ES2933738A1 - HYDROGEL BASED ON DECELLULARIZED EXTRACELLULAR MATRIX AND ITS USES (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Hydrogel based on decellularized extracellular matrix and its uses. The invention relates to a biomimetic hydrogel of decellularized extracellular matrix derived from mesenchymal stem cells, decellularized extracellular matrix derived from mesenchymal stem cells differentiated into adipocytes, fibroblasts, chondrocytes and osteoblasts, and decellularized extracellular matrix derived from cultured fibroblasts, to compositions containing said hydrogel, to methods of producing said hydrogel, and to its preclinical research and medical uses. (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

HYDROGEL BASED ON DECELLULARIZED EXTRACELLULAR MATRIX AND ITS USES

The invention relates to a biomimetic hydrogel from decellularized extracellular matrix, to compositions containing said hydrogel, to methods for producing said hydrogel and to its medical uses. The invention belongs to the technical field of biomaterials, particularly tissue engineering.

STATE OF THE ART

Articular cartilage (AC) is a specialized connective tissue that performs important functions related to the maintenance and enabling of locomotion. Due to its avascular structure and physiology, cartilage has limited intrinsic repair capacity when injured, a condition that often results in degenerative diseases such as osteoarthritis (OA) ( Ryu J., et al, 1984, Arthritis Rheum ., 27, 49).

To promote cartilage regeneration, tissue engineering (IT) strategies have taken a step forward, providing biomaterials as scaffolds in which cells can be placed to locally produce new cartilage-like tissues ( Armiento AR, et al, 2018, Acta Biomater., 65, 1). Among scaffold biomaterials, injectable hydrogels have received much attention not only for their exceptional biological and physicochemical properties (for example, cytocompatibility, high water retention, permeability, and tunable mechanical properties) but also for their ability to fill any shaped wound. ( Jooybar E., et al, 2019, Acta Biomater., 83, 233).

Taking inspiration from the extracellular matrix (ECM), which plays an important role not only as mechanical support, but also as a key functional regulator for tissue maturation, considerable efforts have been made to develop hydrogels that recreate the composition or properties of articular cartilage. native to guide tissue-specific formation ( Luo Y., et al, 2019, J Biomed. Mater. Res. Part B Appl. Biomater., 107, 1695).

Bone injuries are common disorders that affect people of all ages; In the case of knee cartilage lesions, as the cartilage lesion progresses, it spreads to the underlying subchondral bone and an osteochondral defect appears ( Jiménez G., et al, 2018, J Adv Exp Med Biol., 1059 : 63-83)). Current clinical approaches for the repair of joint defects are not fully satisfactory due to the poor quality of the newly formed tissue and the lack of integration ( López-Ruiz E., et al, 2018, Eur Cell Mater., 35: 209-224). Thanks to tissue engineering, in recent decades, different constructions have been developed for this type of injury, but these products are still not capable of imitating all the characteristics of cartilage and bone, mainly due to technological limitations to produce biomimetic structures composed of materials with diverse properties and that allow the spatial arrangement of the cells for their correct integration. To promote the regeneration of bone tissue, the technique known as 3D bioprinting is presented as an alternative for the manufacture of supports made up of various biomaterials and different types of cells distributed in a personalized, structured and spatially defined manner. Therefore, for the creation of constructs for bone lesions, the creation of bioinks and the combination of materials that mimic the structural, physical, and biological properties of bone are important. These materials must be bioactive, biocompatible, biodegradable, and must provide signals for differentiation and proliferation ( López-Ruiz E., et al, 2016, Expert Opin Ther Pat. 26 ( 8):877-90).

The skin is one of the largest organs in the body, comprising 15% of the total body weight of adults, this organ plays a vital role in maintaining the homeostasis of the body, as it protects the internal organs from possible chemical threats, biological and physical. In addition, it exerts many other functions, such as the prevention of excessive water loss, thermoregulation, excretion and perception through specialized receptors (Kanitakis J., et al, 2002, Eur J Dermatology. 12(4): 390-401; Kolarsick, PAJ & Kolarsick MA. 2001, Journal of the Dermatology Nurses' Association. pp. 203-13; Sontheimer RD. 2014, Journal of Investigative Dermatology. Nature Publishing Group. 134, pp. 581-2). Skin-related diseases can be due to several reasons: acute or chronic wounds, skin-related pathologies (such as diabetic foot ulcers), perianal fistulas, epidermolysis bullosa, or the most common cause of significant skin loss, thermal trauma. . Deep skin trauma and full-thickness skin wounds are often difficult to treat because of the high risk of infection, the size of the area involved, and the potential for damage to deeper layers of the skin, including the dermis. A typical approach to treating severe skin lesions is to replace lost tissue with an autograft, allograft, or xenograft. Despite this, these solutions have two major drawbacks: the availability of donors and, especially, large wounds, where neither of these approaches is feasible (Vrana NE, et al. 2013, Tissue Engineering - Part B: Reviews. 19, pp. 529-43; Santema TB, et al. 2016, Cochrane Database of Systematic Reviews. Vol 2016). To overcome these limitations, it is necessary to develop substitutes tissue engineering (IT) is a promising alternative to meet the growing demand for skin substitutes. As a result of their exceptional properties, such as high water storage capacity, mechanical characteristics, and biocompatibility, hydrogels are considered the most favorable choice for application as wound dressings compared to other alternatives. For example, hydrogels have great potential due to their intrinsic 3D network, which mimics the native structure of the ECM, providing mechanical support and porosity. They also show an inherent cellular interaction capacity. Currently, in clinical practice, the use of autografts is the gold standard treatment. However, the availability of tissue from healthy donors is limited in patients with severe tissue loss. To overcome this limitation, tissue-engineered skin substitutes are being developed to offer alternative treatments for wound healing.

Currently, tissue-derived decellularized ECM (t-EMC) is considered a promising biomaterial, as it is a direct way to provide the complex composition of native tissue, which is difficult to reproduce using common biomaterials. However, there are still some challenges and problems that need to be addressed. For example, the limited availability of donor tissue when the dCEM source is autologous, the potential risk of increased immunogenicity and pathogen transmission if it is allogeneic, or long-term protocols for its total decellularization {Zhang W. , et al, 2016, Tissue Eng.- Part B Rev., 22, 193).

Recently, cell culture has emerged as an alternative source of dEMC for IT. Cell-derived matrix (CEMd-c) is also a functional matrix containing a complex mixture of macromolecules and signaling factors that can resemble the native tissue microenvironment. Compared to d-ECM, this cell-derived ECM offers some advantages, such as its large-scale production and easy customization through the use of different cell types or culture conditions, as well as excellent biocompatibility and bioinductive properties ( Cai R., et al, 2015, Biomaterials, 52, 199).

Scaffolds or matrices are needed for the treatment of cartilage, bone, and skin defects that even better reproduce the dynamically changing microenvironment sustained by ECM during tissue development, without exogenous growth factors, providing a more easily translatable product than ECM derived from xenogenic tissues and avoiding invasive surgery.

DESCRIPTION OF THE INVENTION

The present invention relates to a decellularized extracellular matrix (dECM)-based hydrogel derived from cultured mesenchymal stem cells (dCEM) that promotes chondrogenesis and cartilage regeneration without exogenous growth factors, providing an injectable biomimetic dCEM hydrogel for cartilage engineering. tissues. In particular, the present invention relates to a hydrogel based on decellularized extracellular matrix (MECd) derived from differentiated mesenchymal stem cells (MSCs) (MECdmd), preferably from cultured early chondrogenic mesenchymal stem cells (MECdmc) and from differentiated mesenchymal stem cells a cultured adipocytes (MECda), fibroblasts (MECdfd) and osteoblasts (MECdo) or decellularized extracellular matrix derived from cultured fibroblasts (MECdf).

The inventors have developed new biomimetic hydrogels that provide a favorable environment for cells with adequate physicochemical and biological properties and promote specific regeneration of cartilage, bone and skin tissue, in vivo or in vitro, all of which support high cell viability at different concentrations. of MECd.

The inventors have seeded human MSCs from adipose tissue under specific culture conditions to generate early chondrogenic, osteogenic, fibroblastic and adipogenic MECs and have seeded human FBs from dermal tissue to generate dfMECMs. At the end of the culture period, the cells were removed from the underlying matrix by exposure to a decellularization treatment.

Decellularization of dEMCs was assessed by measuring DNA content and composition analyses. The composition, determined by quantitative, histological and mass spectrometric techniques, includes the expression of the main components of native tissue, type II collagen and GAGs, as well as proteins such as fibronectin, type I collagen, versican or tenascin. -C. By means of mass spectrometry, the inventors have identified other components that constitute this rich and complex matrix, including structural proteins such as the fibrillar family and associated collagens (type III, IV, V, VIII, XII), proteoglycans, and glycoproteins. , which confer mechanical properties and cell adhesion, as well as affiliated proteins that contribute to the function and dynamics of the ECM and signaling factors.

In addition, the inventors performed in vivo biocompatibility assays in immunocompetent (ICR) CD1 mice. The results showed that early chondrogenic MECDM possesses excellent biocompatibility and adequate physicochemical and mechanical properties for injectability, is capable of inducing MSC chondrogenesis without complementary factors and, in addition, to form a tissue similar to hyaline cartilage after its implantation in vivo. These results demonstrate the potential of this early chondrogenic MECDM-based hydrogel for applications in tissue regeneration, particularly cartilage repair and regeneration. The inventors observed these effects through an increase in cartilage-specific markers, both at the genetic level (COL2A1, ACAN and SOX9 genes) and protein (type II collagen and proteoglycans) using qPCR and histological techniques, respectively. Rheological assays demonstrated that the early chondrogenic MECDM hydrogel at both concentrations has suitable properties for use as an injectable hydrogel not only for local cartilage repair, but also for the development of biomimetic cartilage-like substitutes using 3D bioprinting.

Thus, a first aspect of the present invention relates to a biomimetic hydrogel comprising a decellularized extracellular matrix derived from differentiated mesenchymal stem cells (dmSC), hereinafter referred to as the hydrogel of the invention.

The term "hydrogel" as used herein refers to a 3D network or scaffold of mixtures of materials, molecules, polymers, or substances that are combined by chemical bonds, including covalent, ionic, and supramolecular bonds, or by any combination thereof. , to form water-swellable but water-insoluble structures with a solid, semi-solid or semi-liquid texture, which are used as tissue engineering scaffolds. Preferably, the hydrogel is a biomimetic hydrogel. The term "biomimetic hydrogel" as used herein refers to a hydrogel or scaffold that mimics the natural extracellular microenvironment to facilitate interaction between hydrogels and surrounding cells through molecular recognition, and further enhance cell-specific response and tissue regeneration.

The hydrogel of the invention is based on the extracellular matrix (ECM). The terms "extracellular matrix" or "ECM" as used herein refer to a natural or artificial scaffold for cell growth that provides a microenvironment for cell/cell communication, cell growth, and tissue remodeling, particularly three basic biofunctions, including cell adhesion, proteolytic degradation, and GF binding. Natural ECMs, ECMs found in multicellular organisms, such as, but not limited to, mammals and humans, are complex mixtures of structural and non-structural biomolecules, including, but not limited to, collagens, elastins, laminins, glycosaminoglycans, proteoglycans , antimicrobials, chemoattractants, cytokines and growth factors. The composition and structure of ECMs vary depending on the source of the tissue.

Particularly, the hydrogel of the invention comprises a decellularized extracellular matrix. The terms "decellularized extracellular matrix" or "dECM" used herein refer to an ECM in which endogenous cells (cell fractions or components) have been substantially removed while preserving their structural and compositional, tissue or organ properties. native, thereby providing tissue- or organ-specific microenvironments for cell growth and function. Preferably, the decellularized ECM is isolated or separated from at least about 60%, 70%, 80%, 90%, 95%, 99%), or more, endogenous cellular material. The presence or extent of endogenous cellular material can be determined using any method known in the art, eg Western blotting, nuclei detection, microscopy, etc.

As is known to one skilled in the art, the decellularization procedure involves cell lysis followed by separation of the cells from the ECM. Various physical, chemical, and biological methods are used during decellularization, and the choice of which one to use depends on several factors, such as the thickness, density, and also the lipid content of the tissue and organ.

In the present invention, the decellularized extracellular matrix (MECd) is derived from differentiated mesenchymal stem cells or "MECdmd". The term "mesenchymal stem cells" or "MSCs" refers to multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells), fibroblasts, and adipocytes. In the present invention, the term "differentiated mesenchymal stem cells" (dMSCs) refers to partially or fully differentiated mesenchymal stem cells, ie, cells of different stages of differentiation or specialization. The differentiation, proliferation, specialization implies a succession of alterations in cell morphology, membrane potential, metabolic activity and sensitivity to signals.

Obtaining mesenchymal stem cells from a subject, preferably a mammal, more preferably a human, is standard practice for one skilled in the art.

The terms "patient,""subject," or "individual" are used interchangeably and refer to a mammal, eg, a human, or a non-human mammal, including primates (for example, example, macaque, Pan troglodytes, Pongo), a domesticated mammal (for example, felids, canids), an agricultural mammal (for example, bovine, ovine, porcine, equine), and a laboratory mammal or rodent (for example, Rattus, murine, lagomorph, hamster).

In a preferred embodiment of the invention, the mesenchymal stem cells are mammalian mesenchymal stem cells, preferably human mesenchymal stem cells (hMSCs).

MSCs can be isolated from tissues such as bone marrow, placenta, cord blood, adipose tissue, adult muscle, corneal stroma, amniotic fluid, endometrium, and teeth. In another preferred embodiment of the invention the mesenchymal stem cells are isolated from adipose tissue.

As described above, the decellularized extracellular matrix (MECd) is derived from differentiated mesenchymal stem cells (MECdmd). In a preferred embodiment of the invention, the differentiated mesenchymal stem cells are selected from the list consisting of: early chondrogenic mesenchymal stem cells, adipocytes, fibroblasts, and osteoblasts.

