associated pathologies, and cause of death). Likewise, the associated variables such as time of death and time between death and taking the cartilage are verified.

chondrocyte isolation

Chondrocyte isolation (see figure 3) is carried out in appropriate facilities for this purpose, for example, in a tissue culture laboratory, under sterile conditions and in a level II laminar flow hood. The tissue is washed, with PBS + 10% antibiotic-antimycotic, at least a couple of times or more to remove donor fluids and debris, in one embodiment 3 or more washes are preferred. The cartilage obtained is weighed and with this the ratio of the number of chondrocytes obtained per milligram of tissue is obtained. Subsequently, mechanical digestion is performed to obtain cartilage fragments smaller than 1 mm (see figure 3B), followed by enzymatic digestion with type 2 collagenase (0.1 to 0.3% w/v), for at least 1 hour at 37 °C, in continuous stirring: in one modality the digestion is carried out for at least 2 hrs., in another equally preferred modality the digestion is carried out for at least 3 hrs., in another equally preferred modality the digestion is carried out out for at least 4 hrs. As is evident, the efficiency of the enzymatic treatment can be obtained with the variation of different parameters, for example, the efficiency increases when there is a greater surface area of the exposed tissue, when the amount or activity of the enzyme increases, and with the time of treatment. digestion. The enzymatic digestion could be carried out continuously or, alternatively, it could be carried out in two or more digestion steps; for example, the digestion is carried out for 60 to 120 minutes, the supernatant containing the cells released by the digestion is recovered, and the undigested fragments are subjected to a second digestion step with a fresh enzyme solution, for at least another 60 to 120 minutes (Figure 3C). The number of cells and their viability are determined by counting in the Neubauer chamber and trypan blue (Figure 3D). The chondrocytes obtained are cryopreserved (figure 3E) without expanding, with the purpose of conserving and maintaining both their viability, their morphology and functionality; various methodologies are known in the state of the art for this purpose. The cryopreservation medium comprises culture medium, serum or combinations thereof, and a cryoprotective substance. In one embodiment, the cryopreservation or freezing medium may be comprised of 10% commercial human serum or 10% autologous serum, although autologous serum is preferred, plus 80% DMEM/F1 2 culture medium and 10% DIVISO. DMSO is a cryoprotective substance that prevents chondrocytes from swelling and bursting on freezing. In an embodiment of the present invention, the obtained chondrocytes were deposited in the aforementioned freezing medium in a vial in which 1 ml of said substance was deposited for a maximum of 500,000 cells and stored in a liquid nitrogen chamber at - 192°C.

Example 2: Formation of the implant or construct©.

autologous serum

A peripheral blood sample (200-250 ml) from the patient to be treated is processed by centrifugation to remove the red fraction and obtain the autologous serum. This is inactivated, filtered and aliquoted into 10 ml tubes and stored under conditions sterile at -4°C, until required for use.

In one modality, the orthopedic surgeon, who requests the graft, provides the tissue culture laboratory or tissue bank with the data of the patient to be treated, who is contacted to come and donate a peripheral blood sample (200-250 mi), to obtain autologous serum, preferably 5 to 7 days prior to surgery.

Implant preparation To prepare an implant or construct, chondrocytes are thawed, through a gradual process to minimize the percentage of cell death, and once thawed, counting and determination of cell viability is performed again. Next, the chondrocytes are seeded on the hyaluronic acid membrane or scaffold, at a density of 1 x 10 ⁶ cells per cm ² . The construct is sealed over its entire surface with fibrin glue to promote adherence of chondrocytes to the membrane and decrease the risk of cell loss. The size of the hyaluronic acid membrane or scaffold, in one modality the hyaluronic acid is of synthetic origin, depends on the requirements of the orthopedic surgeon who will implant it, and can have a size ranging from 10 x 10 mm to 50 x 50mm. The chondrocytes seeded on the membrane are cultured at 37°C, 5% CO2 and 5% humidity, with DMEM-F12 culture medium + 10% autologous serum + 1% antibiotic-antimycotic. Every second or third day a change of culture medium is made, for a period of 5 to 7 days (Figure 4), to allow cell adherence and the formation of extracellular matrix. The autologous serum is obtained from the patient in whom the construct is to be implanted, thus avoiding the use of fetal bovine serum, which can lead to secondary reactions in the patient.

