WO2022063679A1

WO2022063679A1 - Biomaterial for use in the treatment of diseases involving tissue repair or regeneration

Info

Publication number: WO2022063679A1
Application number: PCT/EP2021/075512
Authority: WO
Inventors: Juan Antonio Marchal Corrales; Carlos CHOCARRO WRONA; Elena LÓPEZ RUIZ; Juan DE VICENTE ÁLVAREZ-MANZANEDA; Cristina ANTICH ACEDO; Daniel MARTÍNEZ MORENO; Gema JIMÉNEZ GONZÁLEZ; Macarena PERÁN QUESADA
Original assignee: Universidad De Granada
Priority date: 2020-09-16
Filing date: 2021-09-16
Publication date: 2022-03-31

Abstract

The present invention refers to the use of 1,4-Butanediol thermoplastic polyurethane elastomer (b-TPUe) for the preparation of a biomaterial structure that serves as a substrate or guide for tissue repair and regeneration. Thus, the biomaterial can be used in the treatment of diseases involving tissue repair or degeneration.

Description

BIOMATERIAL FOR USE IN THE TREATMENT OF DISEASES INVOLVING TISSUE REPAIR OR REGENERATION

FIELD OF THE INVENTION

The present invention refers to the medical filed. Particularly, the present invention refers to the use of an elastomer comprising methylene diphenyl diisocyanate [MDI] and 1,4- butanediol, preferably to the use of 1,4-Butanediol thermoplastic polyurethane elastomer (b- TPUe) (which comprises methylene diphenyl diisocyanate [MDI] and 1,4-butanediol), for the preparation of a biomaterial structure that serves as a substrate or guide for tissue repair or regeneration. Thus, the biomaterial can be used in the treatment of diseases involving tissue repair or degeneration.

PRIOR ART

Tissue engineering is the use of a combination of cells, engineering, and materials methods, and suitable biochemical and physicochemical factors to improve or replace biological tissues. Tissue engineering involves the use of a tissue scaffold for the formation of new viable tissue for a medical purpose.

Tissue engineering is closely associated with applications that repair or replace portions of or whole tissues (i.e., bone, cartilage, blood vessels, bladder, skin, muscle etc.). Often, the tissues involved require certain mechanical and structural properties for proper functioning. The term has also been applied to efforts to perform specific biochemical functions using cells within an artificially-created support system (e.g. an artificial pancreas, or a bio artificial liver).

Tissue scaffolds are materials that have been engineered to cause desirable cellular interactions to contribute to the formation of new functional tissues for medical purposes. Cells are often 'seeded' into these structures capable of supporting three-dimensional tissue formation. Scaffolds mimic the extracellular matrix of the native tissue, recapitulating the in vivo milieu and allowing cells to influence their own microenvironments. They usually serve at least one of the following purposes: allow cell attachment and migration, deliver and retain cells and biochemical factors, enable diffusion of vital cell nutrients and expressed products, exert certain mechanical and biological influences to modify the behaviour of the cell phase.

Several synthetic materials such as poly-L-lactic acid (PLA), poly-e-caprolactone (PCL) or polylactic-co-glycolic acid (PLGA) have been used to generate bioprinted scaffolds for tissue engineering applications. However, these materials lack the appropriate elasticity to mimic native tissues. The stiffness of porous scaffolds produced using rigid biomaterials, such as PLA, are in the MPa magnitude order comparable to those found in hard tissues such as porous bone. Therefore, their restricted flexibility makes them not appropriate for engineering flexible tissues that suffer mechanical loading such as ligaments, tendons, cartilage, blood vessels, skin, or muscles.

Thus, the inventors of the present invention have identified that there is an unmet medical need of finding new and effective strategies for generating biomaterial, for instance scaffolds, for tissue engineering applications. The present invention is focused on solving this problem by providing a specific biomaterial wherein the cells, particularly chondrocytes and skin cells, are particularly well adhered and grown, causing desirable cellular interactions, thus contributing to the formation of new functional tissues for medical purposes.

To our knowledge, this is the first time that an elastomer comprising methylene diphenyl diisocyanate [MDI] and 1,4-butanediol is used, precisely, for the generation of biomaterials for tissue engineering applications, thus serving as a substrate or guide for tissue repair or regeneration.

DESCRIPTION OF THE INVENTION

Brief description of the invention

The present invention refers to a biomaterial structure that serves as a substrate or guide for tissue repair or regeneration. Thus, the biomaterial can be used in the treatment of diseases involving tissue repair or degeneration. The elastic 3D printing biomaterial structure comprises an elastomer which in turn comprises or consists of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol. Preferably, the elastic 3D printing biomaterial consists essentially of b-TPUe, comprising or consisting of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol, and shows a combination of mechanical properties which are optimal for tissue engineering purposes.

Particularly, the first embodiment of the present invention refers to the use of an elastomer comprising or consisting of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol, for the preparation of a biomaterial structure that serves as a substrate or guide for tissue repair or regeneration. In a preferred embodiment, the present invention refers to the use of 1,4-butanediol thermoplastic polyurethane elastomer (b-TPUe), comprising or consisting of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol, for the preparation of a biomaterial structure that serves as a substrate or guide for tissue repair or regeneration.

In a preferred embodiment, the biomaterial structure thus generated is a tissue scaffold, a tissue implant, a stent or a valve.

In a preferred embodiment, the elastomer is in the form of filament, powder, electro-spun mesh, sponge or pellets.