The term "early chondrogenic mesenchymal stem cells" used herein refers to stem cells that are in the process of differentiating from mesenchymal stem cells to chondrocytes, ie, chondrocytes of early stages of chondrogenesis. The early stage matrix contains elements and factors that are not present in the mature one. Specifically, the early chondrogenic matrix consisting of the initial expression of the major components of mature cartilage tissue (type II collagen and GAG) as well as proteins that have been implicated in the early stages of chondrogenesis (i.e., fibronectin, collagen type I, versican or tenascin-C).

The term "adipocytes" used herein refers to cells that constitute adipose tissue. They are round cells, from 10 to 200 micrometers, with a lipid content that represents 95% of the cell mass and that forms the constituent element of fatty tissue. Their fundamental characteristic is that they store a large amount of fat (triglycerides), which, in the case of adipocytes, the white adipose tissue (the most abundant in the adult human body), group together to form a large drop that occupies the most of the cell.

The term "fibroblasts" used herein refers to a cell type resident connective tissue. It synthesizes fibers and maintains the extracellular matrix of tissues in many animals. These cells provide a network-like structure (stroma) to many different tissues and play a crucial role in wound healing, being the most common cells of connective tissue. They derive from primitive mesenchymal and pluripotent cells. Stromal cells that can transform into fibroblasts, osteoblasts, adipocytes, and muscle cells are identified in bone marrow cultures as adherent cells.

The term "osteoblasts" used herein refers to bone cells specialized in producing the particular matrix of bone, made up of hydroxyapatite crystals, which are composed primarily of phosphate and calcium. Osteoblasts still retain the ability to replicate, until they become surrounded by matrix and enter a matrix-maintaining state called osteocytes.

Differentiated mesenchymal stem cells can be obtained by isolation from tissue, cell culture (eg, fibroblast culture) and/or differentiation of mesenchymal stem cells.

It is common practice for one skilled in the art to promote the differentiation of mesenchymal stem cells into early chondrogenic mesenchymal stem cells, adipocytes, fibroblasts or osteoblasts.

In another preferred embodiment, the decellularized extracellular matrix (MECd) is developed from fibroblasts (MECdf), mesenchymal stem cells (MECdm), mesenchymal stem cells differentiated into adipocytes (MECda), mesenchymal stem cells differentiated into fibroblasts (MECdfd), cells mesenchymal stem cells differentiated into osteoblasts (MECdo) or early chondrogenic mesenchymal stem cells (MECdmc).

In addition, to provide a more favorable microenvironment for specific tissue formation, differentiated mesenchymal stem cell matrices are used in the present invention to obtain the hydrogel of the invention, i.e., MSC-derived early chondrogenic decellularized extracellular matrix or early chondrogenic dmEM. . The concentration of the MECdmd in the hydrogel of the invention comprises a certain range.

In a preferred embodiment of the invention, the concentration of MECdmd in the hydrogel of the invention varies between 1% w/v and 10% w/v on the total weight percentage of hydrogel. The expressions "% by weight" or "% w" or "% w/v" are considered equivalent and can be used interchangeably in this document and refer to the percentage by weight with respect to the total weight or volume of the solution or dispersion, unless otherwise specified.

In another more preferred embodiment of the hydrogel of the invention, the MECdmd is selected from 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4 % w/v, 4.5% w/v, 5% w/v, 5.5% w/v, 6% w/v, 6.5% w/v, 7% w /v, 7.5% w/v, 8% w/v, 8.5% w/v and 9% w/v, more preferably the percentage of MECdmd is 3 or 6% w/v , more preferably the percentage of MECdmd is 6%.

As a person skilled in the art knows, the hydrogel of the invention may be comprised in a composition. Thus, in another aspect, the present invention relates to a composition, hereinafter "inventive composition", comprising the inventive hydrogel.

In accordance with conventional techniques known to those skilled in the art, the composition according to the present invention can be formulated with an excipient and/or vehicle. Therefore, in a particular embodiment, the composition of the invention comprises an excipient and/or vehicle. In case of therapeutic use of the composition of the invention, it can be formulated with a pharmaceutically acceptable excipient and/or vehicle.

The term "excipient" refers to a substance that helps to absorb any of the components of the composition of the invention, stabilizes said components or helps in the preparation of the composition in the sense of giving it consistency or, if necessary, providing aromas. make it more enjoyable. Therefore, the excipients could have the function of keeping the components together, such as, for example, starches, sugars or celluloses, a sweetening function, a coloring function, the protection function of the drug, such as, for example, isolating it from air and/or moisture, a bulking function for a tablet, capsule or any other form of formulation, such as, for example, dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Therefore, the term "excipient" is defined as any material included in the galenical forms that is added to the active principles or their associations to allow their preparation and stability, modify their organoleptic properties or determine the physicochemical properties of the composition and its bioavailability. . The "pharmaceutically acceptable" excipient must allow the activity of the compounds of the pharmaceutical composition, that is, to be compatible with said components. Examples of excipients are binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, and colorants. More specific, non-limiting examples of acceptable excipients are starches, sugars, xylitol, sorbitol, calcium phosphate, steroidal fats, talc, silica or glycerin, among others.

The term "vehicle" refers to a compound that facilitates the incorporation of other compounds to allow for better dosing and administration or to provide consistency and shape to the composition. Therefore, the vehicle is a substance that is used to dilute any of the components of the composition of the present invention to a determined volume or weight, or even without diluting said components, capable of allowing better dosage and administration or providing consistency. When the presentation form is liquid, the vehicle is the diluent. In a particular embodiment, the vehicle is pharmaceutically acceptable.

Additionally, the composition according to the present invention can be further formulated with embryonic stem cells (ESCs), non-embryonic or adult stem cells, preferably mesenchymal stem cells, progenitor cells, MPi cells, mature adult cells and/or umbilical stem cells, cells tumor stromal cells, tumor cells (of different molecular types and differentiation stages) and adipose and tumor tissue samples. The term "mesenchymal stem cells" has been defined in previous paragraphs of this document and is equally applicable to this aspect of the invention.

The hydrogel or composition of the invention can be placed or incorporated into an implant. Thus, another aspect of the present invention refers to an implant comprising the hydrogel or composition of the invention, hereinafter referred to as the implant of the invention.

The expressions "implant", "medical device", "device", "medical implant" are used synonymously and refer to any object or element that is biocompatible with the physiological state of the patient's body and does not produce adverse side effects and that is structured, designed, or configured for placement in whole or in part within a patient's body for one or more therapeutic or prophylactic purposes, such as tissue augmentation, contouring, restoration of physiologic function, tissue repair or restoration damaged by disease or trauma and/or the delivery of therapeutic agents to normal, damaged or diseased organs and tissues. Although medical devices are typically composed of synthetic, biologically compatible materials (for example, medical-grade stainless steel, titanium, and other metals; polymers exogenous, such as polyurethane, silicon, PLA, PLGA), other materials may also be used in the construction of the medical implant. Specific medical devices and implants that are particularly useful for the practice of the present invention include soft tissue implants for cosmetic and reconstructive surgery.

The inventors obtained the hydrogel of the invention by means of decellularized extracellular matrix derived from differentiated mesenchymal stem cells (dmEC), preferably early chondrogenic mesenchymal stem cells MECd, to obtain an early chondrogenic MECdm. The inventors obtained the hydrogel of the invention using decellularized extracellular matrix derived from cultured mesenchymal stem cells differentiated into adipocytes (MECda), fibroblasts (MECdfd) and osteoblasts (MECdo) or decellularized extracellular matrix derived from cultured fibroblasts (MECdf).

Thus, another aspect of the present invention refers to a method for producing the biomimetic hydrogel of the invention, hereinafter referred to as the "method of the invention", comprising:

(a) culturing mesenchymal stem cells with at least one compound that induces differentiation or culturing differentiated mesenchymal stem cells, from a subject, (b) decellularizing the culture from step (a), obtaining a decellularized extracellular matrix derived from differentiated mesenchymal stem cells (MECdmd), (c) freeze-drying the decellularized extracellular matrix obtained in step (b),

(d) solubilizing the lyophilized decellularized extracellular matrix obtained in step (c), and

(e) gel the solution obtained in step (d) for 15-30 min at 30-40 °C.

The terms "mesenchymal stem cells", "subject", "decellularized extracellular matrix" and "differentiated mesenchymal stem cells" have been defined in previous paragraphs of this document and are equally applicable to this aspect of the invention.

During step (a), mesenchymal stem cells (undifferentiated) from a subject with at least one compound induce differentiation.

As described in previous inventive aspects, the hydrogel of the invention can be obtained by decellularized extracellular matrix derived from cultured fibroblasts (MECdf), where the cells are not cultured with a compound that induces differentiation. It is standard practice for a person skilled in the art to isolate and culture stem cells. mesenchymal or differentiated cells (particularly fibroblasts), preferably the cells are cultured at 25 to 45°C, preferably 30 to 40°C, more preferably 37°C, in a humid atmosphere.

In another more preferred embodiment, the mesenchymal stem cells or differentiated mesenchymal stem cells are cultured in a monolayer or in a 3D scaffold, preferably in a monolayer.

As described above, in another preferred embodiment, the mesenchymal stem cells (or fibroblasts) are mammalian, preferably human.

In another more preferred embodiment, the mesenchymal stem cells are isolated from bone marrow, placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, amniotic fluid, endometrium and teeth, preferably from adipose tissue.

In addition, to allow the formation of ECM, in another preferred embodiment, the MSCs are cultured in monolayer with at least one compound that induces differentiation, preferably with at least one compound that induces chondrogenesis, adipogenesis, fibroblast differentiation or osteogenesis for at least one week, preferably at least two weeks.

The term "chondrogenesis" as used herein refers to the differentiation of any cell into a chondrocyte, particularly, the process by which cartilage is formed from condensed mesenchymal tissue, which differentiates into chondrocytes and begins to secrete the molecules that form the extracellular matrix.

The term "adipogenesis" as used herein refers to the formation of adipocytes (fat cells) from stem cells. It involves 2 phases, determination and terminal differentiation. The determination is that mesenchymal stem cells commit to adipocyte precursor cells, also known as pre-adipocytes which lose the potential to differentiate into other cell types such as chondrocytes, myocytes and osteoblasts. Terminal differentiation is that preadipocytes differentiate into mature adipocytes. Adipocytes may arise from adipose tissue-resident preadipocytes or from bone marrow-derived progenitor cells migrating into adipose tissue.

The term "fibroblast differentiation" used herein refers to the acquisition of fibroblast characteristics and functionalities by stem cells. mesenchymal.

The term "osteogenesis" used herein refers to the formation of bone. There are two main modes of bone formation, or osteogenesis, and both involve the transformation of pre-existing mesenchymal tissue into bone tissue. The direct conversion of mesenchymal tissue to bone is called intramembranous ossification. This process occurs mainly in the bones of the skull. In other cases, the mesenchymal cells differentiate into cartilage, and this cartilage is subsequently replaced by bone. The process by which a cartilage intermediate is formed and replaced by bone cells is called endochondral ossification.

It is widely known by a person skilled in the art that chondrogenesis, adipogenesis, fibroblast differentiation or osteogenesis are promoted in the presence of compounds or supplements. Examples of compounds or supplements that induce chondrogenesis, adipogenesis, fibroblast differentiation, or osteogenesis include, without limitation, dexamethasone, ascorbic acid 2-phosphate, L-proline, insulin-transferrin-selenium, bone morphogenic protein (BMP , particularly BMP-2, BMP-4, BMP-7), TGF-p (particularly TGF-pi or TGF-P3), fibroblast growth factor (FGF, particularly FGF-2), fibroblast growth factor connective tissue (CTGF), p-glycerophosphate, isobutylmethylxanthine or indomethacin.

In another preferred embodiment of the method of the invention, the compound that induces chondrogenesis is dexamethasone and/or TGF-3p. In another more preferred embodiment of the method of the invention, the dexamethasone concentration is 100 nM and/or the TGF-3P concentration is 10 pg/ml.

To obtain the decellularized extracellular matrix derived from differentiated mesenchymal stem cells (MECdmd), particularly, the decellularized extracellular matrix (MECd) derived from early chondrogenic mesenchymal stem cells (MECdmc), from mesenchymal stem cells differentiated to adipocytes (MECda), fibroblasts (MECdfd ) and osteoblasts (MECdo) or decellularized extracellular matrix derived from cultured fibroblasts (MECdf), it is necessary to decellularize the culture from step (a).

The term "decellularize" or "decellularization" as used herein, refers to the process of removing or removing endogenous cells (cellular fractions or components) from the ECM while preserving its native tissue, organ, or structural and compositional properties. , thus providing microenvironments tissue or organ specific for cell growth and function.

Decellularization methods are standard practice for a person skilled in the art. Examples of such methods are, without limitation, decellularization with detergent solution, acids (peracetic acid), alcohol, or alkaline reagent (ammonium hydroxide), to disrupt cell and nuclear membranes, followed by trypsin- or nuclease-based enzymatic treatment, to disrupt the cytoplasmic content and break down the remains of DNA and RNA. Sterilization and decellularization can be simultaneous. For example, and without limitation, peracetic acid sterilization, described above, can also serve to decellularize ECM. Decellularization can also be achieved through the use of other methods such as ultrasonication, heating, application of pressure, and electroporation.

In another preferred embodiment of the method of the invention, the decellularization treatment of step (b) is carried out with a detergent solution at 30-40 °C, preferably 35-39 °C, more preferably at 37 °C, for 5-20 min or 2 to 8°C overnight, preferably 4°C overnight, preferably followed by nuclease treatment at 30-40°C, preferably 35-39°C, more preferably 37°C for 1-2 hours.

Examples of detergents that can be used in step (b) of the method of the invention are, without limitation, Triton X-100 solution, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sodium deoxycholate , sodium dodecyl sulfate (SDS).

In another preferred embodiment of the method of the invention, the detergent solution comprises Triton X-100 and/or NH4OH. In a more preferred embodiment, the concentration of Triton X-100 is between 0.1 and 0.5%, preferably 0.25%, and the concentration of NH4OH is between 10 and 50 mM, preferably 10 mM. . In another preferred embodiment of the method of the invention, the nuclease is deoxyribonuclease, such as DNase, and/or ribonuclease, such as RNase. In another preferred embodiment, the nuclease concentration is 50 U/mL.

