Silica replicas derived from mammalian cells to direct cell lineage decisions of mesenchymal stem cells

Abstract

The invention relates to a method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with the immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type. The invention also relates to scaffolds, silicified cells and kits for the differentiation of cells.

Description

[0001] Silica Replicas Derived from Mammalian Cells to Direct Cell Lineage Decisions of Mesenchymal Stem Cells

[0002] Technical field

[0003] The present invention relates to methods to obtain silica replicas of cells to be used as scaffold for differentiating cells to a specific lineage and / or cell type, scaffolds comprising said silica replicas, and uses thereof.

[0004] Background

[0005] Stem cells (SCs) have the ability to differentiate into different cell types and, as such, they have the potential to enable the patient-specific regeneration of injured or diseased tissues.

[0006] While chemical stimuli in the form of differentiation factors have been widely employed, assessing the effect of physical and mechanical factors is a more recent approach. However, the use of differentiation factors and other smaller chemical compounds has important limitations and risks. For example, usually, combinations of several molecules need to be administered in a controlled manner in terms of dose and kinetics over long periods of time (e.g., days or even weeks). Achieving such a regulated drug release when making use of highly complex scaffolds is not always possible and, often times, to direct the differentiation of stem cells to a specific cell lineage, supra- physiological dosages of the chemical compounds need to be administered (Dobbenga et al., 2016). This results not only in high costs but also in off-target effects. Additionally, once these chemical cues are removed following in vivo transplantation, the effects on the differentiation of the stem cells are rapidly lost. As such, these disappointing clinical results have pushed forward the development of alternative strategies to guide the differentiation of stem cells towards predefined cell lineages.

[0007] Since the fate of stem cells is regulated not only by the chemical factors but also by the biophysical features (i.e. , surface topography, surface smoothness / roughness and flexibility / rigidity) of the extracellular matrix, the engineering of scaffolds mimicking its stiffness and topography has emerged as a promising approach (Nicolas et al., 2020). By making use of micro- and nanofabrication technologies (e.g., soft lithography or 3D printing), precisely engineered substrates with defined micro / nanotopography and variable stiffness have been fabricated. In vivo, the extracellular matrix has a complex hierarchical structure and fabrication of simple geometries consisting of grooves, ridges, dots or pits is not sufficient to recapitulate its architecture. This makes it impossible to prepare an optimal surface topography by making use of micro- and nanofabrication technologies to control cell fate decisions.

[0008] There is a need for methods which allow efficient differentiation of stem cells into cells of a specific, predefined cell lineage of interest, in order to take advantage of the enormous potential of stem cells in tissue engineering and regenerative medicine.

[0009] Summary

[0010] The present invention relates to methods to obtain silica replicas of isolated mammalian cells, which are then used as substrates to direct the differentiation of stem cells (SCs), such as mesenchymal stem cells (MSCs) to specific cell lineages.

[0011] Specifically, herein are disclosed fully synthetic silicified cells replicating the morphology and topography both at the micro- and nano-level of living mammalian cells. This is achieved by the silica bioreplication technique which involves the use of biological samples (i.e. , mammalian cells, such as osteoblast or chondrocytes) to direct the condensation of silicon dioxide (also known as silica) in a solution of tetramethoxylsilane. Following calcination of the resulting cell silica composites, replicas fully made of silica with the unique morphology and surface topography of the biological structures used as templates are obtained. Surprisingly, the silicified cells direct the differentiation of stem cells to the specific cell lineage that had been employed to fabricate the particles. When stem cells are contacted with a scaffold made of a multitude of silicified cells, they differentiate with and without differentiation factors in the cell culture media to the specific cell lineage that had been employed to fabricate the silicified cells. Furthermore, surprisingly, a scaffold made of a monolayer of silicified cells can direct the differentiation fate of stem cells.

[0012] The present invention discloses a silica bioreplication process by creating silica replicas of isolated mammalian cells, which replicas are then used as substrates to direct the differentiation of stem cells to the specific cell lineage that had been employed to fabricate the replicas, even in the absence of differentiation factors. This is advantageous compared to the use of chemical stimuli in the form of differentiation factors, since differentiation factors are expensive and need to be replaced in the cell culture medium regularly.

[0013] The extracellular matrix (ECM) has a complex hierarchical structure, which is known to guide the cell fate decisions of stem cells. The rational design of an optimal topography for each cell type requires many trial and error attempts with the number of possible substrate patterns being virtually unlimited. Compared thereto, silicified cells replicate the morphology and topography both at the micro- and nano-level of living mammalian cells.

[0014] A first aspect of the invention relates to a method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with the immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type.

[0015] A second aspect of the invention relates to a method of obtaining cells of a specific cell lineage of interest and / or of a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type, and obtaining cells of said specific cell lineage and / or of said specific cell type.

[0016] A third aspect of the invention relates to the use of a scaffold comprising or consisting of silicified cells as a substrate to regulate the fate of immature and / or to differentiate immature cells into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined herein, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type of interest are as disclosed herein.

[0017] A fourth aspect of the invention relates to a scaffold comprising or consisting of silicified cells, wherein the scaffold promotes the differentiation of immature into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined herein, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type are as defined herein.

[0018] A fifth aspect of the invention relates to a scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold is obtained by a method comprising the steps of: i) Silicificating cells of a specific cell lineage or a specific cell type to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold, preferably wherein the scaffold, the silicified cells and the specific cell lineage or specific cell type are as defined herein.

[0019] A sixth aspect of the invention relates to a population of differentiated cells of a specific cell lineage or a specific cell type of interest, obtained by the method as described herein.

[0020] A seventh aspect of the invention relates to the use of a scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold, the silicified cells and the specific cell lineage or the specific cell type of interest are as defined herein.

[0021] An eighth aspect of the invention relates to the use of a scaffold comprising or consisting of silicified cells as a substrate to regulate the fate of immature and / or to differentiate immature cells into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined herein, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type of interest are as defined herein. A ninth aspect of the invention relates to an implant comprising the scaffold described herein.

[0022] A tenth aspect relates to a scaffold as described herein for use in bone engineering.

[0023] An eleventh aspect relates to a kit-of-parts comprising: i) The scaffold as defined herein, comprising silicified cells of a specific lineage or specific cell type of interest; ii) Immature cells, preferably stem cells, even more preferably as defined herein; iii) Optionally, instructions for use.

[0024] The invention is defined in the attached claims.

[0025] Description of Drawings

[0026] Figure 1 : Scheme of the fabrication of the silica replicas. The fabrication of the silica replicas involves the silicification of cells of a specific predefined cell type followed by a calcination step to remove the organic matter. The resulting silica replicas are incubated with stem cells to direct their differentiation to a specific predefined cell type.

[0027] Figure 2: A) Bright field images of i) human osteoblasts (HObs) and the corresponding HObs-derived silica replicas (HObs@SiC>2) and ii) human chondrocytes (HCs) and the corresponding HC-derived silica replicas (HCs@SiC>2). B) SEM images and the corresponding EDS spectra at different stages of the preparation of the HObs@SiC>2 (i) and the HCs@SiC>2 (ii).

[0028] Figure 3: A) i) Normalized cell viability (nCV) and ii) alkaline phosphatase (ALP) readings of SaOS-2 cells cultured onto HObs@SiC>2-based substrates containing increasing numbers of silica replicas derived from human osteoblasts (HObs) (i.e., HObs@SiO2) for 1 , 3 and 7 days. As controls, SaOS-2 cells cultured onto glass coverslips and in growth media only (i.e., without the addition of osteogenic inducers) have been considered. B) i) nCV and ii) ALP readings of SaOS-2 cells cultured onto HObs@SiO2-based substrates for 1 , 3, 7 and 14 days. As controls, SaOS-2 cells cultured onto glass coverslips either in growth media or in osteogenic media have been considered, n = 3; ns > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0029] Figure 4: A) Normalized cell viability (nCV) B) and alkaline phosphatase (ALP) readings of hMSCs cells cultured onto HObs@SiC>2-based substrates with increasing numbers of silica replicas derived from human osteoblasts (HObs) (i.e., HObs@SiC>2) for 1 , 7, 14 and 21 days. As controls, hMSCs cells cultured onto glass coverslips either in growth media or in osteogenic media have been considered, n = 3; ns > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0030] Figure 5: Alkaline phosphatase staining of hMSCs cells cultured onto glass coverslips or onto HObs@SiC>2-based substrates containing 20 000 silica replicas derived from human osteoblasts (HObs) (i.e., HObs@SiO2) for 14 days. The cells were cultured either in growth medium or in osteogenic medium.

[0031] Figure 6: Alizarin Red S staining of hMSCs cells cultured onto glass coverslips or onto HObs@SiO2-based substrates containing 20 000 silica replicas derived from human osteoblasts (HObs) (i.e., HObs@SiO2) for 1 and 21 days.

[0032] Figure 7: Expression levels of four different osteogenic markers: RLINX2 (A), ALP (B), COL1A1 (C) and OCN (D) and the chondrogenic marker SOX9 (E) in hMSCs cultured onto glass coverslips, smooth silica (SiO2) surface, SiO2-microspheres (MSs) or HObs@SiO2-based substrates containing 20 000 silica replicas (i.e., HObs@SiO2) for 1 , 3, 7, 14 and 21 days. The hMSCs have been cultured either in growth media or in osteogenic media. The silica replicas have been derived from human osteoblasts (HObs). n = 3; ns > 0.05; *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ****p < 0.0001.

[0033] Figure 8: Expression levels of three different chondrogenic markers: SOX9 (A), COL2A1 (B) and ACAN (C) and the osteogenic marker RUNX2 (D) in hMSCs cultured onto glass coverslips, smooth silica (SiO2) surface, SiO2-microspheres (MSs) or HCs@SiO2-based substrates containing 20 000 silica replicas (i.e., HCs@SiC>2) for 1, 7, 14 and 21 days. The hMSCs have been cultured either in growth media or in chondrogenic media. The silica replicas have been derived from human chondrocytes (HCs). n = 3; ns > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Detailed description

[0034] Definitions

[0035] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise. Thus, for example, reference to “a scaffold” includes a plurality of such scaffolds.