The second embodiment of the present invention refers to a biomaterial structure consisting essentially of an elastomer comprising or consisting of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

In a preferred embodiment, the biomaterial structure consists essentially of 1,4-butanediol thermoplastic polyurethane elastomer (b-TPUe) which comprises or consists of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

In a preferred embodiment, the biomaterial structure is characterized in that it is a tissue scaffold, a tissue implant, a stent or a valve.

The third embodiment of the present invention refers to a tissue scaffold consisting essentially of an elastomer comprising or consisting of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

In a preferred embodiment, the tissue scaffold consists essentially of 1,4-Butanediol thermoplastic polyurethane elastomer (b-TPUe) comprising or consisting of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

The fourth embodiment of the present invention refers to a composition comprising a population of cells, or the secretome secreted from said cells, within the above defied tissue scaffold.

In a preferred embodiment, the cells are mature cells or mesenchymal/pluripotent/embryonic stem cells.

In a preferred embodiment, the cells are selected from the list comprising: chondrocytes, skin cells, endothelial cells, smooth muscle cells, osteoblasts or myocytes. The fifth embodiment of the present invention refers to a pharmaceutical composition comprising the above defined composition and, optionally, pharmaceutically acceptable excipients or carriers.

In a preferred embodiment, the pharmaceutical composition is for use in the treatment of diseases involving tissue degeneration, preferably in the treatment of diseases involving cartilage, vascular, skeletal muscle or skin degeneration, most preferably in the treatment of chondromalacia.

Particularly, the sixth embodiment of the present invention refers to the use of b-TPUe for the preparation of a biomaterial structure that serves as a substrate or guide for tissue repair or regeneration, particularly for the formation of functional tissues. In a preferred embodiment, the biomaterial structure thus generated is a tissue scaffold, a tissue implant, a stent or a valve. In a preferred embodiment, the b-TPUe is in any form suitable for 3D-bioprinting, for instance: filament, powder, electro-spun mesh, sponge or pellets.

In a preferred embodiment, the present invention also refers to the use of a thermoplastic polyurethane structure comprising Methylene diphenyl diisocyanate (MDI) and 1,4- Butanediol for the preparation of the biomaterial structure. In other words, any of the components of the thermoplastic polyurethane structure (preferably MDI or 1,4-Butanediol) can be used, according to the present invention, for the preparation of the biomaterial structure. In a particularly preferred embodiment, the above cited thermoplastic polyurethane structure also comprises the following additives: 2,6-Di-tert-butyl-4-methylphenol, Pentaerythritol tetrakis(3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate) and/or crodamines.

The seventh embodiment of the present invention refers to a biomaterial structure, for instance a tissue scaffold, a tissue implant, a stent or a valve, consisting essentially of b- TPUe. In a particularly preferred embodiment, the present invention refers to a tissue scaffold consisting essentially of b-TPUe.

The eight embodiment of the present invention refers to a composition comprising a population of cells, or the secretome secreted from said cells, within the tissue scaffold of the invention. In a preferred embodiment, the cells are mature cells or mesenchymal/pluripotent/embryonic stem cells. In a preferred embodiment, the cells are selected from the list comprising: chondrocytes, skin cells, endothelial cells, smooth muscle cells, osteoblasts or myocytes. The ninth embodiment of the present invention refers to a pharmaceutical composition comprising a population of cells, or the secretome secreted from said cells, within the tissue scaffold of the invention and, optionally, pharmaceutically acceptable excipients or carriers.

The tenth embodiment of the present invention refers to the pharmaceutical composition of the invention for use in the treatment of diseases involving tissue degeneration. In a preferred embodiment, the pharmaceutical composition of the invention is used in the treatment of diseases involving cartilage (for example chondromalacia), vascular, skeletal muscle or skin degeneration. Alternatively, this embodiment of the present invention refers to a method for treating diseases involving tissue degeneration, preferably diseases involving cartilage (for example chondromalacia), vascular, skeletal muscle or skin degeneration, which comprises administering to the patient a pharmaceutically effective amount of the composition or pharmaceutical composition of the invention.

The eleventh embodiment of the present invention refers to a method for improving the growth and differentiation of cells, said method comprising culturing the cells within the tissue scaffold of the invention.

Thus, the inventors of the present invention propose b-TPUe as a novel 3D bioprinting material for tissue engineering, preferably cartilage and skin tissue engineering. The mechanical behaviour of b-TPUe in terms of friction and elasticity were examined and compared with human articular cartilage and with two different 3D bioprinting polymer materials, poly-e-caprolactone (PCL) and poly-L-lactic acid (PLA). Moreover, adipose tissue-derived human mesenchymal stem cells (MSCs) isolated from infrapatellar fat pad of osteoarthritic (OA) patients were bioprinted together with b-TPUe scaffolds and cytotoxicity, proliferative potential, cell viability and chondrogenic differentiation were analysed by Alamar blue assay, SEM, confocal microscopy, and RT-qPCR, respectively. b-TPUe demonstrated a much closer compression and shear behaviour to native cartilage than PCL and PLA, as well as better tribological properties. Moreover, b-TPUe bioprinted scaffolds were able to maintain proper MSCs proliferative potential, cell viability, and support MSCs chondrogenesis. Finally, in vivo studies in mice revealed no toxic effects and the maintenance of chondrogenic phenotype 21 days after scaffolds implantation.