Lyophilization of the MECdmd obtained in step (b) is performed in order to obtain a stable preparation of the MECdmd. The term "lyophilize" or "lyophilization" as used herein refers to the rapid freezing and dehydration of the frozen product in a high vacuum and is also commonly referred to as "freeze drying".

Alternatively, after the lyophilization is complete, the MECdmd obtained in step (c) of the method of the invention is pulverized into a powder by grinding. In a preferred embodiment of the invention, the method of the invention further comprises an additional step (c1) grinding into powder the lyophilized decellularized extracellular matrix obtained in step (c). In another more preferred embodiment of the method of the invention, the grinding treatment of step (c1) of the method of the invention is carried out using liquid nitrogen.

In order to make the lyophilized decellularized extracellular matrix obtained in step (c) or the powder obtained in step (c1) of the method of the invention more water soluble, a solubilization step is performed.

In another preferred embodiment of the method of the invention, the solubilization of step (d) is performed by enzymatic digestion or chemical treatment. In a further preferred embodiment, the enzymatic treatment is carried out with pepsin, preferably carried out with pepsin dissolved in HCl. In another more preferred embodiment, the solubilization of step (d) is carried out at a concentration of 1 to 10% w/v with pepsin between 1 and 2 mg/mL dissolved in HCI of 0.01 to 0.1 M.

In another preferred embodiment, the solubilization of step (d) is performed by chemical treatment, preferably urea-based solubilization, a less aggressive procedure than digestion with pepsin and which allows better preservation of the ECM components. In another more preferred embodiment, the chemical treatment is carried out with acetic acid and urea, preferably with 5% acetic acid and 3 M urea.

Once the MECdmd powder has been solubilized, a gelling step is performed. In a preferred embodiment of the method of the invention, step (e) is carried out by neutralization with 1 M NaOH and incubation at 30 to 40 °C, preferably 35 to 29 °C, more preferably at 37 °C, for 15-30 minutes.

In another preferred embodiment of the method of the invention, the final concentration of MECdmd varies between 11 and 10% w/v.

In another more preferred embodiment of the method of the invention, the MECdmd is selected from 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4 % w/v, 4.5% w/v, 5% w/v, 5.5% w/v, 6% w/v, 6.5% w/v, 7% w /v, 7.5% w/v, 8% w/v, 8.5% w/v and 9% w/v, more preferably the percentage of MECdmd is 3 or 6% w/v , preferably 6%.

The hydrogel of the invention has various uses and applications. Consequently, one aspect A further feature of the present invention is the hydrogel or the composition or the implant of the invention for use as a medicine, hereinafter, the medicinal use of the invention.

Another aspect of the present invention is the hydrogel of the invention or composition or implant of the invention for its use in the treatment of degenerative and/or traumatic diseases, hereinafter, the treatment use of the invention.

As used herein, the term "treat" (or "treats" or "treatment" or "therapy") refers to processes that involve slowing, interrupting, halting, controlling, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition or disease, but does not necessarily imply a total elimination of all symptoms, conditions or disorders related to the disease. In addition, "treatment" also refers to a process that involves preventing the onset of symptoms that have not yet manifested, but will manifest due to an untreated progression of the disorder, condition, or disease. Treatment of a disorder or disease may lead, for example, to an arrest in the progression of the disorder or disease (for example, without deterioration of symptoms) or to a delay in the progression of the disorder or disease (if the arrest of progression is only transitory in nature). The "treatment" of a disorder or disease can also lead to a partial response (eg, improvement of symptoms) or a complete response (eg, disappearance of symptoms) of the subject/patient suffering from the disorder or disease. . Accordingly, "treatment" of a disorder or disease may also refer to an amelioration of the disorder or disease, which may lead, for example, to an arrest in the progression of the disorder or disease or a delay in the progression of the disorder or disease. . Such a partial or complete response may be followed by a relapse. It is to be understood that a subject/patient may experience a wide range of responses to a treatment (such as the exemplary responses described herein above). In the present invention, the condition or disorder to be treated is degenerative diseases.

The term "degenerative diseases", as used herein, refers to a pathological condition in which a tissue loses its original function due to irreversible quantitative loss of the tissue. Examples of degenerative disease include, but are not limited to, cerebral neurological disease, ischemic disease, skin damage, bone disease, and degenerative arthritis.

The expression "traumatic illnesses", as used in the present document, is It refers to a pathological condition resulting from the effect of an external or internal agent. Examples of traumatic disease include, but are not limited to, skin burns, rashes, skin or bone fractures, ie, any injury that leads to a loss of tissue functionality.

In a preferred embodiment of the treatment of the invention, the degenerative disease is selected from the list consisting of: osteoarthritis, inflammatory arthritis, and crystal-induced arthritis. In a preferred embodiment of the treatment of the invention, the traumatic disease is selected from the list consisting of: bone and skin injuries (such as wounds, wounds, fractures, breaks), soft tissue injuries, and cosmetic surgeries.

Another aspect of the present invention is the use of the hydrogel of the invention or composition in pharmacological development and preclinical screening of chemotherapeutic drugs, the basic study of the fundamental biology of cancer and the processes that mediate proliferation, growth, metastasis and tumor invasion.

The hydrogel or composition of the invention can be used as an in vitro disease model of skin pathologies, lesions or tumors.

The hydrogel of the present invention or the composition of the invention may be comprised in the implant of the invention, to form a tissue regenerative scaffold that can be implanted in a subject.

Another aspect of the present invention refers to the use of the implant of the invention for the regeneration of tissues or organs, hereinafter, the regenerative use of the invention.

The terms "tissue" or "biological tissue" are interchangeable and refer to a collection of interconnected cells and extracellular matrix that perform a similar function or functions within an organism. Biological tissues include, without limitation, connective tissue, muscle tissue, nervous tissue (of the brain, spinal cord, and nerves), epithelial tissue, and organ tissue. Connective tissue includes fibrous tissue, eg, fascia, tendon, ligaments, heart valves, bone, and cartilage. Muscle tissue includes skeletal muscle tissue, smooth muscle tissue, eg, oesophageal, stomach, intestinal, bronchial, uterine, urethral, bladder and blood vascular tissue, and cardiac muscle tissue. Epithelial tissue includes simple epithelial tissue, for example, alveolar epithelial tissue, blood vascular endothelial tissue, and cardiac mesothelial tissue, and stratified epithelial tissue. Biological tissue may further be selected, without limitation, from the group consisting of heart valve, vessel, vascular conduit, artery, vein, skin, dermis, pericardium, dura, intestinal submucosa, ligament, tendon, bone, cartilage, ureter, urinary bladder, liver, lung, umbilical cord, and heart. Organs comprise multiple tissues/tissue types. Organs are included herein with the terms "tissue" and/or "biological tissue". The "intact tissue" phase refers to tissue that has not been minced or homogenized. The intact tissue can be a whole tissue or a whole organ. In a preferred embodiment of the regenerative use of the invention, the tissue is selected from a list consisting of: skin, bone, cardiac tissue, cartilage tissue, and vascular tissue.

The implant of the invention may be used to treat diseases or conditions that may benefit from enhanced angiogenesis, cell proliferation, and/or tissue regeneration and/or strengthening. Such diseases or conditions include, but are not limited to, burns, ulcer, trauma, wound, bone fracture, diabetes, psoriasis, arthritis, asthma, cystitis, inflammation, infection, ischemia, restenosis, stenosis, atherosclerosis, occlusion, stroke, infarction, aneurysm. , abdominal aortic aneurysm, uterine fibroid, urinary incontinence, vascular disorders, hemophilia, cancer, and organ failure (eg, heart, kidney, lung, liver, intestine, etc.). The administration of the hydrogel of the invention or of the composition of the invention or of the implant composed of or consisting of the hydrogel of the invention or the composition of the invention can be carried out by direct injection into the tumor, or by spraying, brushing or other application at the application site. In some embodiments, a delivery vehicle is used, such as collagen. Biocompatible supports, such as mesh, can also be used, wherein the support is coated with the hydrogel. The soluble fraction of the solubilized ECM hydrogel, or the solubilized ECM hydrogel can be administered by any route, including parenteral administration, eg, intravenous, intraperitoneal, intramuscular, intraperitoneal, intrasternal, intraarticular, sublingual, oral, topical injection or infusion. , intranasal or transmucosal, or by pulmonary inhalation. The physician may select the appropriate route of administration based on the requirements of the clinical presentation.

Another aspect of the present invention relates to the cosmetic use of the hydrogel of the invention or the composition of the invention.

The term "cosmetic use" refers to a non-therapeutic use that can improve the aesthetic appearance or comfort of mammalian, preferably human, skin, hair and/or hair. In the present context, cosmetic use may include preventing damage to and/or improving human skin, hair and/or hair.

The hydrogel or composition of the invention may take the form of a topical composition which may be in the form of an aqueous, aqueous-alcoholic or oily solution, dispersion-type solution or dispersions of lotion or serum type, of emulsions having a liquid or semi-liquid consistency of the milk type, obtained by dispersing a lipid phase in an aqueous phase (O/W) or vice versa (W/O), or of a suspension or emulsion having a soft consistency, semi-solid or solid cream, aqueous gel or anhydrous gel, or alternatively microemulsion, microcapsule or microparticle or vesicular dispersion of ionic and/or non-ionic type. Said compositions are prepared by usual methods known to those skilled in the art.

Examples of compositions for topical applications include, without limitation, cleansing and protective treatment or care creams for the face, hands, feet, major anatomical folds, or body (for example, day creams , night creams, make-up removing creams, base creams, sun creams), make-up products, such as liquid foundations, cleansing milks, protective or body care milks, after-sun milks, skin care lotions , gels or foams, such as cleansing or disinfecting lotions, sun lotions, artificial tanning lotions, bath compositions, deodorant compositions containing a bactericidal agent, aftershave gels or lotions, depilatory creams or compositions for insect bites, soaps, cleansing pastes, creams, gels, emulsions or foams, hairspray compositions, etc.

Another aspect of the present invention refers to the study of the reduction in the effectiveness of drugs, which can benefit both in the knowledge of new drugs and in the understanding of the resistance mechanisms exerted by cells or also for the design of Matrix biobonds from 3D cell printing. In addition, using biopsies from the patients themselves, it will be possible to obtain a personalized model that allows a previous High Throughput Screening (HTS) to establish the most appropriate therapeutic regimen in each patient and for the study. of basic molecular processes of various diseases

Another aspect of the present invention is the use of the hydrogel or the composition of the invention as a bioink for 3D bioprinting, hereinafter, the bioprinting use of the invention.

Another aspect of the present invention is the use of the hydrogel or the composition of the invention as a bioink for spraying cells, hereinafter, the spray use of the invention.

Another aspect of the present invention is the use of the hydrogel or the composition of the invention as bioink for organs and tissues-on-a-chip or multiple organs-on-a-chip, hereinafter, the use of organ-on-a-chip of the invention.

The term "bioink" as used herein refers to a hydrogel or a mixture of cells with a hydrogel used for bioprinting. Thus, a bioink composition can be applied for bioprinting and spraying. The term "3D bioprinting" as used herein refers to the use of techniques similar to 3D printing to combine cells, growth factors, and biomaterials to fabricate a 3D object, part, tissue, or structure that closely mimics natural characteristics. and biological. Preferably, 3D bioprinting uses the layer-by-layer method to deposit materials or bioinks to create structures that are later used in the fields of biotechnology, medicine, and tissue engineering. The term "cell spray" as used herein refers to the technique of spraying cells in cell culture dishes with a pump-actuated aerosol nozzle. The term "organ-on-a-chip" used herein refers to a cell culture system that simulates the microenvironment and key functional aspects of living organs on a microscopic scale, using principles of biomimicry, microengineering, and microfluidics technologies. .

As is known to the person skilled in the art, the bioink composition may contain various other components that find use in changing the characteristics of the bioink that are necessary in the bioprinting process or in the final product. Therefore, in a preferred embodiment of the use of the composition of the invention, the composition of the invention additionally comprises at least one of the components selected from a list consisting of: alginate, gelatin of various origins (from fish, bovine, skin porcine), fibrinogen of various sources (bovine, porcine, human plasma), intra-articular sodium hyaluronate, silk fibroin and sericin, xanthan, genipin, agarose, agar, chitosan, nanocellulose, or its methacryloyl variants, or any combination of the same.

In another preferred embodiment of the composition of the invention, the concentration of alginate and/or alginate methacryloyl is between 1 and 15% w/v, preferably 2.0%, 2.5%, 3, 0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, 9.0%, 10.0%, 11.0% , 12.0%, 13.0% or 14.0% w/v. In another preferred embodiment, the concentration of porcine skin gelatin and/or gelatin methacryloyl concentrations are between 5 and 15% w/v, preferably 6.0%, 7.0%, 8.0% %, 9.0%, 10.0%, 11.0%, 12.0%, 13.0% or 14.0% w/v, more preferably 10%. In another preferred embodiment of the composition of the invention, the plasma fibrinogen concentration bovine and/or intra-articular sodium hyaluronate is between 0.5 and 1.5%, preferably 0.6, 0.7, 0.8, 0.9, 1.0, 1 .1, 1.2, 1.3 or 1.4%.

In another preferred embodiment of the composition of the invention, silk fibroin and sericin, xanthan, genipin, agarose, agar, chitosan, nanocellulose, or the concentration of its methacryloyl variants is between 0.2% and 10% w /v.

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as that normally understood by a person skilled in the art to which the present invention pertains. Methods and materials similar or equivalent to those described herein may be used in practicing the present invention. Throughout the description and claims, the word "comprising" and its variations are not intended to exclude other technical features, additives, components or steps. Additional objects, advantages, and features of the invention will be apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Phenotypic characterization and differentiation potential of MSCs. (A) FACS characterization of MSCs showed positive expression of surface markers CD105, CD90, CD73 and negative or low expression for CD45, CD133, and CD34. (B) The differentiation potential of MSCs obtained from liposuction toward osteogenic, adipogenic, and chondrogenic lineage was confirmed by alizarin red S, oil red O, and toluidine blue staining, respectively. Scale bar: 100 pm.