[0036] As used herein, unless indicated otherwise or contradictory in context, the term "cell lineage" refers to the developmental history of a particular cell from its primary state in the fertilized egg or embryo through to its fully differentiated state. The different steps and phases involved in the development of a cell produces many intermediate cells which may be referred to as progenitor or precursor cells in the present application and form an integral part of the cell lineage.

[0037] As used herein, the term "cell type" refers to a group of cells having a distinct set of morphological, biochemical, and / or functional characteristics that define the cell type. One of skill in the art will recognize that a cell type can be defined with varying levels of specificity. For example, osteoblasts and chondrocytes are distinct cell types, which can be distinguished from one another, but differentiated both from mesenchymal stem cells (MSCs). Typically, cells of different types may be distinguished from one another based on their differential expression of a variety of genes which are referred to in the art as "markers" of a particular cell type or types (e.g., cell types of a particular lineage). In some embodiments, cells of different types may be distinguished from one another based on their enzyme activity. A "cell type-specific marker" is a gene product or modified version thereof that is expressed at a significantly greater level by one or more cell types than by all or most other cell types and whose expression is characteristic of that cell type. Many cell type specific markers are recognized as such in the art.

[0038] As used herein, the term “immature cells” refers to cells that are not terminally- differentiated. In some embodiments of the present invention, the immature cells are stem cells. In some embodiments of the present invention, the stem cells are mesenchymal stem cells. In some embodiments of the present invention the immature cells are progenitor cells. In some embodiments of the present invention, the immature cells are progenitor cells or precursor cells of the specific cell lineage or cell type of interest. "Medium", "Cell culture medium", "Culture medium": These terms as used herein refer to a solution containing nutrients that nourish growing cells. In certain embodiments, the culture medium is useful for growing mammalian cells. Typically, a culture medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and / or survival. A culture medium may also contain supplementary components that enhance growth and / or survival above the minimal rate, including, but not limited to, hormones and / or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and / or glucose or other energy source. In some embodiments, the culture medium contains differentiation factors.

[0039] The term "differentiation factor" or "differentiation agent" as used herein, refers to a molecule that induces a stem cell or a progenitor cell to commit to a particular specialized cell type. Exemplary differentiation factors include BMP-2 growth factor. In some embodiment of the present disclosure, differentiation of the cells comprises culturing the cells in a medium comprising one or more differentiation factors.

[0040] The term “differentiation” generally refers to the acquisition of a phenotype of a mature cell type from a less developmentally defined cell type, e.g. from an MSC to an osteoblast.

[0041] As used herein "contacting cells" is defined as exposing the cells to one or more agent or combinations of agents described herein. In some embodiments, the cells are covered by the agents, while in other embodiments, the cells are not fully covered by the agents. Examples for the contacting agents are cell culture medium, Phosphate buffered saline, ethanol, paraformaldehyde, water and orthosilicic acid.

[0042] Method of differentiation

[0043] The present invention relates to a method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type. Silica replicas of cells can be used as scaffold for differentiating cells to said specific cell lineage of interest and / or said specific cell type. The present invention relates to a method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with the immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type.

[0044] In another aspect, the present invention relates to a method of obtaining cells of a specific cell lineage of interest and / or of a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type, and obtaining cells of said specific cell lineage and / or of said specific cell type.

[0045] As used herein, "contacting the scaffold with immature cells" refers to the process by which cells come into physical interaction with a scaffold. This can involve placing the cells in direct contact or in close proximity to the scaffold material, allowing them to attach, adhere, or interact with the scaffold surface.

[0046] In some embodiments, the method is for obtaining osteoblasts, and the present invention relates to a method of differentiating mesenchymal stem cells to osteoblasts, said method comprising: i) Silicificating osteoblasts to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with the mesenchymal stem cells; iv) Culturing the mesenchymal stem cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the mesenchymal stem cells into osteoblasts.

[0047] In some embodiments, the method is for obtaining chondrocytes, and the present invention relates to a method of differentiating mesenchymal stem cells to chondrocytes, said method comprising: i) Silicificating chondrocytes to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with the mesenchymal stem cells; iv) Culturing the mesenchymal stem cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the mesenchymal stem cells into chondrocytes.

[0048] In some embodiments of the present invention, the method further comprises fixing and / or plating said cells of the specific cell lineage or specific cell type prior to silicification, preferably wherein said cells have not reached confluence. In some embodiments of the present invention, the method further comprises fixing said cells of the specific cell lineage or specific cell type prior to silicification, preferably wherein said cells have not reached confluence. In some embodiments of the present invention, the method further comprises plating said cells of the specific cell lineage or specific cell type prior to silicification, preferably wherein said cells have not reached confluence.

[0049] The person skilled in the art will appreciate that different procedures can be performed for the preservation of biological tissues from decay due to autolysis or putrefaction, such as heat fixation, immersion or preferably chemical fixation. The person skilled in the art will also appreciate that different fixatives such as alcohols, oxidizing agents or preferably aldehydes, such as paraformaldehyde can be used.

[0050] The person skilled in the art will appreciate that different plating protocols can be used dependent on the cell type and cell lineage. Furthermore different cell culture plates can be used, such as multi-well plates preferably containing glass coverslips. The person skilled in the art will also appreciate, that the cells can be seeded at different densities depending on the cell type. For example, MSCs can be seeded at a density of at least 500 cells per cm2, such as at least cells 1000 cells per cm2, such as at least 1500 viable cells per cm2.

[0051] By "confluence", in the present invention, it is meant that the growth surface of the culture is being uniformly covered by a single layer of cells.

[0052] Accordingly, in some embodiments, the cells of the specific cell lineage are chondrocytes, and the method comprises fixating chondrocytes prior to silicification.

[0053] In other embodiments, the cells of the specific lineage are osteoblasts, and the method comprises fixating osteoblasts prior to silicification.

[0054] Silicification

[0055] Silicification is the process by which organic or inorganic materials are gradually replaced or impregnated with silica (SiO2).

[0056] In some embodiments of the present invention, step i) of the method comprises or consists of: i) Incubating said cells of said specific cell lineage or specific cell type at a temperature between 25°C and 55°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours in the presence of a silicic acid compound, preferably selected from the group of orthosilicic acid, tetramethoxysilane, tetraethoxysilane and their derivatives such as methyltriethoxysilane, methyltrimethoxysilane, or a mixture thereof, and ii) Optionally, contacting said cells of said specific cell lineage or specific cell type with a compound selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably wherein the compound is ethanol, preferably for a duration of at least 10 minutes; optionally, the compound is first added as a 50% (v / v) solution, then as a 100% (v / v) solution.

[0057] The temperature at which said cells in step i) of the method are incubated (herein referred to as "incubation temperature") may vary. The incubation temperature may be held constant throughout the course of incubation or may be made to vary. For example, the incubation temperature may be at least 25 °C, such as at least 30 °C, such as at least 32 °C, such as at least 35 °C, such as at least 37 °C, such as at least 40 °C.

[0058] In some embodiments of the present invention, the incubation temperature in step i) of the method is at most 55 °C, such as at most 50 °C, such as at most 47 °C, such as at most 45 °C, such as at most 42 °C, such as at most 40 °C.

[0059] In some embodiments of the present invention, the incubation temperature in step i) of the method is in a range of 25 °C to 55 °C, such as 30 °C to 50 °C, such as in the range of 32 °C to 48 °C, such as in a range of 35 °C to 45 °C.

[0060] By 'incubation duration' is in this context of step i) of the method understood the time said cells of said specific cell lineage or specific cell type are incubated in the presence of a silic acid. For example, the incubation duration may be at least 7 hours, such as at least 8 hours, such as at least 9 hours, such as at least 10 hours, such as at least 11 hours, such as at least 12 hours.

[0061] In some embodiments of the present invention, the incubation duration in step i) is at most 17 hours, such as at most 16 hours, such as at most 15 hours, such as at most

[0062] 14 hours, such as at most 13 hours, such as at most 12 hours.

[0063] In some embodiments of the present invention, the incubation duration in step i) is in a range of 7 to 17 hours, such as in a range of 8 to 16 hours, such as in a range of 9 to

[0064] 15 hours, such as in a range of 10 to 14 hours, such as in a range of 11 to 13 hours.

[0065] In some embodiments of the present invention, the incubation temperature in step i) of the method is at least 25 °C and the incubation duration in step i) is at least 7 hours. In some embodiments of the present invention, the incubation temperature in step i) of the method is at most 55 °C and the incubation duration in step i) is at most 17 hours. In some embodiments of the present invention, the incubation temperature in step i) of the method is in a range of 25 °C to 55 °C and the incubation duration in step i) is in a range of 7 to 17 hours. In some embodiments of the present invention, the incubation temperature in step i) of the method is in a range of 35 °C to 45 °C and the incubation duration in step i) is in a range of 11 to 13 hours. In some embodiments of the present invention, the incubation temperature in step i) of the method is 40 °C and the incubation duration in step i) is 12 hours.

[0066] The person skilled in the art will appreciate that different reagents can be used for the silicification of cells. The person skilled in the art will appreciate, that orthosilic acid and its derivates like its esters, preferably its methyl ester can be used for the silification of the cells. For example, the orthosilicic acid used in step i) of the method is tetramethoxysilane, preferably a solution of tetramethoxysilane.

[0067] The person skilled in the art will appreciate that as used herein "solution" refers to homogeneous mixtures and non-homogeneous mixtures of tetramethoxysilane with other reagents, such as Phosphate-buffered saline, Milli-Q water, buffer, preferably Milli-Q water. The person skilled in the art will also appreciate that the pH of the solution can be adjusted with acids, such as carboxylic acids, sulfonic acids, phenols, acetic acid, citric acid, lactic acid, ascorbic acid, preferably hydrochloric acid.