Example 3: Evaluation of the presence of proteoglycans in the formed cartilage

During the development of the invention, viability tests were carried out on the chondrocytes obtained from cadaveric donors (P0), as well as their ability to form hyaline cartilage. These were subjected to comparative studies with chondrocytes obtained from a living donor without expansion and without cryopreservation (P0) and chondrocytes obtained from a living donor expanded, during 2 passages, without cryopreservation (P2), in both cases the donors presented similar demographic conditions. For all groups, chondrocytes were seeded on a collagen membrane, then this implant or construct was implanted in the back of athymic (Nunu) mice, which were monitored for 3 months. After three months, the tissue formed in each of the study groups was evaluated by staining and immunofluorescence for cartilage compared with native hyaline cartilage. The tissue formed in the different groups was analyzed by staining with alcian blue (blue stain) and Safranin-0 (red-brown stain). Alcian blue staining reveals cartilage and is especially useful for differentiating articular hyaline cartilage vs. bone, since the cartilaginous extracellular matrix stains with alcian blue (Figure 5). For its part, staining with the Safranin-0 dye has become a basic guide in the identification of articular cartilage, because safranin O binds to glycosaminoglycans (GAGs), which are terminal structures of proteoglycans such as aggrecan, which is an essential component of articular cartilage and is abundant in the extracellular matrix of articular cartilage, likewise it is considered that the intensity of safranin-0 staining is directly proportional to the content of PG in cartilage (figure 6).

The results are shown in figure 5, in which histological sections stained with alcian blue are observed. In (5A) the control tissue is shown, native hyaline cartilage; in (5B) the tissue generated from the cadaveric chondrocytes (P0) of the present invention is observed, while in (5C) and (5D) the tissue generated from the chondrocytes derived from living donors (P0 and P2, respectively). The images clearly show that the tissues generated from living donor chondrocytes (5C and 5D) show moderate to very low staining, respectively, while the tissue generated from cadaveric chondrocytes (P0) of the present invention show very low staining. marked blue staining (Figure 5B). This blue staining makes it possible to identify an extracellular matrix, with a homogeneous appearance, characteristic of hyaline cartilage. The extracellular matrix is secreted by the chondrocytes generated from the implant or construct of the present invention. In addition, in Figure 5B the chondrocytes that usually associate in pairs or tetrads can be clearly seen, thus forming the so-called isogenic groups.

Likewise, from this result it can be deduced that living donor chondrocytes (P2), which were cultured for 2 passages (figure 5D) practically lose the ability to generate articular hyaline cartilage vs. the control tissue of the histological section (figure 5A). Even more important is the result derived from the comparison of figures (B) vs. (C), in which the tissue formed from the cadaveric chondrocytes of the present invention (P0) is observed vs. the healthy donor chondrocytes. (P0), Under the same conditions; It is evident that when the implant or construct of the present invention is used, the formation of hyaline cartilage is induced, which is evidenced by the marked alcian blue staining that shows the formation of extracellular matrix.

Example 4: Quantitative evaluation of the quality of the cartilage formed

A quantitative analysis of the histological stains was carried out, for which three independent and blinded observers were used, using the modified O'Dhscoll histological scale, through which the percentage of cartilage formation, the quantity and quality of cartilage are evaluated. cells formed and tissue structure. In this evaluation, a maximum score of 14 points is awarded to native cartilage (Figure 6); the closer the score is to 14, which is assigned to histological samples, it means that the tissue formed presents a higher quality and that it is more similar to healthy cartilage. Quantitatively, the tissue formed by the cadaveric chondrocytes (P0) that were chopreserved without expanding, of the present invention, obtained a mean of 9.57 (SD+1.27). On the other hand, the cartilage formed by living donor chondrocytes (P0) that did not expand and were not chopreserved either, obtained a score of 8.71 (SD+3.98), with no significant difference with the tissue of cadaveric chondrocytes (p< 0.05).