Figure 2. Production and characterization of early chondrogenic mECMs. Quantitative analysis of specific components of cartilage GAG (A) and type II collagen (B) in MSC-derived matrix, grown under chondrogenic culture conditions for 1, 2, 3, and 4 weeks, normalized to DNA from cells in culture. Qualitative analysis of principal components, collagen and GAG in MSC-derived matrix and chondrocytes after 2 weeks in culture by histological staining (Syrian red, toluidine O) and immunofluorescence (type II collagen and aggrecan) (C). Quantitative analysis of collagen, collagen II (D) and GAG (E) content in MSC-derived matrix and chondrocytes after 2 weeks in culture. Scale bar: 50 pm. Data represent the mean ± SD (n = 3). **, p <0.01; ***, p < 0.001, Student's T test.

Figure 3. Analysis of MSC differentiation towards adipocytes and FB by means of induction with specific culture medium and subsequent staining with Syrian red, toluidine blue, alcian blue, oil red O staining and vimentin, respectively.

Figure 4. Histological characterization of FB dECM before and after the decellularization process, in comparison with native dermal tissue and decellularized native dermal tissue. Hematoxylin-eosin, Syrian red (collagens), toluidine blue (GAG), alcian blue (PG) and Dapi (DNA) stains showed correct decellularization and similarity to the reference tissue.

Figure 5. Histological characterization of CMM dECM before and after the decellularization process. Hematoxylin-eosin, Syrian red (collagens), toluidine blue (GAG), alcian blue (PG) and Dapi (DNA) stains showed correct decellularization and a matrix rich in native tissue components.

Figure 6. Decellularization of MSC-derived early chondrogenic matrix. (A) Qualitative analysis by light microscope image (phase contrast light) and histological staining: H&E (cell contents), Masson's trichrome (collagen) and toluidine O (GAG). Quantitative analysis of DNA (B), collagen (C) and GAG (D) content. Scale bar: 50 pm (phase contrast) and 200 pm (histological staining). Data represent the mean ± SD (n = 3). ***, p < 0.001, Student's T test.

Figure 7. Formulation and characterization of MECdm hydrogel. (A) Schematic elucidation preparation of early chondrogenic MECdm-based hydrogel. (B) Scanning electron micrographs (SEM) of crosslinked MECdm hydrogel. Magnifications of 10,000x (on the left) and 40,000x (on the right). (C) Steady shear flow behavior of the hydrogel with an isothermal up and down shear rate ramp at 15°C. (D) Transient shear recovery behavior of the hydrogel with stepwise isothermal changes in shear rate at 15°C. Thermally induced gelation of the hydrogel, determined by linear viscoelasticity tests (E) and by the loss factor (G"/G') (F) of the hydrogels from 20 °C to 40 °C. Data represent mean ± SD (n=3).

Figure 8. In vitro biological evaluations of MECdm hydrogel. (A) Representative confocal images of cell viability using the Live/Dead assay at 1, 10, and 28 days (green for live cells, red for dead cells). (B) Cell viability (%) in the MECdm hydrogel after 1, 10 and 28 days. (C) Proliferation rate of MSCs grown on MECdm hydrogel compared to day 0 of culture. (D) Analysis of the Gene expression of specific (COL2A1, ACAN, SOX9) and non-specific (COL1A1) chondrogenic genes from MSCs embedded in 3 and 6% MECdm over time in culture (2.4 weeks) and chondrocytes in culture for 2 weeks (control ). (E) Immunostaining analysis for collagen type II and collagen type I. Scale bar 100 pm. Data are expressed as mean ± SD (n = 3); *, # p < 0.01, Student's T test. (*) Significantly different value compared to values of CMM cultured on day 0; (#) Significantly different value compared to CMM values in MECdm hydrogels at 3 and 16% after 4 weeks in culture (p < 0.01).

Figure 9. In vivo biological evaluations of MECdm hydrogel. (A) Images at 4 weeks postimplantation in CD1 mice. (B) DAPI staining showing cells infiltrating the 3% and 6% cell-free MECdm gels at days 0, 14, and 28 after subcutaneous implantation into CD1 mice. (C) Histological analysis of dmECM gels at 3% and 6% concentrations, in vitro predifferentiated MSC micropellets with chondrogenic medium after subcutaneous implantation into NSG mice at 2 and 4 weeks, and articular cartilage (control). Arrows indicate cells embedded in lacunae. Scale bar 100pm.

Figure 10. Histological characterization of dECM from adipose tissue after three different decellularization processes (protocol 1, 2 and 3). Hematoxylin-eosin, Syrian red (collagens), toluidine blue (GAG) and alcian blue (PG) staining showed correct decellularization and a matrix rich in native tissue components.

Figure 11. Table with the developed bioinks containing 3% tissue-derived ECMd, 10% bovine skin gelatin or gelatin methacryloyl, 2.5% alginate and 1% fibrinogen or intra-articular APM sodium hyaluronate at 2.5%.

EXAMPLES

1. MATERIALS AND METHODS

1.1. Isolation, culture and characterization of cell lines

Human mesenchymal stem cells (hMSC) and human fibroblasts (hFB) were isolated from adipose tissue of patients undergoing a liposuction procedure and from skin tissue during abdominoplasty surgery by enzymatic digestion using collagenase I (Sigma Aldrich), respectively. , after the informed consent and authorization of the Ethics Committee of the Hospital Clínico Universitario de Málaga, Spain.

The characterization of the isolation of hMSCs has been previously published in López Ruiz E., et al, 2013, Osteoarthr. Cartil., 21, 246. Cells were seeded in growth medium [Dulbecco's modified Eagle's medium with high glucose (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), penicillin 100 U /ml and streptomycin 100 mg/ml (Invitrogen Inc.)] at 37°C in a humidified atmosphere with 5% CO ² . The medium was changed every 3 days and when the cells reached 80% confluence, they were subcultured. For all experiments, hMSCs were used between passages 4 and 6.

Dermal tissue was mechanically processed and digested with 2 mg/ml collagenase I (Sigma Aldrich) at 37°C overnight. It was then filtered through a 100 pm CellStrainer (Falcon) and collagenase inactivated with Dulbecco's Modified Eagle's medium (DMEM; Sigma-Aldrich) with 10% fetal bovine serum (FBS; Sigma-Aldrich). Cells were seeded in growth medium [Dulbecco's modified Eagle's medium with high glucose (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 100 U/ml penicillin and 100 mg/streptomycin. ml (Invitrogen Inc.)] at 37°C in a humidified atmosphere with 5% CO ² . The medium was changed every 3 days and when the cells reached 80% confluence, they were subcultured.

The phenotype and differentiation potential of the isolated cells were characterized, as previously described in Dominici M., et al, 2006, Cytotherapy, 8, 315. To examine their immunophenotype, the cells were trypsinized, washed, and resuspended. in PBS with 1% bovine serum albumin (BSA; Sigma-Aldrich). A total of 2*105 cells were incubated in the dark for 30 min with fluorochrome-conjugated monoclonal antibodies [CD34, CD45, CD90, CD73, CD105 and CD133 (Miltenyi Biotec, Auburn, CA, USA)], washed in PBS and analyzed by flow cytometry on a FACSCanto II cytometer (BD Biosciences).

For differentiation assays, mesenchymal stem cells (MSCs) were plated at 1*105 cells/cm2 in DMEM-FBS in 6-well culture plates. After 48 h, the culture medium was replaced with a specific differentiation-inducing medium. For adipogenic, osteogenic and chondrogenic differentiation, cells were cultured for 2 weeks in hMSC Adipogenic Differentiation BulletKit™ (Lonza), hMSC Osteogenic Differentiation BulletKit™ (Lonza) and StemMACS ChondroDiff Medium (Miltenyi Biotec), respectively. Differentiated cell cultures were stained with oil red O (Amresco, Solon, OH, USA) for adipogenic differentiation, oil red alizarin (Lonza) for osteogenic differentiation or toluidine blue (Sigma-Aldrich) for chondrogenic differentiation.

1.2. Isolation and culture of chondrocytes

Articular cartilage chondrocytes were isolated from patients with osteoarthritis during joint replacement surgery after informed consent from all patients and approval by the Ethics Committee of the Hospital Clínico Universitario de Málaga, Spain. The sample was isolated from the femoral side, selecting the non-overload compartment: lateral condyle in varus knees and medial condyle in valgus cases.

None of the patients had a history of inflammatory arthritis or crystal-induced arthritis, and only cartilage that macroscopically appeared relatively normal was used for this study. Cells were seeded in chondrocyte medium [growth medium supplemented with 1% ITS (Insulin-Transferrin-Selenium, Gibco), 50 pg/pl ascorbic acid (Sigma), 40 pg/pl proline (Sigma) and 100U penicillin /ml and streptomycin 100pg/ml], at 37°C in a humidified atmosphere with 5% CO ² . At 80% confluence the cells were subcultured.

1.3. Cell differentiation protocols

After reaching 100% cell confluence, MSCs were induced to differentiate into FB, chondrocytes, osteoblasts, and adipocytes. For this purpose, they were cultured for two weeks with their respective differentiation media (Table 1).

Table 1. Differentiation media complements.

1.4. Matrix generation protocol

When the cultures of sections 1.1, 1.2 and 1.3 reached 100% confluence, treatment with matrix enrichment medium was started for three weeks without subculturing the cells. Cell lines were cultured with DMEM (Sigma-Aldrich) with 10% FBS (Sigma-Aldrich), 1% penicillin/streptomycin (Sigma-Aldrich), 50 µg/mL ascorbic acid (Sigma Aldrich) and 40 µg l-proline. /ml (Sigma Aldrich).

1.5. Early chondrogenic mECM production

At complete confluence, MSCs were released with TryPLE (Invitrogen) and subcultured in Petri dishes to a cell density of 1x10 ⁵ cells/cm 2 . To allow ECM membrane formation, they were cultured in a monolayer for 1, 2, 3, or 4 weeks in chondrocyte medium supplemented with dexamethasone (100 nM) and TGF-P3 (10 ng/mL) at 37 °C in a humidified atmosphere. with 5% CO ² . The medium was refreshed every 2 or 3 days. As a control, MEC was generated from chondrocytes in culture in the same way, but without using supplements.

1.6. Matrix decellularization protocol

At the end of the culture period, cells were detached from the underlying matrix by exposure to a decellularization treatment, selected from a variety of previously published methodologies for preparing cultured cartilage-derived extracellular matrix (CEMd-c) scaffolds. ( Hoshiba T., et al, 2011, Biotechnol. Prog., 27, 788).

Briefly, the chemical procedure consisted of incubation in a detergent solution containing 0.25% Triton X-100 and 10 mM ammonium hydroxide (NH ⁴ OH) at 37 °C for 5 min or at 4 °C overnight, followed by treatment with 50 units/ml deoxyribonuclease I (DNase) and 50 mg/ml ribonuclease (RNase) A (Invitrogen) in 10 mM Tris-HCI (Sigma) for 2h at 37°C. After several washes with PBS, the culture plates were examined by light microscopy to ensure that all cellular material had been removed. Finally, the supernatant was removed and the matrix remaining on the plate was scraped and lyophilized.

1.7. Hydrogel preparation based on a pepsin-based solubilization protocol

Freeze-dried MECd were ground to a powder using liquid N2. The required amount of MECd powder was digested in pepsin solution (1 mg/ml in 0.1 M HCI) for 48 h. MECd were prepared at two concentrations: 3 and 6% w/v. After solubilizing the MECds, the pH was neutralized with the dropwise addition of 1 M NaOH and 10X PBS (up to 1X final dilution), at low temperatures (below 15 °C) to avoid gelation of the MECds. The resulting MECd pregels were mixed with CMM at a concentration of 5 x 10 6 cells/ml for further biological studies. After gelling at 37 °C for 15 min, the cell-embedded ECM hydrogels were maintained in growth medium for a specific time, defined in each experiment.

1.8. Urea-based solubilization protocol

The lyophilized MECds were solubilized in 5% acetic acid (Sigma), at 4°C, and stirred continuously overnight. Then, 3M urea buffer (Sigma) was added, stirring overnight at 4°C. After 24 h, the solution was centrifuged at 13,000 rpm for 10 min and the supernatant was frozen at -20 °C. The pellet was treated again for an additional 48 h in 3M urea buffer, stirred continuously at 4 °C, and the buffer refreshed after 24 h. Afterwards, it was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was frozen at -20 °C. The two supernatants were pooled and dialyzed for 72 hours at 4 °C. The solution was finally lyophilized for storage. The lyophilized MECds were dissolved to a final concentration of 1 to 10% w/v in 5% acetic acid with stirring for 10min at room temperature. To gel, the ECMd was neutralized with 1 M NaOH (Sigma) and incubated at 37 °C for 15 min.

1.9. Theological Essays

Theological properties of MECdm hydrogels (3% and 6% w/v) were investigated with a torsion rheometer (MCR301, Anton Paar, Austria) using a conical plate geometry (20 mm diameter and 2o angle). ). Steady shear tests were performed to determine shear viscosity at 15 °C. These trials consisted of four stages. In the first, the sample was previously sheared at a constant shear rate (500s_1) for 30 seconds. In the second stage, the sample was allowed to settle for 30 seconds in the absence of shear. In the third stage, the shear rate was increased logarithmically from 0.1 s_1 to 1000 s_1. Finally, in the fourth stage, the shear rate was decreased from 1000s_1 to 0.1 s_1 to explore whether the sample is thixotropic or not.

To better understand the thixotropic behavior of the samples, intermittent flow tests were also performed using step changes in shear rate with a logarithmic measurement point duration of 0.01 to 10s at 15°C. The protocol consisted of six stages. In the first stage, the sample was pre-sheared at a constant shear rate (500 s_1) for 30 s. In the second stage, the sample was allowed to settle for 30 s in the absence of shear. The third stage consisted of a start-up test at 1000s'1 while the fourth stage consisted of suddenly reducing the shear rate to Os-1. Steps three and four were repeated at intervals five and six for consistency in order to measure viscosity transients.