[0068] In some embodiments of the present invention, the tetramethoxysilane solution has a tetramethoxysilane concentration of at least 70 mM, such as at least 80 mM, such as at least 90 mM, such as at least 100 mM.

[0069] In some embodiments of the present invention, the tetramethoxysilane solution has a tetramethoxysilane concentration of at most 130 mM, such as at most 120 mM, such as at most 110 mM, such as at most 100 mM.

[0070] In some embodiments of the present invention, the tetramethoxysilane solution has a tetramethoxysilane concentration of 100 mM.

[0071] In some embodiments of the present invention, the tetramethoxysilane solution has a pH of at least 1 , such as at least 1.2, such as at least 1.5, such as at least 1.8, such as at least 2, such as at least 2.2, such as at least 2.5, such as at least 2.8, such as at least 3.

[0072] In some embodiments of the present invention, the tetramethoxysilane solution has a pH of at most 5, such as at most 4.8, such as at most 4.5, such as at most 4.2, such as at most 4, such as at most 3.8, such as at most 3.5, such as at most 3.2, such as at most 3. In some embodiments of the present invention, the tetramethoxysilane solution has a pH of 3.

[0073] In some embodiments of the present invention, the tetramethoxysilane solution has a tetramethoxysilane concentration of at least 70 mM and a pH of at least 1. In some embodiments of the present invention, the tetramethoxysilane solution has a tetramethoxysilane concentration of at most 130 mM and a pH of at most 5. In some embodiments of the present invention, the tetramethoxysilane solution has a tetramethoxysilane concentration of 100 mM and a pH of 3.

[0074] Any of the concentration and pH conditions can be combined with any of the temperature and time conditions described above. In some embodiments of the present invention, the incubation temperature in step i) of the method is at least 25 °C, the incubation duration in step i) is at least 7 hours and the tetramethoxysilane solution has a tetramethoxysilane concentration of at least 70 mM and a pH of at least 1. In some embodiments of the present invention, the incubation temperature in step i) of the method is at most 55 °C, the incubation duration in step i) is at most 17 hours and the tetramethoxysilane solution has a tetramethoxysilane concentration of at most 130 mM and a pH of at most 5. In some embodiments of the present invention, the incubation temperature in step i) of the method is in a range of 25 °C to 55 °C, the incubation duration in step i) is in a range of 7 to 17 hours and the tetramethoxysilane solution has a tetramethoxysilane concentration of 100 mM and a pH of 3. In some embodiments of the present invention, the incubation temperature in step i) of the method is in a range of 35 °C to 45 °C, the incubation duration in step i) is in a range of 11 to 13 hours and the tetramethoxysilane solution has a tetramethoxysilane concentration of 100 mM and a pH of 3. In some embodiments of the present invention, the incubation temperature in step i) of the method is 40 °C, the incubation duration in step i) is 12 hours and the tetramethoxysilane solution has a tetramethoxysilane concentration of 100 mM and a pH of 3.

[0075] Accordingly, in some embodiments, the cells of the specific cell lineage are chondrocytes, and the method comprises silicification of chondrocytes.

[0076] In other embodiments, the cells of the specific lineage are osteoblasts, and the method comprises silicification of osteoblasts. In some embodiments of the present invention, the method further comprises a step of drying said silicified cells of said specific lineage or said specific cell type before step ii) of calcinating a plurality of the silicified cells, such as at a temperature between 25 °C and 55 °C, such as between 30°C and 50°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours.

[0077] It should be understood that "drying" with respect to this method includes the removal of liquid components including water and other solvents from the cells. The temperature at which said cells before step ii) of calcinating a plurality of the silicified cells of the method are dried (herein referred to as "drying temperature") may vary. The drying temperature may be held constant throughout the course of drying or may be made to vary. For example, drying may preferably be performed at at least 25 °C, such as at least 30 °C, such as at least 35 °C, such as at least 40 °C.

[0078] In some embodiments of the present invention, the temperature of the drying step is at most 55 °C, such as at most 50 °C, such as at most 45 °C, such as at most 40 °C.

[0079] In some embodiments of the present invention, the temperature of the drying step is in a range of 25 °C to 55°C, such as in a range of 30 °C to 50 °C, such as in a range of 35 °C to 45 °C.

[0080] In some embodiments of the present invention, the duration of the drying step is at least 7 hours, such as at least 8 hours, such as at least 9 hours, such as at least 10 hours, such as at least 11 hours, such as at least 12 hours.

[0081] In some embodiments of the present invention, the duration of the drying step is at most 17 hours, such as at most 16 hours, such as at most 15 hours, such as at most

[0082] 14 hours, such as at most 13 hours, such as at most 12 hours.

[0083] In some embodiments of the present invention, the duration of the drying step is in a range of 7 to 17 hours, such as in a range of 8 to 16 hours, such as in a range of 9 to

[0084] 15 hours, such as in a range of 10 to 14 hours, such as in a range of 11 to 13 hours. In some embodiments of the present invention, the temperature of the drying step is at least 25 °C and the duration of the drying step is at least 7 hours. In some embodiments of the present invention, the temperature of the drying step is at most 55 °C and the duration of the drying step is at most 17 hours. In some embodiments of the present invention, the temperature of the drying step is in the range of 25 °C to 55 °C and the duration of the drying step is in the range of 7 to 17 hours. In some embodiments of the present invention, the temperature of the drying step is 40 °C and the duration of the drying step is 12 hours.

[0085] Accordingly, in some embodiments, the cells of the specific cell lineage are chondrocytes, and the method comprises drying of the silicified chondrocytes.

[0086] In other embodiments, the cells of the specific lineage are osteoblasts, and the method comprises drying of the silicified osteoblasts.

[0087] Dehydration

[0088] In some embodiments of the present invention, the method further comprises a step of dehydrating said silicified cells between step i) of silicificating cells and step ii) of calcinating a plurality of the silicified cells, wherein said dehydration step comprises or consists of contacting said scaffold with a compound gradient, wherein the compound is selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably wherein the compound is ethanol, preferably for a duration of at least 10 minutes. In some embodiments of the present invention, the method further comprises a step of dehydrating said silicified cells between step i) and ii), wherein said dehydration step comprises or consists of contacting said scaffold with a compound gradient, wherein the compound is ethanol, preferably for a duration of at least 10 minutes.

[0089] The term "dehydrating", as used herein, refers to the removal of water. The term is defined to mean a reaction in which a hydrogen atom and a hydroxyl group on adjacent carbon atoms in the molecule are removed as a water molecule. Any terms including this word at the beginning or end are understood in a similar manner. Dehydrating methods are known to those of ordinary skill in the art. In some embodiments, dehydrating is accomplished by contacting said scaffold with a compound gradient. The dehydrating compound is an compound that undergoes reaction of covalent bonds upon taking up the elements of water. Preferably a dehydrating reagent is an organic compound. Specific examples of a dehydrating compounds are ethanol, isopropyl alcohol, acetone and dioxane.

[0090] In some embodiments of the present invention, the dehydration step further comprises a step of drying said silicified cells, such as at a temperature between 30°C and 50°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours.

[0091] It should be understood that "drying" with respect to this method includes the removal of liquid components including ethanol and other solvents from the cells. The temperature at which said cells in the dehydration step of the method are dried (herein referred to as "drying temperature") may vary. The drying temperature may be held constant throughout the course of drying or may be made to vary. For example, drying may preferably be performed at least 25 °C, such as at least 30 °C, such as at least 35 °C, such as at least 40 °C.

[0092] In some embodiments of the present invention, the temperature of the drying step of the dehydration step is at least 25 °C, such as at least 30 °C, such as at least 35 °C, such as at least 40 °C.

[0093] In some embodiments of the present invention, the temperature of the drying step of the dehydration step is at most 55 °C, such as at most 50 °C, such as at most 45 °C, such as at most 40 °C.

[0094] In some embodiments of the present invention, the temperature of the drying step of the dehydration step is in a range of 25 °C to 55 °C, such as in a range of 30 °C to 50 °C, such as in a range of 35 °C to 45 °C.

[0095] In some embodiments of the present invention, the duration of the drying step of the dehydration step is at least 7 hours, such as at least 8 hours, such as at least 9 hours, such as at least 10 hours, such as at least 11 hours, such as at least 12 hours. In some embodiments of the present invention, the duration of the drying step of the dehydration step is at most 17 hours, such as at most 16 hours, such as at most 15 hours, such as at most 14 hours, such as at most 13 hours, such as at most 12 hours. In some embodiments of the present invention, the duration of the drying step of the dehydration step is in a range of 7 to 17 hours, such as in a range of 8 to 16 hours, such as in a range of 9 to 15 hours, such as in a range of 10 to 14 hours, such as in a range of 11 to 13 hours.

[0096] The method may, in addition to the dehydration step, also comprise a drying step as detailed herein above.

[0097] In some embodiments of the present invention, the temperature of the drying step of the dehydration step is at least 25 °C and the duration of the drying step of the dehydration step is at least 7 hours. In some embodiments of the present invention, the temperature of the drying step of the dehydration step is at most 55 °C and the duration of the drying step of the dehydration step is at most 17 hours. In some embodiments of the present invention, the temperature of the drying step of the dehydration step is in the range of 25 °C to 55 °C and the duration of the drying step of the dehydration step is in the range of 7 to 17 hours. In some embodiments of the present invention, the temperature of the drying step of the dehydration step is 40 °C and the duration of the drying step of the dehydration step is 12 hours.

[0098] Accordingly, in some embodiments, the cells of the specific cell lineage are chondrocytes, and the method comprises dehydration of the silicified chondrocytes.

[0099] In other embodiments, the cells of the specific lineage are osteoblasts, and the method comprises dehydration of the silicified osteoblasts.