The present invention shows the use of b-TPUe as a new 3D bioprinting material for biomedical applications. A rheological characterization was carried out to analyse their mechanical properties (in shear and compression) and a tribological study to evaluate the frictional behaviour in synovial fluid-lubricated b-TPUe-cartilage tribopairs. Moreover, in vitro and in vivo the biocompatibility of b-TPUe 3D bioprinted scaffolds was compared versus PCL and showed the potential application of this biomaterial to produce engineered tissues. Finally, chondrogenic differentiation of MSCs isolated from infrapatellar fat pad was achieved when cultured in 3D bioprinted b-TPUe scaffolds.

In summary, the use of b-TPUe is herein proposed for the automated biofabrication of artificial tissues with tailorable mechanical properties including the great potential for cartilage tissue engineering applications. b-TPUe, was successfully used to fabricate 3D bioprinted scaffolds with improved rheological and tribological properties compared to PCL and PLA. This new printing material can support the growth and chondrogenic differentiation of MSCs. We demonstrated that the mechanical behavior of b-TPUe was more similar to natural cartilage when compared with PCL and PLA. Interestingly, the elastic characteristics of b-TPUe can be tailored by changing the porosity to better mimic natural tissues, and therefore improving the mechanical properties of the constructs. A better tribological performance is exhibited in b-TPUe tribopairs if compared to PLA and PCL. A significantly lower friction was measured for b-TPUe than PLA and PCL, which suggests that b-TPUe is a more appropriate material to be used in cartilage repair to restore joint function than PLA and PCL. Further b-TPUe demonstrated high biocompatibility when MSCs were gown onto b- TPUe scaffolds. In fact, b-TPUe bioprinted scaffolds were found to support MSCs proliferation and the development of a cartilage-like tissue, without in vivo toxic effects. These results support, for the first time, the potential of b-TPUe as a 3D bioprinting material with application in cartilage tissue engineering. In addition, the development of materials such as b-TPUe that allow tailoring the properties of artificial tissues is essential for tissue engineering. Moreover, due to the excellent biomechanical properties and biocompatibility, we have set the basis for further exploration of this novel material for biomedical and tissue regenerative applications.

For the purpose of the present invention the following terms are defined:

• The term "comprising" it is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. • By "consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase "consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

• By “consisting essentially of’ it is meant that further components can be present in the composition, namely those not materially affecting the essential characteristics of the texturizing agent

• “Pharmaceutically acceptable excipient or carrier” refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

• The term “disease involving cartilage degeneration” is any disease or disorder involving cartilage and/or joint degeneration. The term “disease involving cartilage degeneration” includes disorders, syndromes, diseases, and injuries that affect spinal discs or joints (e.g., articular joints) in animals, including humans, and includes, but is not limited to, arthritis, chondr ophasi a, spondyloarthropathy, ankylosing spondylitis, lupus erythematosus, relapsing polychondritis, and Sjogren's syndrome.

• By “therapeutically effective dose or amount” of a composition is intended an amount that, when administered as described herein, brings about a positive therapeutic response in a subject having tissue damage or loss, such as an amount that results in the generation of new tissue at a treatment site. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

• “b-TPUe” refers to this material (as it is), or to any other material or structure comprising thereof, excluding those components which negatively affects cell viability. b-TPUe comprises or consists of methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

• “Biomaterial structure”, according to the present invention, refers to any biocompatible material which serves as a substrate or guide for tissue repair or regeneration, for instance tissue scaffolds, a tissue implants, stents or a valves. Brief description of the figures

Figure 1. b-TPUe scaffolds design. (A) CAD model of the scaffold design. (B) 3D printed b-TPUe scaffold (10 mm in diameter and 3 mm in height). (C, D) SEM images of the top surface of b-TPUe 3D printed scaffolds (scale bars: 1 mm and 300 pm respectively). (E-G) Macroscopic view of b-TPUe, PCL and PLA scaffolds, respectively. (H-J) Scaffold fiber width of b-TPUe, PCL and PLA scaffolds, respectively.

Figure 2. Tribological and rheological characterization. (A) Schematic diagram of the tribological set-up. (B) Frictional behavior of PLA (black), PCL (red) and b-TPUe (green).

(C) Compression curves corresponding to the studied samples (s: solid; p: porous). (D) Linear viscoelastic moduli (G’ and G”) for the materials studied (** = p < 0.01). Graphs created using the Origin 9.0 software.

Figure 3. In vitro biocompatibility of b-TPUe bioprinted scaffolds with MSCs. (A) Proliferative potential of MSCs cultured with control or b-TPUe-conditioned medium up to 7 days (** = p < 0.01). (B) MSCs proliferation cultured in both b-TPUe and PCL bioprinted scaffolds up to 21 days with no significant differences between PCL and b-TPUe (no significance: ns). Significant cell growth was observed in both materials at day 7 of culture in both materials (** = p < 0.01) (RFU: Relative fluorescence units). (C) Representative confocal images of MSCs grown in both b-TPUe and PCL bioprinted scaffolds at day 7 and 21. Live/dead assay was employed, using calcein (green) and ethidium homodimer (red), live cells were stained green while dead cells were stained red. Scale bars: 500 pm. Graphs created using the GraphPad Prism 6.01 software.