Heat-driven gelation of the samples was investigated using small amplitude oscillatory shear (SAOS) tests at a strain amplitude of 1% and a frequency of 1 Hz. The protocol consisted of two steps. In the first stage, the samples were equilibrated at 20 °C for 5 min. Finally, in the second stage, OSA tests were performed while the temperature increased from 20 °C to 40 °C at a rate of 1 °C min_1.

1.10. cell viability

The Live/Dead kit assay (Thermo Fisher Scientific) was used to assess cell viability, following the manufacturer's instructions. Images of the samples were observed using a confocal microscope (Nikon Eclipse Ti-E) and analyzed with Image J software (v. 1.52i, USA). The percentage of viable cells was obtained by counting six regions of each sample (n = 3).

1.11. cell proliferation

The proliferation rate of cells embedded in MECdm hydrogels (n = 3) was assessed by the alamar blue (aB) colorimetric assay (Thermo Fisher Scientific) at different time points, following the manufacturer's instructions. It was normalized to the appropriate control without cells. Absorbance data was plotted as a multiplication factor relative to day 0.

1.12. Gene expression analysis

Total messenger RNA (mRNA) was isolated from MSCs embedded in MECdm hydrogels (n = 3) at different time points using TriReagent (Sigma) and reverse transcribed into cDNA using the Reverse Transcription System kit (Promega), according to the instructions. manufacturer. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR green master mix (Promega), following the manufacturer's instructions. Gene expression levels for collagen type II (COL2A1), aggrecan (ACAN), Sox transcription factor 9 (SOX-9), and collagen type I (COL1A1) were normalized with respect to the gene constitutive of glyceraldehyde 3-phosphate dehydrogenase (GADPH) and were shown as a multiplication factor with respect to the value of embedded MSCs on day 0.

1.13. Histology

Hydrogels of dEMC, micropellets, articular cartilage tissue, dermal tissue, and decellularized dermal tissue were fixed in 4% paraformaldehyde (PFA), serially dehydrated, paraffin-embedded, and sectioned (4 µm thick) with a microtome ( Leica RM2255, Leica Biosystems, USA). Samples were rehydrated and stained with hematoxylin and eosin (H&E), toluidine O, Masson's trichrome, alcian blue, alizarin red/light green (RA/VC). For monolayer staining, fixed cells were directly stained using toluidine O and Syrian red. All samples were photographed using the Leica DM 5500B bright field microscope and analyzed using Image J software.

1.14. Quantitative biochemical assays

Lyophilized mECM and dmCEM were digested with papain and assayed for GAGs and DNA. The GAG content was estimated by a colorimetric assay with dimethyl methylene blue (DMMB). DNA content was quantified by a fluorometric assay using DAPI staining. For collagen quantification, the lyophilized samples were first solubilized in a pepsin solution (2 mg/ml in 0.5 M acetic acid). The total collagen content was measured by staining with Picrosyrian red. Type II collagen content was determined using a commercially available Collagen type II ELISA kit (Chondrex).

The GAG content was estimated by quantifying the amount of sulfated GAG in a colorimetric assay with dimethyl methylene blue (DMMB). For this purpose, the samples were digested in 1 ml of papain solution (125 mg/ml of papain in phosphate 0.1 M sodium with 5 mM Na2-EDTA and 5 mM cysteine-HCl at pH 6.5) for 16h at 60 °C. The resulting extract was mixed with DMMB solution to bind the GAGs. The content was calculated based on a standard curve of shark cartilage chondroitin sulfate (Sigma) at 570 nm in a microplate spectrophotometer.

The total collagen content was measured by staining with Picrosyrian red. For this, it was first solubilized by incubating the matrix with pepsin in 0.5 M acetic acid (2 mg/ml) and stained with 1 ml Syrian red dye for 30 min at room temperature. The stain was then dissolved using 0.5 N sodium hydroxide (0.5 N NaOH) and absorbance was measured at 560 nm using a microplate reader (Berthold technologies, USA). Type I collagen (rat tail) was used as a standard for biochemical assays.

Type II collagen content was determined using a commercially available Collagen type II ELISA kit (Chondrex). For this, the samples were digested with pepsin (1 mg/ml) in 0.5 N acetic acid for 48 h at 4 °C followed by the addition of 1 mg/ml pancreatic elastase solution at 4 °C for 24 h. Finally, these were neutralized with 1 M Tris base. The insoluble material was removed by centrifugation at 10,000rpm at RT for 5min and the supernatant was collected for the assay. Quantitative analysis was performed according to the manufacturer's instructions and was measured in the microplate spectrophotometer at 490 nm.

DNA content was quantified by a fluorometric assay using DAPI staining. For this, the samples were digested in papain solution for 16h at 60 °C. The resulting extract was stained with DAPI. The fluorescence intensity was measured in a fluorescence spectrophotometer (excitation wavelength: 360 nm, emission wavelength: 450 nm). Calf thymus DNA was used to create a standard curve to quantify the DNA in the samples.

1.15. immunostaining assay

Cell monolayers, MECdm hydrogels, micropellets, and articular cartilage tissue were fixed with 4% PFA in PBS for 20 min at RT. For immunofluorescence analysis, dmMEC hydrogels, micropellets, and articular cartilage tissue were embedded in optimal cutting temperature (OCT) compound and sectioned using a 10-mm-thick cryotome. Sections and monolayers were treated with a primary antibody against collagen type I (Santa Cruz Biotechnology, 1:100), collagen type II (Santa Cruz Biotechnology, 1:100), and aggrecan (Sigma, 1:100). The sections were then incubated with secondary antibodies. conjugated with AlexaFluor 488 or 594 (ThermoFisher, 1:500) and counterstained with DAPI. Images were obtained using a Leica DM 5500B microscope and analyzed with Image J software.

1.16. Analysis by mass spectrometry (MS)

Full characterization of protein content in mECM was performed by tandem mass spectroscopy (LC/MS). The samples were resuspended in 50 mM NH ⁴ HCO ³ (pH 8.5) and a small part (1/20) was digested. The dried peptide mixtures were analyzed on a nanoAcquity liquid chromatograph (Waters) coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific). The data was used to search a modified version of the public SwissProt human database. The database search was performed with the Sequest HT search engine using Thermo Proteome Discover (v.1.4.1.14). Results were viewed in Proteome Discoverer (v.1.4.1.14) and the identified protein lists were exported as an Excel file.

1.17. Analysis by scanning electron microscopy (SEM)

The internal microstructure of the hydrogel was examined using SEM. MECdm hydrogel was fixed at different concentrations in cold 2.5% glutaraldehyde. After several washes with PBS, the samples were dehydrated using a graduated series of ethanol (30-100%). These were critical point dried in an Emscope CPD 750 critical point dryer, fixed to aluminum mounting studs for SEM test specimens and sputter coated with a gold palladium alloy using Sputter Coater 108 Auto. . The samples were imaged using an SEM (Quanta 400 (FEI), at 10,000x and 40,000x magnification.

1.18. in vivo experiments

In vivo tests were carried out according to the approved guidelines of the University of Granada and following the institutional and international standards for animal welfare and the experimental procedure. All experiments were approved by the Research Ethics Committee of the University of Granada.

Biocompatibility in vivo was assessed in immunocompetent (ICR) CD1 mice. Acellular gels were embedded in PCL scaffolds (a cylindrical-type porous structure, 8 x 5 mm; 3 mm pore size) to facilitate localization, and transplanted into the posterior subcutaneous tissue of mice anesthetized by isoflurane inhalation (n = 6 ^).

For in vivo assessment of tissue formation and cartilage maturation, cell-loaded 3 and 6% MECdmc hydrogels (5x106 cells/ml), previously cultured in growth medium for 2 weeks in vivo, were injected subcutaneously. vitro, in immunodeficient NOD SCID gamma (NOD.Cg-Prkdcscid H2rgtm1Wjl/SzJ, NSG) mice (n = 8) following the same methodology as in the experiment with CD1 mice. As a positive control (n=6) the present inventors used MSCs pre-differentiated towards the chondrogenic lineage in microbead system for 2 weeks in vitro. For this, 3*105 CMM were centrifuged (1500 rpm, 5 min) in a 15ml conical tube and incubated with loose caps at 37°C and 5% CO ² . The chondrogenic medium was changed every other day for 2 weeks and the tubes were gently shaken to prevent adherence of the pellet to the plastic walls. The animals were kept in a micro-ventilated cage system with food and water on demand. Mice were handled in laminar airflow to maintain sterile conditions. Two and four weeks later, the mice were sacrificed via an overdose injection of anesthetics and the hydrogels were excised for subsequent histological and immunofluorescence analysis.

1.19. Statistic analysis

All data in the graph represent the mean/- SD of at least three experiments. To determine differences between conditions, Student's two-tailed T-test was used for quantitative biochemistry analysis to determine differences between conditions and two-way ANOVA for gene expression analysis using GraphPad software. Prism 8.3.0. Assumptions of homoscedasticity and normality of the data were tested and ensured by using transformed data sets [log(value of dependent variable 1)] when necessary. P values <0.001 (***), <0.01 (**) and <0.05 (*) were considered statistically significant in all cases.

1.20. Bioink formulations

Different formulations of bioinks were made to analyze their properties. The combinations studied are shown in the table below (Table 2).

MECd to Alginate Gelatin from porcine skin Fibrinogen from bovine plasma

3% (Sigma) to 10% (Sigma) to 1% (Sigma)

2.5%

Gelatin Methacryloyl Bovine Plasma Fibrinogen (Sigma) 10% (Sigma) 1%

Sodium Hyaluronate APM Pig Skin Gelatin

intra-articular (Bioiberica) at (Sigma) at 10%

1 %

Sodium Hyaluronate APM Gelatin Methacryloyl

intra-articular (Bioiberica) at (Sigma) at 10%

1 %

Pig skin gelatin 10% human platelet-rich plasma (Sigma) 1% Table 2. Different bioink formulations

The novel addition of MECd in the bioink formulation provides the complex composition of native tissue.

Alginate is an anionic polysaccharide that provides viscosity to the formulation and also acquires adhesive and sealing properties with minimal foreign body reaction on wet surfaces. Gelled using 100 mM -1 M CICa ^2.

Porcine skin gelatin is a mixture of water-soluble and biocompatible proteins present in collagen and is a common compound in coating cell cultures to improve cell adhesion and in tissue engineering for scaffolding. Another option is the use of gelatin methacrylate, common in the formation of cross-linked hydrogels for tissue engineering and 3D printing for the morphogenesis of different cell types.

In addition, fibrinogen is a coagulation protein whose combination with 0.9% thrombin produces fibrin, an insoluble protein capable of forming three-dimensional networks, making it a suitable material as a structural part of the final bioink. As an alternative to fibrinogen, the use of Intra-articular APM Sodium Hyaluronate has been studied, a polysaccharide produced mainly by mesenchymal cells that stimulates the production of collagen, or Platelet Rich Plasma (PRP), cells involved in coagulation rich in growth factors. growth cells that promote cell migration and division and stimulate repair in damaged tissues.

All these compounds had been previously diluted in PBS 1X. Finally, to obtain the final bioink, all the compounds were mixed homogeneously with stirring at 37 °C.

2. RESULTS

2.1. Production and characterization of mECM

MSC isolated from lipoaspirates from patients were characterized following the established criteria of the International Society for Cellular Therapy (ISCT) to define multipotent mesenchymal stromal cells (Figure 1).

In an attempt to produce a hydrogel that better recreates the environment during chondrogenesis, an early chondrogenic matrix was synthesized from CMM. For this, CMM were cultured in monolayer and in chondrogenic medium during different time points (1, 2, 3 and 4 weeks). Quantification of the main components of articular cartilage, such as type II collagen and sulfated GAGs, was performed to investigate the different stages of chondrogenesis. After 1 week in culture, the GAG content per pg DNA of MSCs grown in the chondrogenic medium was at the same level as that grown in the growth medium (Figure 2A). However, the GAG/DNA ratio increased significantly with increasing cell culture time. A similar situation was observed for collagen II, whose levels were seen two weeks after MSC culture under chondrogenic conditions and increased markedly over time, more specifically during the last week (Figure 2B). In view of these results, since the formation of the chondrogenic matrix began after two weeks in culture and this time was sufficient to induce the initial deposition of chondrogenic components, which characterizes the early stages of chondrogenesis, this time was used to subsequent trials.

The production of early chondrogenic matrix was then confirmed by comparing two-week mECM and chondrocyte-derived mature chondrogenic matrix (cECM) in culture for two weeks. Histology and immunofluorescence staining revealed the differential presence and distribution of the major ECM components. Figure 2C shows that both ECM expressed similar levels of collagen and GAG, although chondrocyte-derived ECM had significantly higher expression of specific components of mature cartilage, such as collagen II and aggrecan. These observations were in agreement with the results of quantitative tests of said components, revealing that the matrices were rich in collagen (430 pg/mg for MECc and 760 pg/mg for MECm), with 70% of this collagen II content in chondrocyte matrix, unlike MECm which was only 2 .45% (Figure D). In turn, the GAG content expressed values of approximately 60 pg/mg in MECc and 47 pg/mg in MECm (Figure 2 D, E).