[0100] Calcination

[0101] Calcination refers to the thermal treatment of a solid chemical compound, such as the silicified cells whereby the compound is raised to high temperature without melting under restricted supply of ambient oxygen, generally for the purpose of removing the organic matter. In some embodiments of the present invention, step ii) of calcinating a plurality of the silicified cells of the method comprises or consists of pyrolyzing said silicified cells at a high temperature in an inert atmosphere. In some embodiments of the present invention, the oxygen concentration in the inert atmosphere is at most 15 ppm, such as at most 10 ppm, such as in a range of 1 to 15 ppm, such as in a range of 3 to 12 ppm, preferably in a range of 5 to 10 ppm.

[0102] Pyrolysis of solid carbonaceous materials such as for example biomass or carbonaceous waste materials is carried out by heating the solid material under nonoxidising conditions. The solid carbonaceous materials is first heated and then decomposed into gaseous compounds. In a preferred embodiment, the reactions of the disclosure are carried out under a dry, inert atmosphere. Such an atmosphere may be created using any inert gas. The person skilled in the art will appreciate that different gases can be used for the process, such as nitrogen and argon.

[0103] In some embodiments of the present invention, step ii) of calcinating a plurality of the silicified cells of the method comprises or consists of pyrolyzing said silicified cells at a temperature of at least 400°C, such as at least 450°C, such as at least 500°C, such as at least 525°C, such as at least 550°C in an inert atmosphere.

[0104] In some embodiments of the present invention, step ii) of calcinating a plurality of the silicified cells of the method comprises or consists of pyrolyzing said silicified cells at a temperature of at most 700°C, such as at most 650°C, such as at most 600°C, such as at most 675°C, such as at most 650 °C in an inert atmosphere.

[0105] In some embodiments of the present invention, the step ii) of calcinating a plurality of the silicified cells of the method comprises or consists of pyrolyzing said silicified cells at a temperature of 400 to 700°C, such as 450 to 650°C, such as 500 to 600°C, such as 525 to 575°C in an inert atmosphere.

[0106] In some embodiments of the present invention, the calcination of step ii) of the method is performed for at least 1 hour, such as at least 2 hours, such as at least 3 hours. In some embodiments of the present invention, the step ii) of calcinating a plurality of the silicified cells of the method is performed for 1 to 5 hours, such as 2 to 4 hours, such as 2.5 to 3.5 hours.

[0107] The method may, in addition, comprise a drying step and / or a dehydration step as detailed herein above.

[0108] In some embodiments of the present invention, step ii) of calcinating a plurality of the silicified cells of the method comprises or consists of pyrolyzing said silicified cells at a temperature of at least 400°C for at least 1 hour. In some embodiments of the present invention, step ii) of the method comprises or consists of pyrolyzing said silicified cells at a temperature of at most 700°C for at most 5 hour. In some embodiments of the present invention, step ii) of the method comprises or consists of pyrolyzing said silicified cells at a temperature of 400°C to 700 °C for 1 to 5 hours. In some embodiments of the present invention, step ii) of the method comprises or consists of pyrolyzing said silicified cells at a temperature of 550 °C for 3 hours.

[0109] Accordingly, in some embodiments, the cells of the specific cell lineage are chondrocytes, and the method comprises calcination of the silicified chondrocytes.

[0110] In other embodiments, the cells of the specific lineage are osteoblasts, and the method comprises calcination of the silicified osteoblasts.

[0111] Cultivation of cells

[0112] In the context of the present disclosure, the terms "cultivation" of cells and "cell culture" are to be interpreted broadly, such that they encompass for example conditions in which cells divide, differentiate and / or proliferate, situations in which cells are maintained in a differentiated state with retention of at least one functional characteristic exhibited by the cell type when present in its natural environment, and situations in which stem cells are maintained in an undifferentiated state.

[0113] In some embodiments of the present invention, the scaffold is contacted with the immature cells for at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 5 days, such as at least 7 days, such as at least 10 days, such as at least 14 days, such as at least 21 days. In some embodiments of the present invention, the culture medium comprises differentiation factors.

[0114] In some embodiments of the present invention, the culture medium does not comprise differentiation factors.

[0115] In some embodiments of the present invention, the cells of a specific cell lineage or of a specific cell type are provided as a monolayer cell culture. Thus, in some embodiments, the cells of a specific cell lineage or of a specific cell type are grown in a single, flat layer on a surface.

[0116] In some embodiments of the present invention, the cells of a specific cell lineage or of a specific cell type are not a tissue.

[0117] In some embodiments, the immature cells, that the scaffold of chondrocytes is contacted with are mesenchymal stem cells.

[0118] In some embodiments, the immature cells, that the scaffold of osteoblasts is contacted with are mesenchymal stem cells.

[0119] Expression of marker genes

[0120] The progression of the differentiable cells to the desired cell lineage, or its maintenance in an undifferentiated state can be monitored by quantitating expression of marker genes characteristic of the desired cell lineage as well as the lack of expression of marker genes characteristic of differentiable cell types. One method of quantitating gene expression of such marker genes is through the use of quantitative PCR (q-PCR). Methods of performing q-PCR are well known in the art. Other methods that are known in the art can also be used to quantitate marker gene expression, such as enzymatic assays. Marker gene expression can be detected by using antibodies specific for the marker gene of interest. Furthermore, the person skilled in the art will also appreciate, that also other methods like single-cell sequencing, RNA-Seq and / or immunohistochemistry can be used to determine the cell type and / or cell lineage of said cells. In some embodiments of the present invention, the method further comprises a step comprising or consisting of measuring the increased expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in said differentiated cells.

[0121] As used herein "expression" refers to the process by which a gene or other nucleic acid molecule produces a polypeptide, such as Runx2 or alkaline phosphatase. It includes transcription of the gene or nucleic acid molecule into messenger RNA (mRNA) and translation of such mRNA into polypeptide(s). Methods to determine expression levels are known to the skilled person, and include inter alia Western blotting and Northern blotting.

[0122] As used herein, the term "enzymatic activity" refers to the amount of substrate consumed (or product formed) in a given time under given conditions. Enzymatic activity also may be referred to as "turnover number." For example, enzymatic ALP activity was calculated as picogram (pg) of the reaction product (i.e., p-nitrophenol) formed per minute.

[0123] In some embodiments of the present invention, the relative fold change in gene expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in said differentiated cells is at least 0.25, such as at least 0.5, such as at least 0,75 such as at least 1 , such as at least 1 , such as at least 1.8, such as at least 2, such as at least 2.5, wherein the relative fold change is calculated by reference to the gene expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in cells differentiated on glass coverslips.

[0124] In some embodiments of the present invention, the increased expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in said differentiated cells is measured after 1 day, such as after more than one day, such as after 2 days, such as after 3 days, such as after 4 days, such as after 5 days, such as after 10 days, such as after 14 days, such as after more than 14 days after contacting the scaffold with the immature cells.

[0125] In some embodiments of the present invention, the method further comprises a step comprising or consisting of measuring the absence of increased expression and / or activity of one or more marker associated with a cell lineage or cell type unrelated to the specific cell lineage or specific cell type of interest.

[0126] In some embodiments, the method further comprises a step comprising or consisting of measuring the increased expression and / or activity of one or more marker associated with chondrocytes, such as Collagen Type II (COL2A1), Aggrecan (ACAN), Sox9, Collagen Type IX (COL9A1), CollagenType XI (COL11A1), Cartilage Oligomeric Matrix Protein (COMP), Matrilin-1 (MATN1), Lubricin (PRG4) or Collagen Type X (COL10A1).

[0127] In some embodiments, the method further comprises a step comprising or consisting of measuring the increased expression and / or activity of one or more marker associated with osteoblasts, such as Alkaline Phosphatase (ALP), Osteocalcin (BGLAP), Collagen Type I (COL1A1), Osteopontin (SPP1), Bone Sialoprotein (BSP), Runx2 (CBFA1), Osterix (SP7), Integrin-binding Sialoprotein (IBSP), Dentin Matrix Protein 1 (DMP1) or Osteonectin (SPARC).

[0128] Silicified cells

[0129] This invention regards silicified cells replicating the morphology and topography both at the micro- and nano-level of living mammalian cells. This is achieved by the silica bioreplication technique which involves the use of biological samples (i.e. , mammalian cells, such as osteoblasts and / or chondrocytes) to direct the condensation of silicon dioxide (also known as silica) in a solution of tetramethoxylsilane. Following calcination of the resulting cell or tissue / silica composites, replicas fully made of silica with the unique morphology and surface topography of the biological structures used as templates are obtained.

[0130] In preferred embodiments of the present invention, the calcinated cells do not comprise organic matter as measured by the absence of carbon detection in any suitable spectroscopy technique, such as Energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and / or secondary ion mass spectroscopy (SIMS).

[0131] The person skilled in the art will appreciate, that other spectroscopy techniques can be used to determine the absence of carbon in the silicified cells. The person skilled in the art will appreciate that small amounts of carbon may be present in the silicified cells. In some embodiments, the silicified cells contain at most 15% carbon, such as at most 12% carbon, such as at most 10% carbon, such as at most 5% carbon such as at most 2% carbon, such as at most 1% carbon as measured by any suitable spectroscopy technique.

[0132] Stem Cells

[0133] Cells that are classically termed "progenitor" cells or "precursor" cells fall within the scope of the definition of "multilineage differentiation potential" on the basis that, under appropriate stimulatory conditions, they can give rise to cells of more than one somatic lineage. To the extent that reference to "stem cell" is made herein in terms of the cells generated by the disclosed method, this should be understood as a reference to a cell exhibiting multilineage differentiative potential as herein defined. In all embodiments, the stem cell is not obtained by methods that involve the use of human embryos for commercial or industrial purposes. In all embodiments, the stem cell is not obtained by methods that necessarily involve the destruction of a human embryo.

[0134] In one aspect, the invention concerns a population of differentiated cells of a specific cell lineage or a specific cell type of interest, obtained by the method as described herein.