Figure 4. MSCs chondrogenic differentiation in b-TPUe bioprinted scaffolds. Chondrogenic differentiation was evaluated in MSCs cultured in monolayer, b-TPUe scaffolds (CTL), and b-TPUe scaffolds under differentiation conditions (Diff). (A) RT-PCR analysis of chondrogenic key markers. (B) GAGs quantification. (C) Type II collagen quantification. (D-F) SEM representative images of MSCs growing in b-TPUe bioprinted scaffolds at day 21 (** = p < 0.01). (G-I) SEM representative images of MSCs growing in b- TPUe bioprinted scaffolds under chondrogenic differentiation conditions. Scale bars: 500 pm

(D), 50 pm (E), 10 pm (F), 500 pm (G), 200 pm (H), 100 pm (I). Graphs created using the GraphPad Prism 6.01 software. SEM images false-coloured using the cross-platform image editor GIMP (version 2.10.14). Figure 5. In vivo biocompatibility of b-TPUe. (A) Macroscopic image of the locations of implanted b-TPUe scaffolds in CD1 mice. Scaffolds were implanted in the dorsal region of 8 weeks old CD1 mice and resected 21 days after surgery procedure. (B) Images of b-TPUe scaffolds recovered from CD1 mice. (C) Macroscopic image of the locations of implanted PCL scaffolds in CD1 mice. (D) Images of PCL scaffolds implanted in the dorsal region of CD1 mice.

Figure 6. In vivo biocompatibility of b-TPUe bioprinted scaffolds with MSCs. (A) Macroscopic images for cell-free and cell-laden b-TPUe and PCL scaffolds fabricated by 3D bioprinting. Scaffolds were implanted in the dorsal region of 8 weeks old female NSG mice and resected 21 days after surgery procedure. (B) Histologic analysis of Toluidine blue and Masson's Trichrome staining of cell-free and cell-laden b-TPUe and PCL scaffolds 3 weeks post-implantation. Scale bars: 800 pm for black-labeled images, and 400 pm for red-labeled images.

Detailed description of the invention

Example 1. Material and Methods

Example 1.1. Patients

Human infrapatellar fat pad, cartilage tissue and synovial fluid were obtained from patients with knee OA during joint replacement surgery. Ethical approval for the study was obtained from the Ethics Committee of the Clinical University Hospital of Malaga, Spain. Informed patient consent was obtained for all samples used in this study. None of the patients had a history of inflammatory arthritis or crystal -induced arthritis. Hoffa’s fat pad was harvested from the inside of the capsule excluding vascular areas and synovial regions. Human articular cartilage was obtained from the femoral side, selecting the non-overload compartment. Only cartilage that looked normal macroscopically was used for this study. Samples collected at joint arthroplasty were transported to the laboratory in Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) with 1% penicillin/streptomycin (P/S). Synovial fluid (SF) was pooled from knee joints and mixed on an orbital shaker. Only samples that were free of blood contamination were used, as assessed visually. SF was stored at -20°C between testing sessions.

Example 1.2. Isolation and culture of human MSCs from infrapatellar fat pad

Infrapatellar fat pad tissue was minced and digested with an enzymatic solution of 1 mg/mL collagenase type IA (Sigma) and incubated in shaking at 37°C for 1 h. Once digested, collagenase was removed with a single wash of sterilized phosphate-buffered saline (PBS), followed by two washes of DMEM supplemented with 10% fetal bovine serum (FBS) (Sigma). The cell pellet was resuspended in DMEM supplemented with 10% FBS and 1% P/S, placed into tissue culture flasks, and cultured at 37 °C in a 5% CO2 atmosphere. After 48 h the medium was removed to discard non-adherent and dead cells. When 80% of confluence was reached, cells were released with Tryple Express (Gibco) and sub-cultured.

Example 1.3. Bioprinting process

A Regemat 3D VI bioprinter (REGEMAT 3D S.L., Spain) was used for bioprinting with a direct extruder to print 3D scaffolds. In order to 3D print the scaffolds, commercial nonmedical grade PCL (3D4Makers, 1.75 mm filament) was melted at 75 °C and printed at rate of 1.1 mm/s, commercial non-medical grade PLA (Smart materials 3D, Spain, 1.75 mm filament) was melted at 200 °C and printed at rate of 1.2 mm/s, and b-TPUe (Recreus industries s.l., 1.75 mm filament) was melted at 200 °C and printed at rate of 1.4 mm/s. PCL, PLA and b-TPUe scaffolds were designed to be extruded with triangular patterns for the infill with a pore size of 0.6 mm, solid walls consisting of a perimeter of 0.4 mm width, and 3 solid layers for the bottom, with a 0.2 mm layer height (Figure 1A). The scaffolds were printed as 3D cylindrical frameworks in a triangular inner lattice from alternately stacking filament fibers (Figure IB).

For 3D bioprinting using b-TPUe and PCL filaments, the thermal extruder unit of the bioprinter was used to print the scaffolds, and then the syringe unit of the bioprinter was used to seed the cells into the porous structure with a 200 pm diameter needle (1 x 10⁵ cells/scaffold). For all in vitro assays the scaffold dimensions were designed to fit in a 24- well plate (10 mm in diameter and 3 mm in height), with smaller dimensions for the in vivo assays (5 mm in diameter and 3 mm in height). Once bioprinted, the scaffolds were introduced in a 24-well plate and incubated for at least 1 h to allow the cells to adhere to the fibers. Finally, the scaffolds were submerged in culture medium containing DMEM supplemented with 10% FBS and 1% P/S and, then, stored at 37 °C in a 5% CO2 atmosphere. Scaffolds used to support MSCs chondrogenic differentiation were cultured in DMEM supplemented with 10% FBS, 1% P/S, 50 pg/pL 1-ascorbic acid 2-phosphate (Sigma), 40 pg/mL proline (Sigma), 1% insulin-transferrin-selenium (ITS) (Gibco), 40 pg/pL 1-proline (Sigma), and 10 ng/mL transforming growth factor P3 (TGF-P3).