In order to obtain more details about the mECM components the present inventors performed a characterization by mass spectrometry (Table 3). The results revealed the presence of a complex protein matrix, identifying 100 proteins that included matrisome core and matrisome-associated proteins. The core matrisome, consisting of structural proteins that confer mechanical properties and cell adhesion to the ECM, was the major component. This was rich in collagens, glycoproteins and proteoglycans. A great diversity of collagens was identified, most of them constituents of cartilaginous ECM. There were some members of the fibrillar family (type I, III, and V collagen), fibril-associated collagens with interrupted triple helices (FACIT) (type XII, XIV, and XIV collagens), and network-forming collagens (type IV and XIV collagen). VIII). Other collagen subtypes such as type VI collagen, which is the primary collagen located in the pericellular matrix of cartilage, were also present. Among the glycoproteins, some Emilin-1-like MSC matrix markers were found, but also typical cartilage matrix structural proteins such as cartilage oligomeric matrix protein (COMP), tenascin-C, lumican (LUM), protein core 2 of chondroitin sulfate proteoglycan (CSPG2 or versican), prolargine (PRELP) and protein induced by transforming growth factor beta, although the most detected was fibronectin. Biglycan, decorin and versican core protein, which are also very common in cartilage, were the main proteoglycans in the present mECM. Other affiliated proteins were identified in less abundance, including regulatory ones, for example, matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), ECM-associated proteins (ie, annexins and galectins), and signaling ligands (Table 3).

collagens

Collagen chain alpha-1 (I) NO = Homo

P02452 27.13 16.05 16 sapiens NG = COL1A1 EP=1VS = 5

Collagen chain alpha-2 (I) NO = Homo

P08123 42.96 24.01 22 sapiens NG = COL1A2 EP=1VS = 7

Collagen chain alpha-1 (IV) NO = Homo

P02462 140 3 ⁴ sapiens NG = COL4A1 EP=1VS = 3

Collagen chain alpha-2 (IV) NO = Homo

P08572 370 7 ⁷ sapiens NG = COL4A2 EP=1VS = 4

Collagen chain alpha-3 (VI) NO = Homo

P12111 32978 60 147 sapiens NG = COL6A3 EP=1VS = 5

Collagen chain alpha-1 (III) NO = Homo

P02461 20.18 10.64 11 sapiens NG = COL3A1 EP=1VS = 4

Collagen chain alpha-2 (V) NO = Homo

P05997 486 8 8 sapiens NG = COL5A2 EP=1VS = 3

Collagen chain alpha-1 (V) NO = Homo

P20908 2543 15 18 sapiens NG = COL5A1 EP=1VS = 3

Collagen chain alpha-1 (VI) NO = Homo

sapiens IO = 9606 NG = COL6A1 EP=1VS = P12109 91.69 22.47 16

3

Collagen chain alpha-2 (VI) NO = Homo

sapiens IO = 9606 NG = COL6A2 EP=1VS = P12110 60.68 18.94 14

4

Collagen chain alpha-1 (VIII) NO = Homo

P27658 64 6 3 sapiens NG = COL8A1 EP=1VS = 2

Collagen chain alpha-1 (XII) NO = Homo

sapiens IO = 9606 NG = COL12A1 EP=1VS = Q99715 99.89 18.77 44

2

Collagen chain alpha-1 (Vil) NO = Homo

Q02388 51 1 1 sapiens NG = COL7A1 EP=1VS = 2

Collagen chain alpha-2 (VIII) NO = Homo

P25067 16 7 2 sapiens NG = COL8A2 EP=1VS = 2

Collagen chain alpha-1 (XI) NO = Homo

P12107 360 8 11 sapiens NG = C0L11A1 EP = 1VS = 4

Collagen chain alpha-1 (XIV) NO = Homo

Q05707 2340 42 50 sapiens NG = COL14A1 EP = 1VS = 3

Collagen chain alpha-1 (XVI) NO = Homo

Q07092 58 2 2 sapiens NG = COL16A1 EP = 1VS = 2

Collagen chain alpha-1 (XXI) NO = Homo

Q96P44 93 7 5 sapiens NG = COL21A1 EP = 2VS = 1

glycoproteins

Fibulin-1 NO = Homo sapiens NG = FBLN1

P23142 959 25 13 PE = 1VS = 4

Fibulin-2 NO = Homo sapiens NG = FBLN2

P98095 1468 29 25 EP = 1VS = 2

Philamine-A NO = Homo sapiens IO = 9606 NG

P21333 54.43 15.00 28 = FLNA EP = 1VS = 4

EMILINA-1 NO = Homo sapiens IO = 9606 NG

Q9Y6C2 32.00 12.80 9 = EMILIN1 EP=1VS = 3

EMILINA-2 NO = Homo sapiens NG =

Q9BXX0 316 15 10 EMILINA2 EP = 1VS = 3

Fibronectin NO = Homo sapiens IO = 9606

P02751 294.06 44.17 65 NG = FN1EP=1VS = 4

Fibromodulin NO = Homo sapiens NG =

Q06828 231 21 5 FMOD EP = 1VS = 2

Cartilage oligomeric matrix protein NO

P49747 1060 29 15 = Homo sapiens NG = COMP EP=1VS = 2

Cartilage intermediate layer protein 1 NO

075339 893 27 28 = Homo sapiens NG = CILP EP = 1VS = 4

Cartilage intermediate layer protein 2 NO

Q8IUL8 0 1 1 = Homo sapiens NG = CILP2 EP = 2VS = 2

Chitinase-3-like protein 1 NO = Homo

P36222 88 21 7 sapiens NG = CHI3L1 EP=1VS = 2

Periostin NO = Homo sapiens NG = POSTN

Q15063 7739 66 48 EP = 1VS = 2

Thrombospondin-1 NO = Homo sapiens NG =

P07996 26485 60 64 THBS1 EP = 1 VS = 2

Thrombospondin-2 NO = Homo sapiens NG =

P35442 3219 28 25

Tenascin NO = Homo sapiens IO = 9606 NG

P24821 96.80 26.90 40 = TNC EP = 1VS = 3

Laminin beta-2 subunit NO = Homo

P55268 159 5 5 sapiens NG = LAMB2 EP=1VS = 2

Laminin alpha-4 subunit NO = Homo

Q16363 0 0 1 sapiens NG = LAMA4 EP=1VS = 4

Laminin gamma-1 subunit NO = Homo

P11047 128 2 3 sapiens NG = LAMC1 EP=1VS = 3

SPARC NO = Homo sapiens NG = SPARC EP

P09486 155 17 ⁴ = 1 VS = 1

Extracellular matrix protein 1 similar to

fibulin containing EGF NO = Homo sapiens Q12805 138 15 ⁵ NG = EFEMP1 EP = 1VS = 2

A-like extracellular matrix protein 2

fibulin containing EGF NO = Homo sapiens 095967 120 12 ⁴ NG = EFEMP2 EP = 1VS = 3

Vitronectin NO = Homo sapiens NG = VTN EP

P04004 94 3 1 = 1 V S = 1

Lactadherin NO = Homo sapiens NG =

Q08431 28 6 2 MFGE8 EP = 1VS = 2

Podocano NO = Homo sapiens NG = PODN EP

Q7Z5L7 0 2 1 = 1 V S = 2

Olfactomedin-like protein 3 NO = Homo

Q9NRN5 26 2 1 sapiens NG = OLFML3 EP = 2VS = 1

Secretoglobin 1D family member 2

NO = Homo sapiens NG = SCGB1D2 EP = 2 095969 22 10 1

VS = 1

Semaphorin-3C NO = Homo sapiens NG =

Q99985 0 1 1 SEMA3C EP = 2VS = 2

Thyroglobulin NO = Homo sapiens NG = TG EP

P01266 0 1 1 = 1 V S = 5

proteoglycans

Sulfate proteoglycan core protein

basement membrane specific heparan P98160 64.80 10.18 34 NO = Homo sapiens NG = HSPG2 EP = 1VS =

4

Biglican NO = Homo sapiens 10 = 9606 NG =

P21810 19.54 28.26 8 BGN EP=1VS = 2

Versican core protein NO = Homo

P13611 12.67 1.77 5 sapiens IO = 9606 NG = VCAN EP = 1VS = 3

Decorina NO = Homo sapiens NG = DON EP =

P07585 804 31 8 1VS = 1

Lumicano NO = Homo sapiens NG = LUM EP =

P51884 533 35 9 1VS = 2

Mimecano NO = Homo sapiens NG = NGO EP

P20774 102 17 ⁴ = 1 VS = 1

regulators

Matrix metalloproteinase-14 NO = Homo

P50281 119 13 6 sapiens NG = MMP14 EP = 1VS = 3

A disintegrin and metalloproteinase with

thrombosponin motifs 4 NO = Homo 075173 95 ⁴ 2 sapiens NG = ADAMTS4 EP=1VS = 3

A disintegrin and metalloproteinase with

thrombosponin motifs 3 NO = Homo 015072 41 ² 1 sapiens NG = ADAMTS3 EP = 2VS = 4

A disintegrin and metalloproteinase with

thrombosponin motifs 2 NO = Homo 095450 34 ³ 3 sapiens NG = ADAMTS2 EP = 2VS = 2

Metalloproteinase inhibitor 1 NO = Homo

P01033 30 8 1 sapiens NG = TIMP1 EP = 1VS=1

Metalloproteinase Inhibitor 3 NO = Homo

P35625 55 5 1 sapiens NG = TIMP3 EP = 1VS = 2

Collagenase type IV 72 kDa NO = Homo

P08253 271 22 10 sapiens NG = MMP2 EP = 1VS = 2

Interstitial collagenase NO = Homo sapiens NG

P03956 48 3 1 = MMP1 EP = 1VS = 3

Protein 5 associated with cell remodeling

matrix NO = Homo sapiens NG = MXRA5 EP = Q9NR99 1.90 0.35 1

2VS = 3

Protein 7 associated with remodeling of P84157 1.82 5.39 1

matrix NO = Homo sapiens NG = MXRA7 EP =

1VS = 1

Hyaluronan-binding protein and inducer of

cell migration NO = Homo sapiens NG = Q8WUJ3 74 ^{4 4}

CEMIPEP = 1 VS = 2

Protein 14-3-3 epsilon NO = Homo sapiens

P62258 28.79 66.67 16 NG = YWHAE EP=1VS = 1

14-3-3 zeta/delta protein NO = Homo sapiens

P63104 21.58 57.96 12 NG = YWHAZ EP= 1 VS= 1

Protein 14-3-3 beta/alpha NO = Homo sapiens

P31946 13.40 60.16 8 NG = YWHAB EP=1VS = 3

Protein 14-3-3 theta NO = Homo sapiens NG

P27348 12.00 45.71 6 = YWHAQ EP = 1VS=1

Protein 14-3-3 gamma NO = Homo sapiens

P61981 9.92 49.80 5 NG = YWHAG EP=1VS = 2

Protein 14-3-3 eta NO = Homo sapiens NG =

Q04917 8.97 39.02 5 YWHAH EP = 1VS = 4

Protein 1 containing triple repeat

collagen helix NO = Homo sapiens NG = Q96CG8 36 3 1

CTHRC1 EP= 1 VS= 1

Dermcidin NO = Homo sapiens NG = DCD EP

P81605 3.33 20.00 2 = 1 V S = 2

Serine protease HTRA1 NO = Homo sapiens

Q92743 8.57 18.75 5 NG = HTRA1 EP= 1 VS= 1

ECM affiliated proteins

Growth factor binding protein 1

beta latent transformer NO = Homo sapiens Q14766 269 7 8

NG = LTBP1 EP = 1VS = 4

Prolargine NO = Homo sapiens NG = PRELP

P51888 204 23 7 EP = 1VS = 1

Microfibril-associated protein 2 NO = Homo

P55001 457 27 4 sapiens NG = MFAP2 EP = 2 VS=1

Microfibril-associated protein 5 NO = Homo

Q13361 157 18 3 sapiens NG = MFAP5 EP = 1 VS=1

Ig-h3 protein induced by Q15582 factor 47.51 48.90 24

beta transformant growth NO = Homo

sapiens NG = TGFBI EP = 1VS = 1

Cartilage-associated protein NO = Homo

075718 13.32 21.95 7 sapiens NG = CRTAP EP = 1 VS=1

Procollagen C-endopeptidase enhancer 1

NO = Homo sapiens NG = PCOLCE EP=1VS Q15113 6.33 17.82 5

= 2

Annexin A6 NO = Homo sapiens NG = ANXA6

P08133 71.69 63.00 35 EP = 1VS = 3

Annexin A2 NO = Homo sapiens NG = ANXA2

P07355 52.03 72.86 34 EP = 1VS = 2

Annexin A5 NO = Homo sapiens NG = ANXA5

P08758 47.48 70.00 27 EP = 1VS = 2

Annexin A1 NO = Homo sapiens NG = ANXA1

P04083 36.12 60.98 18 EP = 1VS = 2

Annexin A4 NO = Homo sapiens NG = ANXA4

P09525 22.43 47.65 12 EP = 1VS = 4

Annexin A7 NO = Homo sapiens NG = ANXA7

P20073 16.54 20.70 9 PE = 1VS = 3

Annexin A11 NO = Homo sapiens NG =

P50995 10.33 14.65 6 ANXA11EP=1VS=1

Galectin-1 NO = Homo sapiens NG = LGALS1

P09382 14.45 70.37 8 PE = 1VS = 2

Galectin-3 NO = Homo sapiens NG = LGALS3

P17931 1.88 14.00 3 PE = 1VS = 5

CYR61 protein NO = Homo sapiens NG =

000622 2.13 3.41 1 CYR61 EP = 1VS = 1

secreted factors

Myeloid-derived growth factor

NO = Homo sapiens NG = MYDGF EP = 1VS Q969H8 11.46 37.57 6

= 1

factor mRNA-binding protein 2

insulin-like growth 2 NO = Homo Q9Y6M1 6.52 10.85 5 sapiens NG = IGF2BP2 EP = 1VS = 2

Hepatoma-derived growth factor P51858 3.36 17.92 3

NO = Homo sapiens NG = HDGF EP = 1VS =

1

Connective tissue growth factor NO =

P29279 2.68 6.30 2 Homo sapiens NG = CTGF EP = 1VS = 2

Guanylate binding protein 1 NO = Homo

P32455 2.19 5.07 2 sapiens NG = GBP1 EP = 1VS = 2

Pentraxin-related protein PTX3 NO

P26022 5.69 13.39 ⁴ = Homo sapiens NG = PTX3 EP = 1VS = 3

Leukocyte elastase inhibitor NO = Homo

P30740 4.82 7.92 ³ sapiens NG = SERPINB1 EP = 1VS = 1

Astrocyte-derived neurotrophic factor

mesencephalic NO = Homo sapiens NG = P55145 3.19 21.43 ³ MANF EP = 1VS = 3

Spondin-1 NO = Homo sapiens NG = SPON1

Q9HCB6 30 1 1 PS = 1VS = 2

SRPX protein containing sushi repeats

NO = Homo sapiens NG = SRPX EP = 1VS = P78539 90 8 3

1

SRPX2 protein containing repeats

sushi NO = Homo sapiens NG = SRPX2 EP = 1 060687 23 3 1 VS = 1

Table 3. Detectable proteins in early chondrogenic mECM.