[0135] In some embodiments of the present invention, the immature cells are stem cells.

[0136] In some embodiments of the present invention, the stem cells are pluripotent stem cells, such as wherein the stem cells are mesenchymal stem cells, such as wherein the stem cells are induced pluripotent stem cells, such as wherein the stem cells are embryonic stem cells, such as wherein the stem cells are hematopoietic stem cells, such as wherein the stem cells are neural stem cells, such as wherein the stem cells are epidermal stem cells, such as wherein the stem cells are intestinal stem cells, such as wherein the stem cells are hair follicle stem cells, such as wherein the stem cells are muscle stem cells, such as wherein the stem cells are cardiac stem cells, such as wherein the stem cells are liver stem cells, or such as wherein the stem cells are dental stem cells.

[0137] In a preferred embodiment of the present invention, the stem cells are mesenchymal stem cells. In some embodiments of the present invention the immature cells are progenitor cells.

[0138] In some embodiments of the present invention, the immature cells are progenitor cells or precursor cells of the specific cell lineage or cell type of interest. For instance, the immature cells are mesenchymal cells, which are precursors of chondrocytes, or which are precursors of osteoblasts.

[0139] Thus, in some embodiments, the stem cells are mesenchymal stem cells and the method comprises steps i) of silicification and ii) of calcination as detailed herein above, and may further comprise a drying step, a dehydration step or both a drying step and a dehydration step prior to step ii) of calcination.

[0140] In some embodiments of the present invention, the invention regards a method of differentiating mesenchymal stem cells to osteoblasts, said method comprising: i) Silicificating osteoblasts to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with mesenchymal stem cells; iv) Culturing the mesenchymal stem cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the mesenchymal stem cells into osteoblasts.

[0141] In some embodiments of the present invention, the invention regards a method of differentiating mesenchymal stem cells to chondrocytes, said method comprising: i) Silicificating chondrocytes to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with mesenchymal stem cells; iv) Culturing the mesenchymal stem cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the mesenchymal stem cells into chondrocytes.

[0142] In some embodiments of the present invention, the invention regards a method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type, said method comprising: i) Fixating and / or plating cells of said specific cell lineage of interest and / or of specific cell type as described herein; ii) Silicificating the cells of said specific cell lineage of interest and / or of a specific cell type of step i) to obtain silicified cells; iii) Drying the silicified cells of said specific cell lineage of interest and / or of a specific cell type as described herein; iv) Dehydrating the silicified cells of said specific cell lineage of interest and / or of a specific cell type as described herein; v) Calcinating a plurality of the silicified cells to obtain a scaffold; vi) Contacting the scaffold with immature cells; vii) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into said specific cell lineage of interest and / or to a specific cell type.

[0143] In some embodiments of the present invention, the invention regards a method of differentiating mesenchymal stem cells to osteoblasts, said method comprising: i) Fixating and / or plating osteoblasts as described herein; ii) Silicificating the osteoblasts of step i) to obtain silicified osteoblasts; iii) Drying the silicified osteoblasts as described herein; iv) Dehydrating the silicified osteoblasts as described herein; v) Calcinating a plurality of the silicified osteoblasts to obtain a scaffold; vi) Contacting the scaffold with mesenchymal stem cells; vii) Culturing the mesenchymal stem cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the mesenchymal stem cells into osteoblasts.

[0144] In some embodiments of the present invention, the invention regards a method of differentiating mesenchymal stem cells to chondrocytes, said method comprising: i) Fixating and / or plating chondrocytes as described herein; ii) Silicificating the chondrocytes of step i) to obtain silicified chondrocytes; iii) Drying the silicified chondrocytes as described herein; iv) Dehydrating the silicified chondrocytes as described herein; v) Calcinating a plurality of the silicified chondrocytes to obtain a scaffold; vi) Contacting the scaffold with mesenchymal stem cells; vii) Culturing the mesenchymal stem cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the mesenchymal stem cells into chondrocytes.

[0145] Cell lineage

[0146] As used herein, unless indicated otherwise or contradictory in context, the term "cell lineage" refers to the developmental history of a particular cell from its primary state in the fertilized egg or embryo through to its fully differentiated state. The different steps and phases involved in the development of a cell produces many intermediate cells which may be referred to as progenitor or precursor cells in the present application and form an integral part of the cell lineage. For example MSCs derived from pluripotent cells differentiate in vitro into osteogenic, chondrogenic and adipogenic cell lineages.

[0147] In some embodiments of the present invention, the immature cells are progenitor cells or precursor cells of the specific cell lineage or cell type of interest. In a preferred embodiment, the immature cells are progenitor cells or precursor cells of the osteogenic and / or chondrogenic cell lineage. In a preferred embodiment, the immature cells are progenitor cells or precursor cells of osteoblasts and / or chondrocytes.

[0148] In some embodiments of the present invention, said specific cell lineage or cell type is a mammalian cell lineage or cell type.

[0149] In some embodiments of the present invention, said specific cell lineage is selected from the group consisting of: epithelial lineage, neural lineage, hematopoietic lineage, muscle lineage, connective tissue lineage, adipocyte lineage, osteogenic lineage, germ cell lineage, endothelial lineage, pancreatic lineage, hepatic lineage, epidermal lineage, intestinal lineage.

[0150] Osteoblasts

[0151] The term "osteoblast" refers to a terminally-differentiated bone forming cell that deposits osteoid. The term "osteoid" refers to immature, unmineralized bone that is comprised primarily of type-l collagen. The term "osteogenic cells" refers to cells capable of mineralizing. Osteogenic cells include osteoblasts, osteoblast like cells, mesenchymal cells, fibroblast cells, fetal embryonic cells, stem cells, bone marrow cells and dura cells.

[0152] In some embodiments of the present invention, said specific cell lineage is osteogenic.

[0153] In some embodiments of the present invention, said specific cell type is selected from the group consisting of: osteoblasts, osteocytes and osteoclasts. I a preferred embodiment of the invention, said specific cell type is osteoblasts.

[0154] In some embodiments of the present invention, the specific cell lineage is osteogenic, such as wherein the specific cell type is osteoblasts, and the marker is selected from alkaline phosphatase (ALP), RLINX2, COL1A1 and OCN. The expression of the marker is determined as described in the section “Expression of markers”. The person skilled in the art will appreciate, that other markers can be used to determine osteoblasts.

[0155] In some embodiments of the present invention, the method further comprising a step comprising or consisting of determining enzymatic activity associated with said specific cell lineage or said specific cell type, preferably ALP activity. It is known in the art how to perform ALP assays to determine ALP activity.

[0156] In some embodiments of the present invention, the expression and / or activity of the one or more marker is performed by a cell lineage-specific or cell type-specific staining of the differentiated cells.

[0157] In some embodiments of the present invention, the cell lineage is osteogenic, such as wherein the specific cell type is osteoblasts, and wherein the staining is selected from the group consisting of: Alkaline phosphatase staining (ALP) and Alizarin red staining (ARS) staining.

[0158] Chondrocytes

[0159] The term "chondrocyte" as used herein refers to the art recognized use of the term for a cell type involved in cartilage formation and repair. The chondrocyte functions to produce extracellular matrix of proteoglycans and collagen. In some embodiments of the present invention, said specific cell lineage is chondrogenic. In some embodiments of the present invention, the said specific cell type is chondrocytes.

[0160] In some embodiments of the present invention, the specific cell lineage is chondrogenic, such as wherein the specific cell type is chondrocytes, and the marker is SOX9. The expression of the marker is determined as described in the section “Expression of markers”. The person skilled in the art will appreciate, that more marker can be used to determine chondrocytes.

[0161] In some embodiments of the present invention, the method further comprising a step comprising or consisting of determining enzymatic activity associated with said specific cell lineage or said specific cell type, preferably ALP activity.

[0162] In some embodiments of the present invention, the expression and / or activity of the one or more marker is performed by a cell lineage or cell type-specific staining of the differentiated cells.

[0163] In some embodiments of the present invention, the cell lineage is osteogenic, such as wherein the specific cell type is osteoblasts, and wherein the staining is selected from the group consisting of: Alkaline phosphatase staining (ALP) and Alizarin red staining (ARS) staining.

[0164] Cell differentiation

[0165] The invention further concerns a method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type. The term “differentiation” generally refers to the acquisition of a phenotype of a mature cell type from a less developmentally defined cell type, e.g. from a MSC to an osteoblast. During differentiation, a cell undergoes changes in gene expression, protein synthesis, and morphology, allowing it to perform specific roles within an organism. The person skilled in the art will appreciate, that differentiation can be determined by analysing specific markers, such as the expression of lineage-specific genes and proteins, changes in cell morphology, or the presence of distinct functional traits associated with a particular cell type. The process is regulated by genetic signals and environmental factors, and once differentiation occurs, cells typically retain their specialized state to contribute to the overall function of tissues and organs. For instance differentiation of chondrocytes can be determined by analysing markers specific to chondrocytes, while differentiation of osteoblasts can be determined by analysing markers specific to osteoblasts.

[0166] In some embodiments of the present invention, the immature cells are partially- differentiated cells. In some embodiments, the immature cells are not cancer cells. In some embodiments, the immature cells are cells that grow and divide controlled by the normal regulatory mechanisms that govern cell division, growth, and death. In some embodiments of the present invention, the differentiated cells are partially-differentiated cells. In some embodiments of the present invention, the differentiated cells are terminally-differentiated cells.