Example 1.4. Tribological tests

A ball-on-three plates tribometer was adapted to a rheometer (Anton Paar, Austria) to interrogate the lubricating behavior of the different materials. The contact consisted in a plastic ball (made of PLA, PCL or b-TPUe) that slides along three cartilage surfaces (cartilage disks with a diameter of 5 mm) lubricated by synovial fluid. The MCR501 rheometer head (Anton Paar) was used to calculate the friction coefficient. A schematic diagram of the test set-up is shown in Figure 2A. In this set-up, a ball is pressed at a given normal force FN against three plates that are mounted on a movable stage.

The experimental protocol was as follows. First, the test rig was assembled, and 400 pL of SF was added. This amount was enough to fully immerse the three-point contacts to a depth of 1 mm. Next, temperature was stabilized at 25 °C and the plastic ball was loaded against the cartilage plates. Then, the ball was made to slide over the plates at a controlled (decreasing) speed V from 2500 to 0.1 rpm under a normal force of F_N = 1 N (5 s per data point), while the resulting torque T sensed by the ball was monitored. The friction coefficient /J. was computed with g = T / F_NR) being R the radius of the ball.

Example 1.5. Rheological assays

Specimens for rheological assays were printed with 20 mm in diameter and 5 mm in height, solids and porous to analyze the effect of the infill over the mechanical characteristics of the scaffold. A MCR302 (Anton Paar) head was used to carry out rheological measurements at 25 °C. A three-step test was designed in order to obtain information on the compression and shearing characteristics of specimen. First, the scaffold was placed onto the base of the rheometer. Then, the rheometer head was approached at a constant speed (10 pm/s) up to a normal force of 40 N. Next, the specimen was oscillatory sheared according to a strain amplitude of 0.00001% at a frequency of 1 Hz and normal force of 40 N to determine the shear viscoelastic moduli and, finally, the upper plate was separated at a constant speed (10 pm/s).

Example 1.6. Cell viability assay

Cell viability in the 3D bioprinted scaffolds was determined on days 7 and 21 after bioprinting using Live/Dead™ Viability/Cytotoxicity Kit (Invitrogen). The printed constructs were incubated in PBS containing calcein AM (2pM) and ethidium homodimer (4 pM) at 37 °C for 30 min to stain live and dead cells. Scaffolds were imaged by confocal microscopy (Nikon Eclipse Ti-E Al, Amsterdam, Netherlands) and analyzed using NIS-Elements software (Amsterdam, Netherlands).

Example 1.7. Scanning electron microscopy (SEM)

The morphology and structure of b-TPUe scaffolds were analyzed using a variable-pressure and environmental scanning electron microscope (ESEM) FEI, mod. Quanta 400 (Oregon, USA). The analysis was performed in high vacuum mode to characterize the surface structure of scaffolds and cell growth. Samples were fixed with 2% glutaraldehyde and, then, were rinsed in 0.1 M cacodylate buffer and incubated overnight at 4 °C. For critical point the samples were then maintained with Osmium tetroxide 1% RT during Ih and dehydrated in a series of ethanol solutions (50%, 70%, 90%, 100%, 100%, 100%), by soaking the samples in each solution for 15 min. Subsequently, samples were critical point dried (Anderson, 1951) in a desiccator (Leica EMCPD300), and covered by evaporating them in a carbon evaporator (Emitech K975X).

Example 1.8. In vitro cytotoxicity test

MSCs culture medium aliquots were conditioned with b-TPUe samples as previously described. Briefly, b-TPUe sterilized scaffolds for a total mass of 3 g were placed in T-75 tissue culture flasks and soaked in 100 mL of complete cell culture medium for 10 days at 37 °C in a cell culture incubator on a rocking platform. Control medium was incubated in parallel, but without the b-TPUe scaffolds. MSCs were plated in a 6-well plate at 1 x 10⁵ cells/well. After 24 h the medium was replaced with a mix of a 1 : 1 fresh medium: b-TPUe- conditioned medium or with fresh control medium. Cell growth was analyzed at different time points: 1, 3, 5 and 7 days using AlamarBlue® assay (Bio-Rad Laboratories, Inc., manufactured by Trek Diagnostic System. U.S.). Cells were incubated with AlamarBlue® solution at 37°C for 3 h. Fluorescence of reduced AlamarBlue® was determined at 530/590 nm excitation/emission wavelengths (Synergy HT, BIO-TEK).

Example 1.9. Cell proliferation assay

Cell proliferation was analyzed using AlamarBlue® assay after 1, 3, 5, 7, 14 and 21 days. The scaffolds were incubated with AlamarBlue® solution at 37 °C for 3 h. Fluorescence of reduced AlamarBlue® was determined at 530/590 nm excitation/emission wavelengths.