2.2. Cell differentiation

Figure 3 shows the successful differentiation of cell lines. In it you can see the results of different stains that serve to characterize the existing cell type.

In the first column, there is positive labeling of collagen by Syrian red staining in differentiating ^FB MSCs, with a pattern similar to that of undifferentiated MSCs and FBs. In the second column, toluidine blue staining images are shown, indicating the existence of proteoglycans, which generally exist in young cartilage or immature matrix, standing out in MSCs above the rest. Alcian blue is used for the labeling of glycosaminoglycans, which are expressed to a greater extent in CMM, CMM ^ FB and Facebook. In fat tissue, however, the stain used is oil red, a marker of triglycerides and lipoproteins that can be seen in the differentiation of MSC ^ A . And as an immunofluorescent marker, vimentin is more intensely expressed in FB and MSC ^ FB since it is a marker of cells of mesenchymal origin that are in epithelial-mesenchymal transition.

23. Decellularization of the matrix

Early chondrogenic matrix cell removal was performed to maximize removal of cellular material while minimizing ECM loss and damage. The treatment containing Triton X-100 and NH4OH successfully removed cell contents, as demonstrated by the absence of nuclei in phase contrast images and in H&E-stained sections of decellularized matrix (Figure 6A), as well as ~98% reduction. of the DNA content (Figure 6B).

In parallel, it was shown that the composition of the ECM was adequately preserved. Comparative information on protein content by mass spectrometry revealed similar patterns before and after decellularization treatment (Table 4). Similarly, a histological analysis using Masson's trichrome and toluidine O staining and quantitative biochemical assays for collagen and GAG, respectively, also showed non-significant differences in remnant content between mECM and dmECM (Figure 6C,D).

The MECds obtained from FB and MSC differentiated into adipocytes and FB were characterized histologically, showing strong staining for collagen, glycosaminoglycans (GAG) and proteoglycans (PG), as well as being correctly decellularized, since no notable cellular traces remained ( Figures 4 and 5). In the comparison of the native and decellularized dermis, a slight difference in the staining of collagens, GAG and PG can be observed, since the decellularization process had a minor impact on the tissue structure. Similarly, the comparison between decellularized matrices and native tissue shows that MECdm and MECdf were rich in components of their corresponding native tissues.

collagens

Collagen chain alpha-1 (I) NO = H om o sapiens NG = COL1A1 EP = 1

XX VS = 5

Collagen chain a alpha-2 (1) NO = H om o sapiens NG = C O L1A 2 EP = 1

X X VS = 7

Alpha-1 collagen chain (IV) NO = H om o sapiens NG = COL4A1 EP =

X X

1 V S = 3

Collagen chain alpha -2 (IV) NO = H om o sapiens NG = C O L4A 2 EP =

X X

1 V S = 4

Collagen chain alpha -3 (VI) NO = H om o sapiens NG = C O L6A 3 EP =

X X

1 V S = 5

Collagen chain alpha-1 (III) NO = H om o sapiens NG = COL3A1 EP =

X X

1 V S = 4

Collagen chain a alpha-2 (V) NO = H om o sapiens NG = C O L5A 2 EP =

X X

1 V S = 3

Alpha-1 collagen chain (V) NO = H om o sapiens NG = COL5A1 EP =

X X

1 V S = 3

Collagen chain alpha-1 (VI) NO = H om o sapiens IO = 9606 NG =

X X C O L6A1 EP = 1 V S = 3

Collagen chain alpha -2 (VI) NO = H om o sapiens IO = 9606 NG =

X X C O L6A 2 EP = 1 V S = 4

Collagen chain alpha-1 (V III) N O = H om o sapiens NG = COL8A1 EP

X X

= 1 V S = 2

Collagen chain alpha-1 (XII) NO = H om o sapiens IO = 9606 NG =

X X C O L12A1 EP = 1 V S = 2

Collagen chain alpha-1 (V il) NO = H om o sapiens NG = C O L7A1 EP =

X X

1 V S = 2

Collagen chain alpha-2 (V III) N O = H om o sapiens NG = C O L8A 2 EP

X

= 1 V S = 2

Alpha-1 collagen chain (XI) NO = H om o sapiens NG = COL11A1 EP

X X

= 1 V S = 4

Collagen chain alpha-1 (X IV ) NO = H om o sapiens NG = COL14A1

X X PE = 1 V S = 3

Collagen chain alpha-1 (XVI) NO = H om o sapiens NG = COL16A1

X X PE = 1 V S = 2

Collagen chain alpha-1 (XXI) NO = H om o sapiens NG = COL21A1

X PE = 2 V S = 1

glycoproteins

Filamin-A NO = Homo sapiens IO = 9606 NG = FLNA EP = 1VS = 4 X X EMILINA-1 NO = Homo sapiens IO = 9606 NG = EMILIN1 EP = 1VS =

X X

3

EMILINA-2 NO = Homo sapiens NG = EMILIN2 EP = 1VS = 3 X X ^{fibronectin NO} = ^{H om sapiens IO} = ^{9606 NG} = ^{FN1 EP = 1 V S = 4} X X ^{F ib rom odu lina NO} = ^{H om sapiens NG} = ^{FM OR D EP = 1 V S} = ² X X Cartilage oligomeric matrix protein NO = Homo sapiens NG

X X

= COMP EP = 1VS = 2

^{P ro tein 1 of the middle layer of cartilage NO} = ^{H om sapiens NG} =

X X ^{C ILP EP} = ^{1 VS} = ⁴

^{P ro tein 2 of the middle layer of cartilage NO} = ^{H om sapiens NG} =

X X ^{C ILP2 EP} = ^{2 VS} = ²

Chitinase-3 similara protein 1 NO = Homo sapiens NG = CHI3L1 EP

X X

= 1 VS = 2

^{periostin NO} = ^{H om sapiens NG} = ^{PO STN EP = 1 V S} = ² X X Thrombospondin-1 NO = Homo sapiens NG = THBS1 EP = 1VS = 2 X X Thrombospondin-2 NO = Homo sapiens NG = THBS2 EP = 1VS = 2 ^{X X} Tenascin NO ⁼ Homo sapiens IO ⁼ 9606 NG ⁼ TNC EP = 1VS = 3 ^{X X} Laminin beta-2 subunit NO ⁼ Homo sapiens NG ⁼ LAMB2 EP ⁼

X X

1VS = 2

Laminin alpha-4 subunit NO ⁼ Homo sapiens NG ⁼ LAMA4 EP ⁼

X X

1VS = 4

Lamin ina gamma-1 subunit NO = H om o sapiens NG = LAMC1 EP =

X X

1 V S = 3

SPARC NO ⁼ Homo sapiens NG ⁼ SPARC EP = 1VS = 1 ^XX Fibulin-like extra cellular matrix protein 1 containing EG F NO

X X

= H om o sapiens NG = EFEMP1 EP = 1 V S = 2

e xtra cellular matrix protein 2 fibulin-like containing EG F NO

X X

= H om o sapiens NG = EFE M P 2 EP = 1 V S = 3

V itronectin NO = H om o sapiens NG = VTN EP = 1 VS = 1 XX Lactadherin NO ⁼ Homo sapiens NG ⁼ MFGE8 EP ⁼ 1 VS = 2 ^XX P odocano NO = H om o sapiens NG = PO DN EP = 1 VS = 2 XXP ro te ina 3 sim ila rao lfac tom edina NO = H om o sapiens NG = O LFM L3

XX PE = 2 VS = 1

M em ber 2 of family 1D of secret toglobin NO = H om o sapiens NG =

X X S C G B 1D 2 PE = 2 V S = 1

S emforin -3C NO = H om o sapiens NG = SEMA 3C EP = 2 VS = 2 XX Thyroglobulin NO ⁼ Homo sapiens NG = TGEP = 1VS ⁼ 5 ^XX Proteoglycans

Proteoglycan core protein from heparan sulphate specific for

X X

basement membrane NO = H om o sapiens NG = H SP G 2 EP = 1 V S = 4

B ig lycan NO = H om o sapiens IO = 9606 NG = BG N EP = 1 V S = 2 X X Vers ican core protein NO = H om o sapiens IO = 9606 NG =

X X VC A N PE = 1 V S = 3

D ecorina NO = H om o sapiens NG = DON EP = 1 VS = 1 XX Lumicano NO ⁼ Homo sapiens NG ⁼ LUM EP ⁼ 1VS ⁼ 2 ^XX M im ecano NO = H om o sapiens NG = ONG EP = 1 VS = 1 XXR egu side res

Matrix metalloproteinase-14 NO ⁼ Homo sapiens NG ⁼ MMP14 EP

X X

⁼ 1VS ⁼ 3

A disintegration and metal proteinase due to thrombosponin 4

X X N O = H om o sapiens NG = A D A M T S 4 EP = 1 V S = 3

A disintegrin and metaloproteinase with motives of thrombosponin 3

X X N O = H om o sapiens NG = A D A M T S 3 EP = 2 V S = 4

A disintegration and metal proteinase with thrombosponin 2 motives

X X N O = H om o sapiens NG = A D A M T S 2 EP = 2 V S = 2

Metalloproteinase inhibitor 1 NO ⁼ Homo sapiens NG ⁼ TIMP1 EP

X X

⁼ 1VS ⁼ 1

Metalloproteinase inhibitor 3 NO ⁼ Homo sapiens NG ⁼ TIMP3 EP

X X

⁼ 1VS ⁼ 2

Collagenase type IV ⁷² kDa NO = Homo sapiens NG = MMP2 EP

X X

= 1 VS = 2

Interstitial collagenase NO = Homo sapiens NG = MMP1 EP = 1VS =

X X

3

^{P rotein 5 associated with remodeling of the matrix N O} = ^{H om sapiens NG}

X X

= ^{M X R A 5 PE = 2 V S = 3}

^{P rotein 7 associated with remodeling of the matrix N O} = ^{H om sapiens NG}

X

= ^{M X R A 7 EP = 1 V S = 1}

^{Protein that binds ah ia luronan and induces cell migration NO} = ^XX

^{H om sapiens NG} = ^{C E M IP EP = 1 V S} = ²

^{Protein 14-3-3 epsilon NO} = ^{H om sapiens NG} = ^{Y W H A E EP = 1 V S} =

X X

1

14-3-3 zeta/delta protein NO = Homo sapiens NG = YWHAZ EP = 1

X X

Protein 14-3-3 beta/alpha NO = Homo sapiens NG = YWHAB EP = 1

X X VS = 3

14-3-3 theta protein NO = Homo sapiens NG = YWHAQ EP = 1VS =

X X

1

14-3-3 gamma protein NO = Homo sapiens NG = YWHAG EP = 1VS

X X

= 2

14-3-3 eta protein NO = Homo sapiens NG = YWHAH EP = 1 VS = 4 X X ^{P ro tein 1 that contains the repeat of the triple helix of collagen N O} =

X X

^{H om sapiens NG} = ^{CTHRC1 EP = 1 V S} = ¹

Dermcidin NO = Homo sapiens NG = DCD EP = 1VS = 2 X X ^{Serine protease HTRA1 NO} = ^{H om sapiens NG} = ^{HTRA1 EP} = ^{1 VS} = ¹ X ECM affiliated proteins

dormant beta transforming growth factor binding protein 1

X X ^NO = ^{H om sapiens NG} = ^{LTBP1 PE} = ^{1 VS} = ⁴

^{P ro la rg ina NO} = ^{H om sapiens NG} = ^{PR E LP EP} = ^{1 VS} = ¹ X X ^{P rotein 2 associated with m icro fibrils NO} = ^{H om sapiens NG} = ^{M FAP2 PE}

X X

^{= 2 VS} = ¹

^{P rotein 5 associated with m icro fibrils NO} = ^{H om sapiens NG} = ^{M FAP5 EP}

X X

= ^{1 VS} = ¹

Ig-h3 protein induced by transforming growth fac tor beta NO

X X

= ^{H om sapiens NG} = ^{TG FBI EP = 1 V S = 1}

Cartilage-associated protein NO = Homo sapiens NG = CRTAP EP =

X X

1VS = 1

Procollagen C-endopeptidase enhancer 1 NO = Homo sapiens

XX NG = PCOLCE EP = 1VS = 2

Annexin A6 NO = Homo sapiens NG = ANXA6 EP = 1VS = 3 ^{X X} Annexin A2 NO = Homo sapiens NG = ANXA2 EP = 1VS = 2 ^{X X} Annexin A5 NO = Homo sapiens NG = ANXA5 EP = 1VS = 2 ^{X X} Annexin A1 NO = Homo sapiens NG = ANXA1 EP = 1VS = 2 ^{X X} Annexin A4 NO = Homo sapiens NG = ANXA4 EP = 1VS = 4 ^X

Annexin A7 NO ⁼ Homo sapiens NG ⁼ ANXA7 EP ⁼ 1VS = 3 ^X

Annexin A11 NO ⁼ Homo sapiens NG ⁼ ANXA11 EP = 1VS = 1 ^X

G alectin-1 NO = H om o sapiens NG = LGALS1 EP = 1 V S = 2 X X

G a lectin -3 NO = H om o sapiens NG = LG A LS 3 EP = 1 VS = 5 XX CYR61 protein NO ⁼ Homo sapiens NG ⁼ CYR61 EP = 1VS = 1 ^XX Secreted factors

Growth factor derived from myeloids N O = H om o sapiens NG =

X M Y D G F PE = 1 V S = 1

Insulin type 2 growth factor mRNA binding protein 2 NO

X X

= H om o sapiens NG = IG F2BP 2 EP = 1 V S = 2

Growth factor derived from hepatoma NO = H om o sapiens NG =

X H D G F PE = 1 V S = 1

Growth factor of connective tissue NO = H om o sapiens NG =

X C TG F EP = 1 V S = 2

Guanyla-binding protein 1 NO = H om o sapiens NG = GBP1 EP = 1

X VS = 2

Pentraxin-related protein PTX3 NO = H om o sapiens NG =

X X PTX3 EP = 1 V S = 3

Leukocyte elastase inhibitor NO ⁼ Homo sapiens NG ⁼

X SERPINB1 EP ⁼ 1VS ⁼ 1

Neurotrophic factor derived from mesencephalic astrocytes NO = H om o

X

sapiens NG = M AN F EP = 1 V S = 3

Spondin-1 NO = H om o sapiens NG = SPON1 EP = 1 V S = 2 X

P rotein S R P X containing repeats sushi i NO = H om o sapiens NG

X X

= S R P X EP = 1 V S = 1

SR P X2 protein containing sushi repeats NO = H om o sapiens NG

X X

= S R P X 2 PE = 1 V S = 1

Table 4. Detectable proteins in mECM before and after the decellularization process.

dECMs obtained from adipose tissue using the three decellularization protocols show strong staining for collagen, glycosaminoglycans (GAGs), and proteoglycans (PGs), as well as decellularizing correctly, with no noticeable cellular traces remaining (Figure 10). However, a greater presence of GAG can be observed in the MECd obtained by protocol 2, suggesting that this protocol preserves native tissue components better than the others.