[0167] Totipotent cells can form an entire organism autonomously. Pluripotent cells can form almost all of the body’s cell lineages (endoderm, mesoderm, and ectoderm), including germ cells. Multi potent cells (e.g., MSCs) can form multiple cell lineages but cannot form all of the body’s cell lineages. Oligopotent cells can form more than one cell lineage but are more restricted than multipotent cells. Partially differentiated or lineage- committed cells can be called progenitor cells or precursor cells. Partially differentiated cells can be cells that have begun the process of differentiation, meaning they have started to acquire specific characteristics and functions of a particular cell type but have not yet fully completed this process. These cells may exist in a transitional state between multipotent stem cells and fully differentiated cells, retaining some potential to differentiate further. For example, they may express certain markers or perform some functions of their target cell type but are not yet fully specialized. Unipotent cells or monopotent cells can form a single differentiated cell lineage. Immature cells are cells, as defined herein, are all cells, which are not terminally differentiated. Terminally differentiated cells, as defined herein, are cells that lost their ability to proliferate permanently, although they continue to perform their specialized functions for a long time.

[0168] In some embodiments of the present invention, the invention regards a method of differentiating immature cells to partially-differentiated cells, said method comprising: i) Silicificating partially-differentiated cells to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into said partially-differentiated cells.

[0169] In some embodiments of the present invention, the invention regards a method of differentiating immature cells to terminally-differentiated cells, said method comprising: i) Silicificating terminally-differentiated cells to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into said terminally- differentiated cells.

[0170] In some embodiments of the present invention, the invention regards a method of differentiating partially-differentiated cells to terminally-differentiated cells, said method comprising: i) Silicificating terminally-differentiated cells to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with partially-differentiated cells; iv) Culturing the partially-differentiated cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the partially-differentiated cells into said terminally-differentiated cells.

[0171] In some embodiments of the present invention, the invention regards a method of differentiating immature cells to partially-differentiated cells, said method comprising: i) Fixating and / or plating partially-differentiated cells as described herein; ii) Silicificating the partially-differentiated cells of step i) to obtain silicified cells; iii) Drying the silicified cells as described herein; iv) Dehydrating silicified cells as described herein; v) Calcinating a plurality of the silicified cells to obtain a scaffold; vi) Contacting the scaffold with immature cells; vii) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into said partially- differentiated cells. In some embodiments of the present invention, the invention regards a method of differentiating immature cells to terminally-differentiated cells, said method comprising: i) Fixating and / or plating terminally-differentiated cells as described herein; ii) Silicificating the terminally-differentiated cells of step i) to obtain silicified cells; iii) Drying the silicified cells as described herein; iv) Dehydrating the silicified cells as described herein; v) Calcinating a plurality of the silicified cells to obtain a scaffold; vi) Contacting the scaffold with immature cells; vii) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into said terminally- differentiated cells.

[0172] In some embodiments of the present invention, the invention regards a method of differentiating partially-differentiated cells to terminally-differentiated cells, said method comprising: i) Fixating and / or plating terminally-differentiated cells as described herein; ii) Silicificating the terminally-differentiated cells to obtain silicified cells; iii) Drying the silicified cells as described herein; iv) Dehydrating the silicified cells as described herein; v) Calcinating a plurality of the silicified cells to obtain a scaffold; vi) Contacting the scaffold with partially-differentiated cells; vii) Culturing the partially-differentiated cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the partially-differentiated cells into said terminally-differentiated cells.

[0173] Scaffold

[0174] In one aspect, the invention concerns a scaffold comprising or consisting of silicified cells, wherein the scaffold promotes the differentiation of immature into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined herein, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type are as defined herein. The invention concerns a silica replica-containing scaffold, wherein the scaffold is used as a substrate to regulate the fate of immature and / or to differentiate immature cells into differentiated cells. Therefore, the scaffold comprises or consists of a multitude of silicified cells. For example, the scaffold of steps ii) and / or iii) comprises or consists of at most 50000 silicified cells / cm2, such as at most 40000 silicified cells / cm2, such as at most 30000 silicified cells / cm2, such as at most 25000 silicified cells / cm2, such as at most 20000 silicified cells / cm2cm2, such as at most 15000 silicified cells / cm2, such as at most 10000 silicified cells / cm2, such as at most 5000 silicified cells / cm2.

[0175] As used herein, “regulation of cell fate” may involve a combination of genetic, molecular, and environmental signals that guide cells toward specific pathways of differentiation, proliferation, or death. Key factors that regulate cell fate include transcription factors, signaling molecules, and epigenetic modifications, all of which influence the activation or repression of genes that drive cells to adopt particular roles. The gene expression can be determined with methods as described in the section “Expression of marker genes”.

[0176] In some embodiments of the present invention, the scaffold of steps ii) and / or iii) comprises or consists of 5000 to 50000 silicified cells / cm2, such as 10000 to 40000 silicified cells / cm2, such as 15000 to 30000 silicified cells / cm2, such as 20000 to 25000 silicified cells / cm2.

[0177] In some embodiments of the present invention, the silicified cells have a roughness as measured by atomic force microscopy characterized by a kurtosis value of in a range of 0.1 to 5.9, such as in a range of 0.5 to 5.5, such in a range of 1 to 5, such as in a range of 1 .5 to 4.5, such as in a range of 2 to 4.

[0178] In some embodiments of the present invention, the silicified cells have a roughness as measured by atomic force microscopy characterized by a skewness value of in a range of -3 to 3, such as in a range of -2.5 to 2.5, such as in a range of -2 to 2, such as in a range of -1 .5 to 1 .5, such as in a range of -1 to 1 , such as in a range of -0.5 to 0.5.

[0179] The person skilled in the art will appreciate, that several techniques can be used to perform a quality analysis of surface samples, such as geometrical analysis and surface roughness analysis. In a preferred embodiment of the invention, a surface roughness analysis is performed.

[0180] The person skilled in the art will appreciate, that several parameters can be used to evaluate the three-dimensional surface roughness of silicified cells. In a preferred embodiment, the kurtosis and skewness parameters is used to evaluate the three- dimensional surface roughness of said silicified cells. In a preferred embodiment, atomic force microscopy is used to quantitatively measure all 3 dimensions of a surface: lateral (x and y) and height (z). Kurtosis gives information on the tails of the distribution of z values on the surface. The skewness of a sample gives a measure of the asymmetry of the surface topography. The person skilled in the art will appreciate, that several tools are able to quantitatively measure surface roughness and can be used to determine the roughness of said silicified cells.

[0181] In one aspect, the invention concerns a scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold is obtained by a method comprising the steps of: i) Silicificating cells of a specific cell lineage or a specific cell type to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold, preferably wherein the scaffold, the silicified cells and the specific cell lineage or specific cell type are defined herein.

[0182] In some embodiments of the present invention, the method to obtain a scaffold further comprises fixing and / or plating the cells of a specific lineage or specific cell type prior to silicification.

[0183] In some embodiments of the present invention, step i) of the method to obtain a scaffold further comprises or consists of a step of contacting said cells with a silicic acid compound selected from the group of orthosilicic acid, tetramethoxysilane, tetraethoxysilane, or a mixture thereof for silicification, preferably wherein the silic acid tetramethoxysilane is used for silicification.

[0184] In some embodiments of the present invention, the method to obtain a scaffold further comprises a step of dehydrating said silicified cells between step i) and ii), wherein the dehydration comprises or consists of contacting said silicified cells with a compound selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably ethanol.

[0185] In some embodiments of the present invention, the method to obtain a scaffold further comprises drying said silicified cells overnight prior to step ii), preferably between steps i) and ii).

[0186] In some embodiments of the present invention, step ii) of the method to obtain a scaffold further comprises pyrolyzing said silicified cells at a high temperature in an inert atmosphere.

[0187] In some embodiments of the present invention, step ii) of the method to obtain a scaffold further comprises pyrolyzing said silicified cells at a temperature of at least 450°C, such as at least 500°C, such as at least 525°C, such as at least 550°C, in an inert atmosphere.

[0188] In some embodiments of the present invention, step ii) of the method to obtain a scaffold further comprises pyrolyzing said silicified cells at a temperature of 400 to 700°C, such as 450 to 650°C, such as 500 to 600°C, such as 525 to 575°C, in an inert atmosphere.

[0189] In some embodiments of the present invention, step ii) of the method to obtain a scaffold further comprises calcinating the silicified cells for at least 1 hour, such as at least 2 hours, such as at least 3 hours.

[0190] In some embodiments of the present invention, step ii) of the method to obtain a scaffold further comprises calcinating for 1 to 5 hours, such as 2 to 4 hours, such as 2.5 to 3.5 hours.

[0191] In some embodiments of the present invention, the scaffold comprises or consists of a monolayer of silicified cells.

[0192] In some embodiments of the present invention, the scaffold comprises or consists of chondrocytes. In some embodiments of the present invention, the scaffold comprises or consists of osteoblasts.

[0193] In some embodiments of the present invention, the scaffold comprises or consists of 0.1x104cells / cm2, such as 0.5x104cells / cm2, such as 1x104cells / cm2, such as 2x104cells / cm2, such as 5x104cells / cm2.

[0194] In some embodiments of the present invention, the scaffold comprises or consists of at least 0.1x104silicified cells / cm2, wherein said cells were pyrolyzed at a temperature of at least 450°C and calcinated for at least 1 hour. In some embodiments of the present invention, the scaffold comprises or consists of at least 5x104silicified cells / cm2, wherein said cells were pyrolyzed at a temperature of at most 700°C and calcinated for at most 5 hours. In some embodiments of the present invention, the scaffold comprises or consists of between 0.1x104and 5x104silicified cells / cm2, wherein said cells were pyrolyzed at a temperature in the range of 450°C to 700 °C and calcinated for 1 to 5 hours. In some embodiments of the present invention, the scaffold comprises or consists of between 0.1x104and 5x104silicified cells / cm2, wherein said cells were pyrolyzed at a temperature of 550°C and calcinated for 3 hours.

[0195] Use of a scaffold

[0196] Scaffolds comprising or consisting of silicified cells as a substrate can be used to fabricate inorganic replicas of intact mammalian tissue and can be used for bone tissue engineering applications.

[0197] In one aspect, the invention concerns the use of a scaffold comprising or consisting of silicified cells as a substrate to regulate the fate of immature and / or to differentiate immature cells into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined herein, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type of interest are defined herein.