However, with the present invention, the source of obtaining the chondrocytes becomes significantly important when considering that when the chondrocytes are obtained from a cadaveric donor, the requirement to perform a first surgery on the patient to be treated is eliminated, thereby avoiding the risks of morbidity in the donor area and significantly reducing the costs and times of treatment and recovery. Likewise, another great advantage is that a greater quantity of tissue is obtained from the cadaveric donor source and with it a greater quantity of cells for the implants or constructs, which eliminates the need for prolonged cultures that carry the risk of generate fibrocartilage instead of hyaline cartilage, this also allows treating lesions of any size, this also impacts treatment costs and one of the most important aspects is that it allows the generation of a tissue more similar to hyaline cartilage compared to the one that is formed with living donor chondrocytes, which are not cultured or cryopreserved. Living donor chondrocyte implants or constructs, which were cultured for 2 passages (P2) formed a tissue with little resemblance to cartilage, on the contrary, they presented a much more fibrous appearance vs. hyaline cartilage, probably due to dedifferentiation that chondrocytes undergo when being expanded several times in monolayer cultures. The mean score of this group on the O'Driscoll scale was 4.37 (SD+4.7), which clearly indicates that the tissue formed presents characteristics far removed from hyaline cartilage.

In autologous chondrocyte implantation (ACI), the source of cartilage is very limited since they come from the same individual, for this reason it is mandatory to culture the cells, for at least two passages (8 weeks), to expand them with the purpose of increase the number of cells, to have a sufficient number of cells for the repair of a cartilage lesion. With the results obtained (figures 5 and 6) previously presented, it is shown that there is a very high risk, practically unacceptable, that the patients treated with this technique generate fibrocartilage, with the consequent risk of premature failure. a tissue as close as possible to hyaline cartilage, as demonstrated in the present invention.

Allogenic Chondrocyte Implantation (ALCI, for its acronym in English; Allogenic Chondrocyte Implantation) is a technique that, like Autologous Chondrocyte Implantation, repairs a joint lesion with tissue very similar to native cartilage, reducing treatment times, costs, number of surgeries, involvement of the autologous cartilage donor site, greater availability of the number of cells required to treat a sizeable lesion, elimination of the need to culture and expand chondrocytes in the laboratory, selection of chondrocytes from young cadaveric donors to improve the quality of the formed tissue. This invention allows high availability of cadaveric chondrocytes, which allows them to be used at any time and in the place that is required (any hospital in any state of the Mexican Republic and even to be marketed internationally). This availability as an implant or construct made in the tissue bank eliminates the need for the hospital center, where the patient is to be treated, to have a laboratory and/or equipment sophisticated to process the sample, in such a way that the attending physician only has to request it from the Tissue Bank.

The quality of the repair tissue formed in a cartilage lesion depends on many factors that are covered with this innovation, such as the seeding of cells in a three-dimensional hyaluronic acid membrane (it forms better tissue than without a scaffold), the number of cells that are implanted in the lesion (the more chondrocytes, the better tissue; a characteristic that is not available when it comes to autologous chondrocytes), age of the donor (the younger, the better quality and greater durability of the tissue formed). In the traditional ACI technique, it is difficult to cover the last two aspects. Table 1 contrasts the differences between the traditional technique (ACI) versus the proposed innovation (ALCI).

Table.1: Technical advantages of the invention vs. traditional technique

The product of the present invention is useful for the treatment of cartilage lesions of any joint, in a preferred embodiment it is used for the treatment of knee cartilage lesions.

The product is provided as a kit comprising a sterile culture box, which in turn contains the implant or construct with culture medium, antibiotic-antimycotic autologous serum; where the implant is made up of a hyaluronic acid membrane, with dimensions ranging from 10 x 10 mm to 50 x 50 mm, where cadaveric allogeneic chondrocytes are attached, at a density of 1 x10 ⁶ cells per cm ² , covered with a layer of a fibrin cell adhesive. Once the graft or construct has 7 days of in-vitro culture, it is transported, under sterile conditions, to the hospital where the surgery will be performed.

In one embodiment of the present invention, the tissue engineer from the Tissue Bank (Biograft) laboratory, attended the Hospital to deliver the graft or construct and remained in the surgical procedure until the implant was placed. In another embodiment or modality, there is also assistance from an orthopedic surgeon specialized in arthroscopy and sports injuries, an expert in cartilage repair, for support and advice during the surgical procedure for orthopedists who are not familiar with the technique. The kit containing the graft of the present invention has been used by an orthopedic surgeon through surgery open or minimally invasive surgery (arthroscopy) in an operating room, under sterile conditions. In this regard, it is recommended that prior to the use of the implant the treating physician should evaluate the lesion during surgery, identifying the location (femoral condyle, patella or trochlea) and size.