2.4. MECdm Hydrogel Formulation

After decellularization, the early chondrogenic MECdm was lyophilized, ground to a fine white powder, and then solubilized with pepsin to liquefy to final concentrations of 3 and 6% w/v (Figure 7A). The resulting homogeneous acidic MECdm solutions were adjusted to physiological pH using a basic solution (NaOH) before encapsulating the cells. Neutralized MECdm solutions appeared viscous-like in appearance with high opacity that increased with concentration. These solutions were heat sensitive; behaving as a viscous liquid at low temperatures (<15 °C) and transforming into a gel when incubated for 20 minutes at 37 °C.

2.5. Hydrogel characterization of MECdm

The present inventors used SEM to examine the ultrastructure of the MECdm hydrogel at 37°C. The micrographs showed that the generated gel had a 3D sponge-like appearance with a randomly oriented nanofibrillar structure, typical of decellularized ECM-based gels (Figure 7B). The 3% and 6% MECdm hydrogels appeared visually similar to a homogeneous microporous system, although fibrillary density and interconnectivity were greater with increasing concentration (Figure 7E). Pores 2.72 ± 1.54 pm in diameter were formed within the 3% MECdm and 1.35 ± 0.77 pm in the 6% MECdm.

The hydrogel injectability and gelling abilities of 3% and 6% MECdm were tested using steady shear, batch flow, and dynamic oscillation experiments. Figure 7C shows the viscosity curves for the MECdm hydrogels at 15°C. As observed, the hydrogels exhibit a very strong shear thinning behavior. This is evidenced by the decrease in viscosity with shear rate, with values ranging from 39.65 Pas and 40.1 Pas at a shear rate of 0.01 s'1 to 0.0057 Pa s and 0.0113 Pa s at a shear rate of 1000 s_1 for MECdm at 3 and 6%, respectively. As expected, the higher the concentration of the matrix, the higher the viscosity. It is also important to note that the curves overlap when performing up and down shear rate sweeps. This indicates that the sample is thixotropic.

Figure 7D shows the viscosity transients with abrupt changes in shear rate at 15 °C. The results show that the transients are extremely short and the samples respond very quickly (relaxation times <1 s) to excitation; to both large and small shear rates. This is consistent with the fact that the rising and falling steady shear curves overlap in Figure 7C.

Heat driven gelation of the suspensions is shown in Figure 7E. This Figure represents the linear viscoelastic moduli (storage modulus G' and loss modulus G") as a function of temperature over the range from 20 °C to 40 °C. Regardless of concentration and temperature, the modulus of storage remains always higher than the loss modulus, which evidences the existence of a gel-like structure in the investigated temperature range.However, at a particular temperature of about 35 °C, the viscoelastic moduli increase further due to self-assembly of the main components of the matrix, such as collagen fibers or fibronectin, among others.

Figure 7F shows the loss factor (G"/G') of the hydrogels as a function of temperature. The loss factor is a measure of the relative importance of viscous dissipation on the elastic recovery of the samples. Regardless of concentration, the loss factor is always less than one, as expected from gel formation (G1 > G"). The loss factor drops drastically for temperatures above 35°C. It should be noted that, contrary to what occurs with viscoelastic moduli, the loss factor is independent of concentration.

2.6. MECdm Hydrogel Biocompatibility

Cellular viability and proliferation of encapsulated MSCs within 3 and 6% MECdm hydrogels, respectively, were evaluated for 28 days of culture. Cell survival was assayed using Live/Dead staining using confocal microscopy. The images showed that the cells were distributed homogeneously throughout the hydrogel and maintained the rounded morphology in both concentrations of MECdm and throughout the culture period (Figure 8A). The results showed that more than 90% of the MSCs embedded within the 3% and 6% MECdm hydrogels were viable (day 1), with no notable differences between them. This high viability was maintained over time in the 6% MECdm hydrogel, meanwhile, in the 3% MECdm a lower number of living cells was observed at the end of the culture period, although this value (83.3 %) was also indicative of high cell viability (Figure 8B).

On the other hand, the assay with alamar blue revealed a tendency to maintain cell proliferation throughout the culture time at both concentrations. In 3% dmECM this trend occurred from the beginning, showing a slight reduction in proliferation. cells between day 5 and day 7. In turn, the rate of proliferation in the 6% MECdm was maintained after three days of culture and decreased until day 14 to a level similar to that of the 3% MECdm ( Figure 8C). Taken together, these results indicate that the novel MECdm-based biomimetic hydrogel provided a favorable environment for cells, supporting high cell viability at different concentrations of MECdm.

2.7. Chondrogenic differentiation of MSC-loaded MECdm biomimetic hydrogel

The chondroinductivity of the MECdm hydrogel was investigated by analyzing the gene expression of the embedded MSCs for 1 month in culture without any inducing factors in the medium (Figure 8D). The qPCR results showed a constant increase in the expression of chondrogenic markers and a decrease in non-chondrogenic markers over time. High levels of the COL2A1 and ACAN genes were observed on day 14 in MSCs embedded in 3% and 6% MECdm hydrogels, with no significant differences between concentrations, compared to these cells on day 0 of culture. On day 28, the increased gene expression of these chondrogenic markers was even more evident. Elevated chondrogenic-inducing factor SOX9 and COL2A1 values were similar to or even higher than positive control chondrocytes cultured for 2 weeks in vitro, respectively. However, ACAN expression in chondrocytes was significantly higher than that of MSCs in MECdm hydrogels at day 28. Although this upregulation appears to be in a concentration-dependent manner, differences in MECdm expression levels between 3 % and 6 % were not statistically significant. A decrease of the fibrotic marker gene COL1A1 was also observed from day 14, and much more significant after 28 days in both hydrogels compared to day 0, while the expression of the hypertrophic marker gene COL10A1 was undetectable from the beginning.

Furthermore, to confirm the gene expression results, the present inventors assessed chondrogenesis at the protein level using immunofluorescence techniques (Figure 8E). Immunofluorescence staining revealed the presence of type II collagen and type I collagen after 14 days of culture. In accordance with the qPCR results described above, a good evolution of the hyaline cartilage matrix was observed over time, with more positive staining for type II collagen and less for type I collagen on day 28, with no differences. appreciable between concentrations. Taken together, these results indicated that the MECdm environment promoted chondrogenic engagement of MSCs and tissue-specific formation regardless of concentration.

2.8. In vivo biocompatibility and development of cartilage-like tissue from MECdmc hydrogel

To assess whether the MECdmc would have biocompatibility and good integration in vivo, hydrogels at both concentrations were implanted subcutaneously in dorsal regions of immunocompetent CD1 mice over 4 weeks. Figure 9A shows good integration of acellular MECdmc hydrogels without the appearance of redness or swelling. DAPI staining also revealed surrounding cell infiltration (Figure 9B). The increase in the number of nuclei over time indicated that the cells were capable of adhering to, penetrating, and growing in the 3D structure of the hydrogels.

Tissue formation in immunodeficient NOD SCID gamma mice was determined by histological analysis of MECdm hydrogels loaded with 3% and 16% MSCs compared to MSCs predifferentiated towards the micropellet chondrogenic lineage (Figure 9C). H&E staining showed that cells in MECdmc hydrogel acquired the typical lacunae-embedded chondrocyte shape after 2 weeks in vivo, being more evident at the highest concentration. Using toluidine O staining, an increase in GAG deposition was evidenced, showing intense staining in 3% and 6% MECdm gels implanted at four weeks, which was higher than that observed in the control pellet.

By immunostaining, high levels of type II collagen and low levels of type I collagen were determined at both concentrations of MECdmc over time. In both histological and immunostaining assays, staining levels were higher in the MECdmc gels than in the control pellet. Alizarin red/light green staining also did not reveal the presence of mineralization in any condition. These results indicate the formation of a mature cartilage matrix in MECdm hydrogels, especially in 6% MECdmc, whose matrix was more similar to cartilage tissue, and confirm the ability of MECdmc hydrogels to induce cartilage-like tissue in vivo.

2.9 Formulations of bioinks based on MECd

After decellularization, the adipose tissue-derived ECMd was lyophilized, ground to a fine white powder, and then solubilized to liquefy to a final concentration of 10% w/v. The resulting homogeneous acidic MECd solutions were adjusted to physiological pH using a basic solution (NaOH). The neutralized MECd solution was mixed with different components to evaluate its long-term stability. term, injectability and bioprintability: porcine skin gelatin, human or bovine plasma fibrinogen, alginate, gelatin methacryloyl and intra-articular sodium hyaluronate (Figure 11). The hydrogels were stable for two weeks at 37 °C in a humidified atmosphere with 5% CO ² .

Claims (29)

1. A biomimetic hydrogel comprising a decellularized extracellular matrix derived from differentiated mesenchymal stem cells (MECdmd). The biomimetic hydrogel according to claim 1, wherein the mesenchymal stem cells are mammalian mesenchymal stem cells, preferably human mesenchymal stem cells. The biomimetic hydrogel according to claims 1 or 2, wherein the mesenchymal stem cells are isolated from adipose tissue. The biomimetic hydrogel according to any of claims 1 to 3, wherein the differentiated mesenchymal stem cells are selected from the list consisting of: early chondrogenic mesenchymal stem cells, adipocytes, fibroblasts, and osteoblasts. 5. The biomimetic hydrogel according to any of claims 1 to 4 comprising a percentage of MECdmd between 11 and 10% w/v. 6. The biomimetic hydrogel according to claim 5, wherein the percentage of MECdmd is 3 or 16% w/v. A composition comprising the biomimetic hydrogel according to any of claims 1 to 6. The composition according to claim 7, further comprising embryonic stem cells and/or non-embryonic or adult stem cells, preferably mesenchymal stem cells. 9. An implant comprising the hydrogel according to any of claims 1 to 6 or the composition according to claim 7 or 8. A method for producing the biomimetic hydrogel according to any of claims 1 to 6 comprising: (a) culturing mesenchymal stem cells from a subject with at least one compound that induces differentiation, (b) decellularize the culture from step (a), obtaining an extracellular matrix decellularized derived from differentiated mesenchymal stem cells (MECdmd), (c) lyophilizing the decellularized extracellular matrix obtained in step (b), (d) solubilizing the lyophilized decellularized extracellular matrix obtained in step (c), and (e) gel the solution obtained in step (d) for 15 to 30 min at 30 to 40 °C. 11. The method according to claim 10, further comprising an additional step (c1) of grinding into powder the lyophilized decellularized extracellular matrix obtained in step (c). The method according to claims 10 or 11, wherein the mesenchymal stem cells are mammalian, preferably human. The method according to claims 10 or 12, wherein the mesenchymal stem cells are isolated from adipose tissue. The method according to any of claims 10 to 13, wherein step (a) is carried out for at least one week, preferably at least two weeks. 15. The method according to any of claims 10 to 14, wherein the compound that induces mesenchymal stem cell differentiation is selected from the list consisting of: dexamethasone, TGF-3p. The method according to claim 15, wherein the concentration of dexamethasone is 100 nM and/or the concentration of TGF-3P is 10 pg/ml. The method according to any of claims 10 to 16, wherein the decellularization treatment of step (b) is performed with a detergent solution, preferably followed by nuclease treatment. 18. The method according to claim 17, wherein the detergent solution is Triton X-100 and/or NH4OH and the nuclease treatment is deoxyribonuclease and/or ribonuclease. The method according to any of the claims 10 to 18, wherein the grinding treatment of step (d) is performed by liquid nitrogen. 20. The method according to any of claims 10 to 19, wherein the solubilization of step (e) is performed by enzymatic digestion, preferably with pepsin. The method according to any of claims 10 to 19, wherein the solubilization of step (e) is performed by treatment with urea. 22. The method according to any of claims 10 to 21, wherein the percentage of MECdmd is between 1 and 10% w/v. 23. The method according to claim 22, wherein the percentage of MECdmd is 3 or 16% w/v. The biomimetic hydrogel according to any of claims 1 to 6 or the composition according to claim 7 or 8 or the implant according to claim 9 for use as a medicament. The biomimetic hydrogel according to any of claims 1 to 6 or the composition according to claim 7 or 8 or the implant according to claim 9 for use in the treatment of degenerative diseases. The biomimetic hydrogel or composition for use according to claim 25, wherein the degenerative disease is selected from the list consisting of: osteoarthritis, inflammatory arthritis, and crystal-induced arthritis. 27. Use of the implant according to claim 9 for the regeneration of tissue or organs. 28. Use according to claim 27, wherein the tissue is skin, bone, heart tissue, cartilage tissue, vascular tissue. 29. Use of the biomimetic hydrogel according to any of claims 1 to 6 or the composition according to claim 7 or 8 for 3D bioprinting.