[0198] In another aspect, the invention concerns the use of a scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold, the silicified cells and the specific cell lineage or the specific cell type of interest are defined herein. In some embodiments of the present invention, the scaffold is implanted into a body. In some embodiments of the present invention, the scaffold is embedded in a collagen matrix. In some embodiments of the present invention, the scaffold is used for bone tissue engineering applications.

[0199] In some embodiments of the present invention, the scaffold is used in a method of repairing tissue defect in a mammal. A tissue defect can refer to an area of damage, loss, or dysfunction within a specific tissue in the body, typically resulting from injury, disease, or surgery. This defect may involve the destruction or absence of cells, extracellular matrix, or both, impairing the tissue's normal structure and function. Tissue defects can vary in severity and may affect skin, muscle, bone, cartilage, or internal organs.

[0200] Purification moiety

[0201] The invention regards a scaffold comprising or consisting of silicified cells, wherein the scaffold promotes the differentiation of immature into differentiated cells. To reuse the scaffold, the silica replica comprise a purification moiety in some embodiments.

[0202] In some embodiments of the present invention, the method further comprises a step of conjugating the cells of said specific cell lineage or specific cell type to a purification moiety.

[0203] In some embodiments of the present invention, the purification moiety is magnetic, for example magnetic beads. The magnetic beads can be of any shape, including but not limited to spherical, rod, elliptical, cylindrical, disc, and the like. As used herein, the term "magnetic beads" refers to a nano- or microscale particle that is attracted or repelled by a magnetic field gradient or has a non-zero magnetic susceptibility. The magnetic bead can be paramagnetic or super-paramagnetic. The person skilled in the art, will know which materials can be used as magnetic beads. The person skilled in the art will also appreciate that other purification moieties can be used.

[0204] In some embodiments of the present invention, the step of conjugating is performed during or preferably prior to the step i) of silicification. In some embodiments of the present invention, the scaffold is reusable. In some embodiments of the present invention, the scaffold is sterilizable, preferably wherein the scaffold is autoclavable.

[0205] As used herein, the term "sterilize" or "sterilization" refers to a wide variety of techniques employed to attenuate, kill or eliminate harmful or infectious agents. Examples of sterilization procedures include, for example, gas plasma sterilization, steam sterilization, autoclaving, ozone sterilization, hydrogen peroxide sterilization, ethylene oxide sterilization, and irradiation. The term "sterilizable" as used herein refers to an item that can be sterilized.

[0206] Kit-of-parts

[0207] In one aspect, the invention concerns a kit-of-parts comprising: i) The scaffold as defined herein comprising silicified cells of a specific lineage or specific cell type of interest; ii) Immature cells, preferably stem cells, even more preferably as defined herein; iii) Optionally, instructions for use.

[0208] In some embodiments of the present invention, the kit of parts further comprises a cell culture medium comprising differentiation factors inducing differentiation into said specific lineage or said specific cell type.

[0209] Examples

[0210] Example 1 - Introduction

[0211] The present methods are not based on the traditional rational design and combinatorial screening, but nevertheless allow to direct the fate of SCs based on direct biomimicry. Specifically, fully synthetic silica particles replicating the morphology and topography both at the micro- and nano-level of living mammalian cells were generated. This was achieved by the silica bioreplication technique which involves the use of biological samples (e.g., mammalian cells, tissues but also small organisms) to direct the condensation of silicon dioxide (or silica) in a solution of tetramethoxylsilane (Kaehr et al., 2012 and Huang et al., 2015) Following calcination of the resulting cell or tissue / silica composites, replicas fully made of silica with the unique morphology and surface topography of the biological structures used as templates were obtained. Here, silica replicas of isolated mammalian cells were generated and then used as substrates to direct the differentiation of MSCs to the specific cell lineage that had been employed to fabricate the replicas (Figure 1).

[0212] Example 2 - Materials and methods

[0213] Materials

[0214] Phosphate buffered saline (PBS), paraformaldehyde (PFA), ethanol (EtOH), alizarin red S (ARS), BCIPO / NBT Liquid Substrate System, Alcian Blue 8GX, tetramethyl orthosilicate (TMOS), silica monodisperse, non-porous, 50 pm microspheres (SiC>2- MS), McCoy’s 5A Medium, sodium pyruvate, penicillin-streptomycin, fetal bovine serum (FBS), trypsin, PrestoBlue® cell viability reagent kit were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Mesenchymal stem cell growth medium BulletKit™ (MSCGM™) and mesenchymal stem cell growth medium osteogenic differentiation medium BulletKit™ were purchased from Lonza (Basel, CH). RNeasy Mini Kit was purchased from Qiagen (Hilden, GER). iTaq universal SYBR Green One- Step kit and mammalian protein extraction reagent (M-PER®) were purchased from ThermoFisher Scientific (Waltham, MA, USA). SensoLyte® pNPP Alkaline Phosphatase Assay Kit was purchased from Anaspec (Fremont, CA, USA). The human MSCs (hMSCs derived from bone marrow, PCS-500-012™) and the sarcoma osteogenic-2 (SaOS-2) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA).

[0215] Human Osteoblasts cells (HObs), human chondrocytes (HCs), chondrocyte growth medium C-27101 (ready-to-use), osteoblast growth medium C-39615 (ready-to-use) and chondrocyte differentiation medium (with inducers) C-28012 were purchased from Promocell (Heidelberg, GER).

[0216] Runt-related transcription factor 2 (RUNX2, Unique Assay ID: qHsaCID0006726), alkaline phosphatase (ALP, Unique Assay ID: qHsaCED0045991), osteocalcin (OCN, Unique Assay ID: qHsaCED0038437), collagen type I alpha 1 (COL1A1 , Unique Assay ID: qHsaCEP0050510), SRY-related HMG-box 9 (SOX9, Unique Assay ID: qHsaCEP0051345), collagen type II alpha 1 (COL2A1, Unique Assay ID: qHsaCEP0023931), aggrecan (ACAN, Unique Assay ID: qHsaCIP0026649) primers were purchased from Bio-Rad (CA, USA). The buffer solutions were prepared with ultrapure water (Milli-Q (MQ) gradient A 10 system, resistance 18 MV cm, TOC < 4 ppb, EMD Millipore, USA).

[0217] Cell culture

[0218] HObs and HCs

[0219] HObs and HCs were cultured in human osteoblast growth medium or human chondrocyte growth medium, respectively, at 37 °C and 5% (v / v) CO2. During cell culture, the medium was exchanged every 2 days and only cells between passages 2 and 7 were used for the experiments. Sub-confluent cells were detached from the culture flask by adding trypsin followed by 5 min of incubation at 37 °C and 5% (v / v) CO2. The cells were centrifuged at 220g for 5 min and re-suspended in fresh cell media.

[0220] SaOS-2.

[0221] SaOS-2 cells were cultured in McCoy’s 5A medium supplemented with 10% (v / v) fetal bovine serum, 1 % (v / v) sodium pyruvate, 1 % (v / v) penicillin-streptomycin (10 000 U mL-1and 10 pg mL-1) at 37 °C and 5% (v / v) CO2. The medium was exchanged every 2 days and only cells between passages 13 and 21 were used for the experiments. Subconfluent SaOS-2 cells were detached from the culture flask by adding trypsin followed by 5 min of incubation at 37 °C and 5% (v / v) CO2. The cells were centrifuged at 300g for 5 min and re-suspended in fresh cell media. hMSCs hMSCs were cultured in MSCGM™ BulletKit™ consisting of MSCBM™ basal media supplemented with MSCGM™ SingleQuots Supplement Kit and containing 10% FBS. Sub-confluent hMSCs were detached from the culture flask by incubation in trypsin for 5 min at 37 °C and 5% (v / v) CO2. The hMSCs suspension was then centrifuged at 200g for 5 min and re-suspended in new culture media at a density of 1500 viable cells cm-2in a T-175 flask. Only cell passages 1 to 4 were used for experiments. and characterization of the silica i-based substrates

[0222] Silicification and carbonization.

[0223] The substrates containing the silica replicas were fabricated following a previously reported protocol (Kaehr et al., 2012 and Wang et al., 2017). Briefly, before reaching confluence, the cells (HObs or HCs) were detached from the culture flask with trypsin and different cell aliquots were prepared (i.e. , containing 5 000, 10 000, 20 000 or 30 000 cells suspended in 0.5 mL). The different cell aliquots were added to 48-well plates containing clean and sterilized glass coverslips (1 cm in diameter) and incubated overnight at 37 °C and 5% CO2. Next, the cells were washed in PBS (2x), fixed with a 4% PFA solution for 30 min and washed again in PBS (3x). The now fixed cells were immersed in a solution of TMOS (100 mM, pH 3) and incubated overnight at 40 °C followed by the sequential incubation in MQ, 50% EtOH in MQ and pure EtOH for 10 min each. Then, the samples were dried at 40 °C and incubated for an additional 3 h at 550 °C for calcination. Silica replicas of HObs (i.e., HObs@SiC>2) and HCs (i.e., HCs@SiC>2) were obtained.

[0224] Dual interference contrast (DIC) and scanning electron microscopy (SEM) imaging. The morphology of the cells and the corresponding silica replicas was evaluated by DIC using an Olympus Inverted IX83 microscope (Olympus, TYO, JPN) equipped with a 63x oil immersion objective. SEM imaging was conducted making use of a FEI Quanta 200 ESEM FEG (FEI-Company, USA) equipped with an energy-dispersive X- ray spectroscopy detector (EDS, Oxford Instruments, UK). Images of the HObs@SiO2 and HCs@SiO2 were analyzed using Imaged software in order to assess the area (mm2) covered by the silica replicas which was subsequently normalized to the total area of the coverslip (%).

[0225] Atomic force microscopy (AFM) analysis.