Example 1.10. RNA isolation and real time-PCR analysis

Total cellular RNA was isolated using TriReagent (Sigma) and reverse transcribed using the Reverse Transcription System kit (Promega). Real-time PCR was performed using the SYBR-Green PCR Master mix (Promega) according to the manufacturer's recommendations. PCR reactions were performed as follows: an initial denaturation at 95 °C for 2 min, 40 cycles of 95 °C for 5 s followed by 60 °C for 30 s, and final cycle of dissociation of 60 - 95 °C. The gene expression levels were normalized to corresponding GAPDH values and are shown as relative fold expression to the control sample. All samples were analyzed in triplicate for each gene. Primer sequences used are shown in Table 1. Table 1. Primer sequences

Gene Forward Reverse

Col 1 SEQ ID NO: 1 SEQ ID NO: 2

Col 2 SEQ ID NO: 3 SEQ ID NO: 4

Acan SEQ ID NO: 5 SEQ ID NO: 6

Sox 9 SEQ ID NO: 7 SEQ ID NO: 8

Gapdh SEQ ID NO: 9 SEQ ID NO: 10

Example 1.11. Glycosaminoglycan quantification

The dimethylmethylene blue (DMMB) assay was used to study the glycosaminoglycans (GAGs) content as previously described. Briefly, 50 pL of papain-digested sample harvested at day 21 were added in triplicate to a 96-well plate and combined with 200 pL of DMMB dye, and the absorbance at 540 nm was immediately read. To determine the GAGs content of the samples chondroitin sulphate from shark cartilage (Sigma) was used as standard.

Example 1.12. Type II Collagen quantification

Type II collagen content produced in the scaffolds was quantified by ELISA (Type II Collagen Detection kit #6018; Chondrex, Redmond, WA) according to manufacturer’s instruction. Briefly, samples were digested using pepsin in 0.5 M acetic acid: collagen ratio of 1 :10 (w/w) for 2 days. Once digested, samples were incubated at 4 °C overnight in elastase: collagen ratio of 1 :10 (w/w). Then, standard and samples were placed in a precoated 96-well plate with capture antibodies and incubated for 30 min. The detection antibody was added and incubated for 1.5 h and then washed. The plate was incubated with streptavidin peroxidase for 1 h, washed, and incubated with ortho-phenyldiamine (OPD) solution for 30 min. A solution of 2N sulphuric acid was added to stop the reaction, and the content of type II collagen was quantified by absorbance at 490 nm.

Example 1.13. In vivo assays

In vivo assays were carried out in accordance with the approved guidelines of University of Granada following institutional and international standards for animal welfare and experimental procedure. The Research Ethics Committee of the University of Granada approved all experimental protocols.

Experiments were performed in immunocompetent CD-I mice and immunodeficient NOD SCID (NOD.CB17-Prkdcscid/NcrCrl) (NSG) mice purchased from Charles River (Barcelona, Spain). In order to evaluate the biocompatibility, PCL and b-TPUe cell-free scaffolds were transplanted into two independent small subcutaneous pockets made on the back of CD-I mice anesthetized by isoflurane inhalation (n = 5 per group). In addition, MSCs cell-laden scaffolds cultured for 21 days were implanted into two independent small subcutaneous pockets created on the back of NSG mice anesthetized by isoflurane inhalation. Cell-laden or cell-free scaffolds were implanted in each pocket with a single biomaterial per mouse (b- TPUe or PCL) (n = 5 per group). Animals were maintained in a microventilated cage system with a 12-h light/dark cycle with food and water ad libitum. Mice were manipulated within a laminar airflow hood to maintain pathogen-free conditions. Three weeks later, mice were sacrificed via an overdose injection of an aesthetic, and the scaffolds were photographed to evaluate the implantation within the surrounding mouse tissue and recovered for histological analyses.

For the histological analysis, samples were dehydrated, embedded in Technovit 7200 and polymerized. The blocks were sectioned with a diamond-coated band saw (Exakt 310 CP) and, then, grounded and polished with a high precision grinder (Exakt 400). The total histological processing, including Toluidine Blue and Masson staining, were performed by Histology Unit of BIONAND (Malaga, Spain) following the Donath and Bruener cutting/grinding technique.

Example 1.14. Statistical analysis

Statistical calculations were performed using SPSS 13.0 software for Windows (SPSS, Chicago, IL, USA). All graphed data represent the mean +/- SD from at least three experiments. Differences between treatments were tested using the two-tailed Student's T test. Assumptions of Student's T test (homoscedasticity and normality) were tested and assured by using transformed data sets [log(dependent variable value + 1)] when necessary. P -values < 0.05 (*) and P-values < 0.01 (**) were considered statistically significant in all cases.

Example 2. Results

Example 2.1. Fabrication of scaffolds of the invention

Polyurethane elastomers are an adaptable category of materials broadly used for biomedical purposes thanks to their biocompatibility, elasticity and strength. In this study, a novel elastic polyurethane-based 3D printing material, b-TPUe, was successfully used to fabricate live scaffolds by 3D bioprinting.

As is shown in Figure 1A, scaffolds were designed with a regular geometry and structure to enable an adequate cell bioprinting. Figure IB shows that 3D b-TPUe scaffolds were fabricated with the desired shape and dimensions, like the CAD model. SEM images (Figure 1C and ID) show scaffold pores and filament surfaces and demonstrate that the thickness of the fibers of the b-TPUe printed scaffolds (200 - 400 pm) is maintained during the fabrication process (Figure 1E-J). The porosity and interconnectivity of the scaffold plays a significant role in nutrient supply, gas diffusion and metabolic waste removal. As can be clearly seen, the pores are large, ranging from 500 to 700 pm (Figure 1C and ID) and have a regular structure, uniformly distributed, and interconnected. Therefore, cells can penetrate the pores following their growth on the scaffold.