In order to have a good integration of the graft or construct of the present invention, it is recommended that before the placement of the product, an adequate debridement of the lesion is carried out (Fig. 8A and Fig. 8B) removing all the damaged tissue and leaving borders. stable from healthy cartilage (Fig. 8C). It is important that the lesion be measured again after debundation, since it is very certain that the size of the lesion is now larger than it was initially, that is, before removing the injured and unstable edges. If the surgery is open, the measurement can be made with a surgical ruler; If the procedure is arthroscopic, the palpator hook or a flexible ruler is used to determine the exact size of the lesion in the proximal-distal and mediolateral plane (figure 8D and 8E). Likewise, it is highly recommended that the lesion be given a square or rectangular shape (figure 8F), to facilitate measurement and adaptation of the size and shape of the product. Equally important is not to damage the subchondral bone.

Once the final measurements of the lesion are obtained, after the excision of the lesion, it is recommended to trim the graft or construct with an excess of at least 2 mm on each edge, preferably at least 5 mm more than the estimated measurement. . It is preferable to have a larger construct, which can be compressed and adapt to the lesion, than to leave a tight or smaller construct, with the consequent risk that one or more of its ends will not be able to integrate with the edges of the healthy cartilage.

The product can be fixed by any means appropriate for this purpose, for example, by means of biodegradable anchors or with fibrin glue. In a preferred modality, he recommends the use of fibrin glue, as it is less invasive, less expensive, and much easier to place. For fixation with anchors, the bone exposed in the lesion must be drilled and an anchor placed for every 10 mm of lesion, the sutures are recovered and the construct is slipped through them until the lesion is already debrided, finally knots are made to ensure the fixation. At the time the construct was placed in the lesion, the water inlet into the arthroscopy was closed to prevent loss of chondrocytes. A cannula was placed in the portal with the best access to the lesion, and through this the construct was introduced into the joint (Fig. 9A), placed and extended over the entire surface (Fig. 9B) until the lesion was covered. in its entirety and ensuring contact of the graft with all edges of the adjacent native cartilage (Fig. 9C). Finally, fibrin glue was applied to the edges and surface of the construct to ensure fixation of the implant to the lesion, (Fig. 9D). The knee must remain in extension for at least a couple of days to allow adherence of the product to the subchondral bone and adjacent cartilage edges.

Example 6: Evaluation of the integration of the construct by means of NMR

In the present invention, the study of nuclear magnetic resonance was used, an imaging technique that is widely used to evaluate the integration of the construct in the area of the defect, in the follow-up of a patient, treated with the implant of the present invention. Figure 10 shows the images obtained by NMR at 3 and 6 months post-op, respectively (Fig. 10A & Fig. 10B), in which it is clearly observed that from 3 months post-op there is good integration. of the graft and at 6 months changes in the intensity of the construct are observed, which reflects the presence of extracellular matrix formation by the seeded chondrocytes, as well as integration into the subchondral bone and adjacent cartilage.

Example 7: Cartilage lesion repair at 12 months: ACI vs ALCI

Figure 11 shows the arthroscopic evaluation, 12 months after treatment of cartilage lesion in the lateral trochlea of both knees in the same patient. In this patient, the right knee (Fig. 1 A, B and C), with a 20x15 mm lesion, was treated with autologous chondrocyte implantation (ACI); This shows repair tissue with 80% partial thickness filling (Fig. 11 A), with an irregular surface, predominant presence of fibrocartilage (Fig. 1 1 B) and areas of fibrillation (Fig. 1 1 C). In contrast, the left knee (Fig. 1 1 D, E and F), with a 18x15 mm lesion, was treated with the allogeneic chondrocyte implant of the present invention (ALCI), in which repair tissue with 95% partial thickness filling is observed (Fig. 1 1 D), integration with the adjacent cartilage (Fig. 11 E) , smooth surface with minimal fibñlation.

Example 8: Formation of fibrocartilage in the donor site in the ICA

Figure 12 shows the arthroscopic evaluation of the osteochondral biopsy in autologous chondrocyte implantation (Fig. 12A and B). Subsequently, by means of a second arthroscopic view 12 months after autologous chondrocyte implantation, filling of the donor area with abundant presence of fibrous, irregular and fibrillar tissue is clearly observed (Fig. 12C and D); process that is avoided in cadaveric chondrocyte implantation.

Example 9: Follow-up of some patients treated with the graft of the present invention.

Below is a summary of the data from 4 patients treated with the graft or construct of the present invention and the evaluation of different lesions, parameters and evolution times.

Table 2: Demographic report of patients implanted with the present invention.