Example 2.2. Frictional test

The frictional behavior of the different plastics used in this work is exemplified in Figure 2A and Figure 2B. For this, plastic-cartilage point contacts were lubricated by synovial fluid and data are plotted in terms of a Stribeck curve, where friction coefficient is represented as a function of the sliding speed for a constant normal load of 1 N. Only for b-TPUe, the contact operates in the full film lubricated regime as demonstrated by the increase in friction for large sliding speeds. As observed, a significantly lower friction was measured for b-TPUe followed by PCL and PLA. A selected biomaterial for treating joint replacements is expected to preserve the remaining native cartilage against degradation while maintaining the frictional properties of the joint. Summarizing, b-TPUe showed better frictional characteristics than PLA and PCL.

Example 2.3. Compression test

The mechanical properties of a scaffold are important for engineering tissues, especially for cartilage, which is subjected to cyclic mechanical forces. Printed scaffolds produced with thermoplastics possess higher Young’s modulus than scaffolds based on hydrogels, which mimic more adequately the mechanical properties found in native tissues.

Figure 2C shows the compression curves of PLA, PCL, b-TPUe and cartilage. These strongly non-linear curves clearly demonstrate that b-TPUe is significantly softer that the other materials investigated (PLA and PCL). Also, unlike PLA and PCL, results for solid (s) and porous (p) b-TPUe scaffolds showed differences in compression. Interestingly, porous b- TPUe scaffolds were significantly softer than their solid counterparts, suggesting that b-TPUe scaffold elasticity can be tailored by changing the inner pore size. So, b-TPUe scaffolds with greater porosity present a mechanical behavior closer to the one of native cartilage. In addition, for low strains 8, the mechanical behaviour of b-TPUe was similar to that observed in natural cartilage when compared with PCL or PLA.

Moreover, the shear moduli obtained in the second interval of the test showed a clear correlation with the compression data, again demonstrating that b-TPUe exhibited a much lower storage modulus in contrast to the conventional plastics, PCL and PLA (Figure 2D).

Example 2.4. Effects of b-TPUe-conditioned medium on MSCs proliferation

Since b-TPUe is a recently developed polyurethane-based 3D printing filament, no previous data concerning the possible cytotoxicity of this material on cell growth has been published. Polyurethanes are considered to have good biocompatibility properties and are widely used for long-term medical implants, such as cardiac pacemakers and vascular grafts. ^[39] Biocompatibility must be a priority when selecting biomaterials for TE,^[40] thus, as a novel used for this material we conducted a proliferation assay to evaluate if the exposure to b- TPUe could have a negative effect in the proliferative potential of MSCs. Results showed no adverse effects in the proliferative potential of MSCs cultured in b-TPUe-conditioned medium for 7 days when compared with MSCs cultured with control medium (Figure 3A).

Example 2.5. Proliferation and viability of MSCs cultured in b-TPUe bioprinted scaffolds

Cell proliferation of MSCs cultured in b-TPUe bioprinted scaffolds was evaluated with an AlamarBlue® assay. PCL filament was used as a control material since it is an FDA- approved biocompatible material and has shown to promote cell proliferation, cell attachment and ECM production. As can be observed in Figure 3B cell proliferation increased from day 1 until day 21 with a significant increase at day 7 of culture in both bioprinting materials. At day 21 no significant differences were observed in the proliferation rate between cells bioprinted in b-TPUe and those cultured in PCL control scaffolds (Figure 3B).

Besides, the viability of MSCs was evaluated to validate the biocompatibility of b-TPUe bioprinted scaffolds using a live/dead assay. Confocal images (Figure 3C) show MSCs covering both b-TPUe and PCL scaffold fiber surfaces at day 7 and 21 after bioprinting. These results indicate that b-TPUe bioprinted scaffolds can provide an environment that supports MSCs growth in a same manner as PCL. Although other TPU-based materials have been tested to be biocompatible, this is the first assessment to demonstrate the biocompatibility of b-TPUe bioprinted scaffolds. Example 2.6. Chondrogenic differentiation of MSCs cultured in b-TPUe bioprinted scaffolds

To investigate the capacity of b-TPUe scaffolds to support the induction of cartilage-like phenotype, chondrogenic key markers were evaluated by RT-PCR after 21 days of culture of bioprinted cell-seeded scaffolds under chondrogenic conditions. Cells extracted from b-TPUe bioprinted scaffolds cultured under chondrogenic media showed a significant increment in type II collagen, aggrecan and Sox9 gene expression when compared with cells grown in monolayer and onto b-TPUe scaffolds without chondrogenic media (Figure 4A). Type II collagen and aggrecan are the main proteins of the hyaline cartilage ECM and Sox9 is a known transcription factor of chondrogenesis, which acts in the early stages of chondrogenic differentiation inducing type II collagen production. In addition, non-increased expression of collagen type I in b-TPUe scaffolds under chondrogenic media (Diff) compared with their counterparts cultured in non-differentiated media (CTL) or cells cultured in monolayer without chondrogenic media was observed. Type I collagen has been described in fibroblastic differentiation, and could indicate the formation of fibrous cartilage. The upregulation of chondrogenic genes together with the low expression of collagen type I of MSCs bioprinted in b-TPUe scaffolds, indicate the ability of this elastomeric material to support the formation of hyaline-like cartilage.