From the information contained in the table it is clear that patients of various ages were treated, from young people (15 years) to patients adults (43 years old) with injuries to different joints such as ankle, knee and hip; with lesions averaging 37 mm (25-60 mm) in size. Figures 13 and 15 show the intervention with the implant or construct of the present invention for hip and knee, respectively.

During the follow-up of these patients, the intensity and frequency of pain was evaluated, before and after treatment. Pain intensity was evaluated with the Visual Analog Scale (VAS), which allows measuring the intensity of pain described by the patient with maximum reproducibility, which consists of 10 points; the higher the numerical value, the greater the pain intensity. Likewise, the frequency of pain was identified in 5 categories: never, rarely, sometimes, often or always. This measurement was made before surgery and 3 months after it. In the first two patients the pain decreased considerably from a maximum of 9 points to a value of 2 points.

Table 3 Evaluation of pain intensity and frequency, before and after treatment.

VAS=Visual Analogue Scale

The results in the table show that, just 3 months after the intervention, with the graft of the present invention, there is a significant decrease in both intensity and frequency of pain in adult patients with hip and knee intervention. , respectively. Pain decreased on average 6.5 points (before versus at 3 and 6 months of follow-up) from a maximum value of 9.

Likewise, in the follow-up of these patients, the mobility arcs were also evaluated before and after treatment, using conventional techniques, avoiding mobility associated with the lumbar spine, in the supine and prone or lateral decubitus positions. Table 4: Improvement of the range of motion in the hip after 3 and 6 months of the implant.

Comparison of the range of motion of the operated hip versus the healthy hip before surgery, 3 and 6 months after the implant was placed, in which a significant improvement was observed in all ranges of motion. The results show a clear improvement in all the values for the different types of hip movement.

Additionally, the success of a treatment is also determined through the joint function of the patients, evaluated by means of internationally validated clinical scales, where parameters such as pain level, inability to perform activities of daily living, activities sports and quality of life. The scales are different for hip and knee and provide a numerical score that was evaluated before and after surgery.

Figure 17 shows the evaluation of hip joint function before and after (6 months) the intervention with the implant of the present invention.

In the same way, the mobility arcs were evaluated before and after knee treatment: Table 5: Improvement of the range of motion in the knee after 3 months of the implant.

Comparison of the range of motion of the operated knee versus the healthy one before surgery and 3 months after the implant of the present invention was placed, in which a significant improvement in flexion is observed, only a deficit of only 5 ^S (130°) with respect to the healthy knee (135°), at 3-month follow-up.

Similarly, Figure 18 shows the evaluation of knee joint function before and after the intervention (3 months), in all of them the improvement of various functional parameters is clearly appreciated.

In addition, in these patients the quality of repair tissue vs. healthy cartilage was evaluated at 3 months, the results are shown in the following table 6. One of the main non-invasive methods to assess the quality of repair tissue in treatment of cartilage lesions is T2-mapping. This is one of the world-renowned techniques for the study of cartilage, with which the values of physical parameters are mapped, which show the relaxation time of the water observed in the structure of interest. This value is established for cartilage, ranging from 20 to 50 milliseconds in normal cartilage; values within the normal range indicate that the repair tissue has a quality very similar to that of native cartilage (hyaline cartilage) and its durability is better, on the contrary, values outside this range indicate that the tissue is of poor quality (very fibrous or with a tendency to form bone), with a high susceptibility to deteriorate over time.

Table 6: Values of repaired cartilage vs. native cartilage

Figure 14 shows the corresponding T2 mapping for the hip, while Figure 16 shows the corresponding T2 mapping for the knee. The values of the relaxation time of the water, at 3 months post-operation in the first two patients, were found to be elevated with respect to the normal values of healthy cartilage (ROI-1 vs ROI-2), which is an expected behavior. , the same behavior has been observed in the previously reported Autologous Chondrocyte Implantation techniques, since the repair tissue begins to mature and transform into cartilage at around 12 months. It is expected that in subsequent evaluations (6 and 12 months) these values will decrease, approaching the values of healthy cartilage.

While some of the preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of illustration only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the spirit of the invention, and should therefore be considered within the scope of protection of the present invention. It is to be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents are thus covered. BIBLIOGRAPHIC REFERENCES

1. Olivos-Meza A, Cortés-González S, Ferniza-Garza JJ, Pérez-Jiménez FJ, Villalobos-Córdova, Ibarra JC. Arthroscopic Treatment of Patellar and Trochlear Cartilage Lesions with Matrix Encapsulated Chondrocyte Implantation versus Microfracture: Quantitative Assessment with MRI T2-Mapping and MOCART at 4-Year Follow-up. Cartilage 2019:1-3.