The ECM produced under induction of chondrogenic differentiation was evaluated assessing GAGs and type II collagen concentration in cell culture supernatants of MSCs monolayers and bioprinted MSCs b-TPUe scaffolds cultured with (Diff) or without (CTL) chondrogenic medium at day 21. The GAGs analysis showed that b-TPUe bioprinted scaffolds in chondrogenic conditions produced a high significant number of GAGs compared to control b- TPUe scaffolds or monolayer conditions (Figure 4B). Similarly, collagen type II production was also markedly greater in b-TPUe bioprinted scaffolds cultured under chondrogenic conditions at 21 days compared to control b-TPUe scaffolds and monolayer conditions (Figure 4C). This increased GAGs and collagen type II deposition in the ECM of b-TPUe MSCs bioprinted scaffolds cultured under chondrogenic conditions indicates the development of a cartilaginous-like matrix.

Moreover, SEM images showed cell growth and wide cell spread throughout the scaffold confirmed the formation of ECM over the b-TPUe filament after 21 days of cell growth with and without differentiation conditions. It is relevant to note that cells attached to the filament surface and junctions via formation of filopodia and start to form a network of cell and matrix (Figure 4D-F). Also, it was observed an enhanced cell growth that covered the pore spaces (Figure 4G-H) and over the filament surfaces (Figure 41).

Example 2.7. In vivo assay

Biocompatibility of cell-free b-TPUe scaffolds was assessed in vivo by subcutaneous in situ implantation in the back of immunocompetent CD-I mice using PCL as comparative gold standard material (Figure 5A-D). During the study, no cases of mice showing pain behaviour that could be induced by the scaffold implantation or infection were observed. The scaffolds were excised 21 days after implantation and photographed to evaluate their appearance and integration within the subcutaneous surrounding tissue of the mouse. As shown in Figure 5B and 5D, both b-TPUe and PCL scaffolds were firmly anchored and integrated within the subcutaneous tissue maintaining their shape and integrity, and the presence of blood vessels proximal to and inside of the scaffolds was observed. Moreover, no sign of edema or inflammatory response was detected. All these characteristics indicate the in vivo biocompatibility of b-TPUe as previously described for other 3D polyurethanes.

Finally, both b-TPUe- and PCL- MSCs bioprinted scaffolds cultured for 21 days were transplanted into subcutaneous tissue on the flanks of immunodeficient NSG mice and harvested 3 weeks later for subsequent analysis. The implanted bioprinted cell-laden scaffolds were well tolerated by the mice showing the biocompatibility and the integration of both polymer scaffolds (Figure 6A). Toluidine blue staining showed the presence of GAGs in both b-TPUe and PCL scaffolds. In cell-free scaffolds it was observed the ability of b- TPUe to promote the formation of new tissue since host cells infiltrated, adhered and grew into the scaffold, which confirm again the biocompatibility of b-TPUe. This was confiremed by Masson's Tri chrome staining showing that the deposition of collagenous fibers occurred in both materials and in both cell-free and cell-laden conditions (Figure 6B). These results suggest that b-TPUe can allow in vivo GAGs and collagenous fiber production as well as PCL. Both polymer scaffolds, b-TPUe and PCL, showed good in vivo integration a biocompatibility.

Claims

1. Use of an elastomer comprising methylene diphenyl diisocyanate [MDI] and 1,4- butanediol for the preparation of a biomaterial structure that serves as a substrate or guide for tissue repair or regeneration.

2. Use, according to claims 1, of 1,4-butanediol thermoplastic polyurethane elastomer comprising methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

3. Use, according to any of the previous claims, wherein the biomaterial structure thus generated is a tissue scaffold, a tissue implant, a stent or a valve.

4. Use, according to any of the previous claims, wherein the elastomer is in the form of filament, powder, electro-spun mesh, sponge or pellets.

5. Biomaterial structure consisting essentially of an elastomer comprising methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

6. Biomaterial structure, according to claim 5, consisting essentially of 1,4- butanediol thermoplastic polyurethane elastomer which comprises methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

7. Biomaterial structure, according to any of the claims 5 or 6, characterized in that it is a tissue scaffold, a tissue implant, a stent or a valve.

8. Tissue scaffold, according to claim 7, consisting essentially of an elastomer comprising methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

9. Tissue scaffold, according to claim 8, consisting essentially of 1,4-Butanediol thermoplastic polyurethane elastomer comprising methylene diphenyl diisocyanate [MDI] and 1,4-butanediol.

10. A composition comprising a population of cells, or the secretome secreted from said cells, within the tissue scaffold of claims 8 or 9.

11. A composition according to claim 10, wherein the cells are mature cells or mesenchymal/pluripotent/embryonic stem cells.

12. A composition according to claims 10 or 11, wherein the cells are selected from the list comprising: chondrocytes, skin cells, endothelial cells, smooth muscle cells, osteoblasts or myocytes.

13. Pharmaceutical composition comprising the composition of any of the claims 10 to 12 and, optionally, pharmaceutically acceptable excipients or carriers.

14. Pharmaceutical composition according to claim 13, for use in the treatment of diseases involving tissue degeneration. Pharmaceutical composition for use, according to claim 14, in the treatment of diseases involving cartilage, vascular, skeletal muscle or skin degeneration.

Pharmaceutical composition for use, according to claim 15, in the treatment of chondromalacia.