2. Arredondo R, Olivos-Meza A, Villalobos E, Cortés S, Pérez-Jiménez FJ, et. to the. A new arthroscopic technique of encapsulated matrix autologous chondrocyte implantation (ICAME) in patella: clinical evaluation and T2 mapping at 4-year follow-up. Spanish Journal of Arthroscopy and Joint Surgery 2018; 25(61):30-41.

3. Orth P, Gao L, Madry H. Microfracture for cartilage repair in the knee: a systematic review of the contemporary literature. Knee Surgery, Sports Traumatology, Arthroscopy 2019:1-8.

4. Holt K, Sorhaindo M, Coady C, Ho-Bun Wong I. Arthroscopic Treatment of Medial Femoral Knee Osteochondral Defect Using Subchondroplasty and Chitosan-Based Scaffold. Arthrosc Tech. 2019 Apr; 8(4): e413-e418.

5. Martin AR, Patel JM, Zlotnick HM, Carey JL, Mauck RL. Emerging therapies for cartilage regeneration currently excluded Ted knee' populations. Regenerative Medicine 2019;12:1-10.

6. Mistry H, Connock M, Pink J, Shyangdan D, Ciar C, Royle P, et al. Autologous chondrocyte implantation in the knee: systematic review and economic evaluation. Health Technol Assessment 2017;21 (6).

7. Taylor A, Lee P. Single Treatment Autologous Chondrocyte Implantation: The Next Generation of ACI. Can J Biomed Res & Tech 2019;2(2): 1-4.

8. Davies RL, Kuiper N. Regenerative Medicine: A Review of the Evolution of Autologous Chondrocyte Implantation (ACI) Therapy. Bioengineering 2019;6(22): 2-16.

9. Olivos-Meza A, Velasquillo-Martínez C, Olivos-Diaz B, Landa-Solis C, Brittberg M, et. to the. Co-culture of dedifferentiated and primary human chondrocytes obtained from cadaveric donor enhance the histological quality of repair tissue: an in vivo animal study. Cell Tissue Bank 2017;14(3):1-12.

Claims

CLAIMS An implant or construct for the treatment of cartilage lesions in joints of a human animal subject characterized in that it comprises cadaveric donor-derived chondrocytes (P0) seeded on a hyaluronic acid scaffold and sealed with a fibrin adhesive. The implant or construct for the treatment of cartilage lesions in joints of a human animal subject in accordance with the previous claim, further characterized in that the chondrocytes are seeded at a density of 1 x 10 ⁶ cells per cm ² . The implant or construct for the treatment of cartilage lesions in joints of a human animal subject in accordance with the previous claim, further characterized in that the cadaveric donor-derived chondrocytes are cryopreserved without expanding (P0). The implant or construct for the treatment of cartilage lesions in joints of a human animal subject in accordance with claim 1, further characterized in that it induces the formation of an extracellular matrix with a high content of specific molecules of articular cartilage, specifically aggrecan and collagen II. The implant or construct for the treatment of cartilage lesions in joints of a human animal subject in accordance with claim 1, further characterized in that the chondrocytes seeded on a hyaluronic acid scaffold are cultured for a period of between 5 to 7 days, in DMEM-F12 culture medium, supplemented with autologous serum (10%) and 10% antibiotic-antimycotic, in an incubator at 37°C, 5% CO2 and 5% humidity. The use of chondrocytes from a cadaveric donor for the treatment of cartilage lesions in joints of a human animal subject. A process for making an Implant or construct for the treatment of cartilage lesions in joints of a human animal subject, characterized in that it comprises the following steps: a) Obtaining chondrocytes from a sample of cartilage tissue from a cadaveric donor; optionally the chondrocytes can be used immediately or cryopreserved; b) Seed the chondrocytes at a density of 1x10 ⁶ cells per 10mm of scaffold, each scaffold will be individualized in size, depending on the size of the patient's lesion to be treated, in a DMEM-F12 culture medium, autologous serum (10% ) and antibiotics-antifungals (10%); ec) Incubate the construct for a period of between 5 and 7 days, to promote cell adherence to the scaffold and the formation of the extracellular matrix, under culture conditions of 37°C, 5% CO? and 5% humidity, making a medium change every 2 or three days.