[0226] Surface roughness of both HObs@SiO2 and HCs@SiO2 at a density of 20 000 replicas per well, was measured with an atomic force microscope (AFM; Multimode8 Nanoscope V controller, Bruker Corporation, Billerica, MA, USA). Imaging was performed in contact mode in air in ambient conditions, using standard Si cantilevers (Scanasyst-air model) with a tip radius of ~80 nm and a spring of 0.4 N / m. No dislodgement of materials on the surfaces was noted in these experiments, suggesting that there were no loosely bound contaminants that would create artefacts in images.

[0227] Osteogenic Differentiation of SaOS-2.

[0228] Cell viability.

[0229] The biocompatibility of the HObs@SiO2-based substrates was first evaluated by cell viability assays with SaOS-2 cells. Different numbers of HObs@SiO2 (i.e., 5000-30 000 HObs@SiO2) placed in glass coverslips (of 10 mm in diameter) and in a 48-well plate, were first sterilized with 70% EtOH and rinsed in PBS. Next, the HObs@SiC>2-based substrates were pre-incubated in 300 pL medium for 6 h at 37 °C and 5% CO2. Then, the medium was removed and a suspension of SaOS-2 at a density of 2 x 104cells in 300 pL was added to each well. The well plates were incubated at 37 °C and 5% CO2 for different pre-defined time intervals (up to 14 days) refreshing the medium every second day. After the incubation time, the wells were washed in PBS (2x) and a mixture of cell medium (135 pL) and PrestoBlue cell viability reagent (15 pL) was added to each well. After 1 h at 37 °C and in the dark, the fluorescence intensity of the reduced resazurin product was evaluated using a multimodal plate reader (Aex / Aem= 535 / 615 nm, Tecan Spark, TECAN, CH). As controls, SaOS-2 without HObs@SiC>2 (i.e., cells in glass coverslips) cultured in osteogenic media (containing 500 ng mL-1of human bone morphogenetic protein 2 (BMP-2)) and in growth media were considered. Cells in glass coverslips in growth media and PrestoBlue-containing cell media only were used as the positive and negative controls, respectively. The normalized cell viability (nCV) was calculated as follows:

[0230] (1) nCV (%) = (experimental value - negative control value) / (positive control value - negative control value) x 100.

[0231] Each condition was evaluated in triplicate in at least three independent experiments.

[0232] ALP activity.

[0233] The ALP activity of SaOS-2 cells depending on the amount of HObs@SiC>2 (i.e., 5000- 30 000 HObs@SiC>2) was evaluated for up to 14 days. As controls, SaOS-2 without HObs@SiC>2 (i.e., cells in coverslips) cultured in osteogenic media (containing 500 ng mL'1BMP-2) and in growth media only, were considered. The HObs@SiC>2were placed onto glass coverslips in 6-well plates and the SaOS-2 cells were seeded following the same procedure as for the cell viability assays (see section 2.4.1). At the different time intervals, the cells were first rinsed in PBS (3x) and then lysed with M-PER®. The cell lysates were then incubated with a solution p-nitrophenol phosphate (pNPP) at room temperature (RT) in the dark for 30 min using a SensoLyte® pNPP Alkaline Phosphatase Assay Kit. The absorbance (Abs) readings of the resulting p-nitrophenol were recorded at 405 nm using the multimodal plate reader. The total amount of p- nitrophenol was obtained using a calibration curve. The enzymatic ALP activity was calculated as pg of the reaction product (i.e., p-nitrophenol) per minute (30 min in total) and normalized to the number of cells obtained in a cell proliferation assay. For that, SaOS-2 cells were seeded onto HObs@SiC>2-based substrates as explained in section “Cell viability” above and incubated for the different time intervals. After incubation with the PrestoBlue-containing cell media, the number of cells was assessed by correlating the fluorescence intensity readings (Aex / Aem= 535 / 615 nm) to a calibration curve that had been generated by seeding increasing numbers of cells (data not shown).

[0234] Osteogenic Differentiation of hMSCs.

[0235] Cell viability.

[0236] The biocompatibility of the HObs@SiO2 was evaluated by cell viability assays with hMSCs after 1 , 7, 14 and 21 days of cell culture. As controls, hMSCs without HObs@SiO2 (i.e. , cells seeded onto glass coverslips) cultured in growth media only or in osteogenic media (containing 500 ng mL-1BMP-2), were considered. The cell viability was evaluated as previously described in section “Cell viability” but using hMSCs seeded in each well at a density of 20 000 cells in 300 pL. Different numbers of HObs@SiC>2 (i.e., 5000-30 000) were considered and each condition was evaluated in triplicate in at least three independent experiments.

[0237] ALP activity and staining.

[0238] The ALP activity was assessed as described in section “ALP activity” but using hMSCs instead of SaOS-2 cells which were seeded in 48-well plates at a density of 20 000 cells in 300 pL for each well. As controls, hMSCs without HObs@SiC>2 (i.e., cells seeded onto glass coverslips) cultured in osteogenic media (containing 500 ng mL-1BMP-2) and in growth media only, were considered. To normalize the ALP activity to the number of cells, a calibration curve was also generated by seeding increasing numbers of hMSCs (data not shown).

[0239] For the ALP staining, hMSCs were seeded onto 48-well plates containing glass coverslips with or without HObs@SiC>2 at a density of 20 000 cells per well. The well plates were incubated at 37 °C and 5% CO2 for 14 days refreshing the cell medium every second day. hMSCs cultured either in growth media only or osteogenic media (containing 500 ng mL-1BMP-2) were considered. After the cell culturing period, the ALP staining was conducted by using a BCIPO / NBT Liquid Substrate System kit and following the manufacturer’s protocol. Briefly, the cell-seeded wells were fixed using a 4% (v / v) PFA solution at RT for 30 min. The cells were then rinsed in PBS (3x) and incubated in a mixture of 300 pL BCIPO / NBT Liquid Substrate System at RT for 30 min. After washing with MQ (3x), the cells were observed by optical microscopy using the Olympus inverted microscope. The area of hMSCs displaying ALP signal (mm2) was analyzed using Image J and normalized to the total area of the coverslip (%). Each condition was also evaluated in triplicate in at least three independent experiments.

[0240] ARS staining.

[0241] The mineralized cell matrix was evaluated by ARS staining after culturing hMSCs for 1 and 21 days. Specifically, hMSCs (20 000 cells in 300 pL) were seeded onto 48-well plates containing 20 000 HObs@SiO2 placed onto a glass coverslip and incubated in growth media at 37 °C and 5% CO2 refreshing the cell medium every second day. After the incubation period, the cells were washed in PBS (3x) and fixed using a 4% (v / v) PFA solution for 20 min at RT. The coverslips were washed again in MQ (3x) and incubated in 300 pL of an ARS solution (40 mM in MQ, pH 4.2) for 20 min at RT under continuous shaking. Several washes in MQ were conducted to remove the excess stain and the samples were evaluated by optical microscopy using the inverted microscope. The area of hMSCs displaying ARS signal (mm2) was analyzed using Image J and normalized to the total area of the coverslip (%). Each condition was also evaluated in triplicate in at least three independent experiments.

[0242] Osteogenic gene expression by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

[0243] The osteogenic differentiation of hMSCs was further evaluated by gene expression analysis after 1 , 3, 7, 14 and 21 days of cell culture. The hMSCs were seeded in 48- well plates at a density of 20 000 cells in 300 pL for each well. Silica replica-based substrates containing 20 000 HObs@SiO2were evaluated. As controls, hMSCs seeded onto glass coverslips, smooth SiO2 surface and onto SiO2-MSs were considered. The smooth SiO2 surface and substrates containing the SiO2-MSs were prepared as shown in the Supporting Information. Successful fabrication of smooth SiO2 surface was verified by contact angle measurements. The hydrophilicity of the glass coverslips significantly increased after silanization, with the water contact decreasing from 70 ± 3.9° to 19.7 ± 3.1°, indicating that smooth SiO2 surfaces were successfully obtained (Data not shown).

[0244] The RLINX2, ALP, COL1A1 and OCN osteogenic-related genes and the SOX9 chondrogenic-related gene were considered. The total mRNA was extracted from the samples at the different time intervals using the RNeasy Mini Kit following the manufacturer's protocol. In brief, the hMSCs were resuspended in RLT buffer (the buffer was added directly to the wells) and lysed by passing the suspension at least 5 times through a 20-gauge needle using an RNAse-free syringe. Prior loading them onto the RNeasy columns provided in the kit, one volume of 70% EtOH was added, and the solution was mixed by pipetting. The RNA was thoroughly washed on the column, the DNA removed using DNase I and the RNA was eluted using 15 pL of RNase-free water. Abs measurements at 260 nm were conducted using a spectrophotometer (Nanodrop ND-1000, NanoDrop Technologies, Montchanin, DE, USA) to confirm the purity of the RNA and to calculate its concentration. The extracted RNA was stored at -80 °C until RT-qPCR procedure. 100 ng of RNA were reverse transcribed into cDNA and directly used as RT-qPCR template by using the Biorad iTaq Universal SYBR Green One-step kit following the manufacturer’s instructions. The RT-qPCRs were conducted by adding 20 pL of a reaction mixture containing 10 pL of iTaq universal SYBR Green One-Step mix, 100 ng of RNA template, 0.25 pL iScript reverse transcriptase, 1 pL of primers (300 nM) and nuclease-free water up to 20 pL of reaction mix volume. The specific primers to amplify specific genes of osteoblast differentiation were selected from Bio-Rad Laboratories (Bio-Rad, Hercules, CA, USA). For the reaction conditions, an initial activation was conducted by heating at 50 °C for 10 min, a second heating of 95 °C for 1 min, followed by 40 cycles of thermal denaturation at 95 °C for 15 s and then annealing and elongation 55 °C for 30 s.

[0245] All samples were normalized to the corresponding expression levels of the housekeeping gene p-actin (reference gene) and fold changes were calculated with respect to hMSCs cultured directly onto the wells (i.e. , without any additional substrate) at day 1 of culture as follows:

[0246] Cq corresponds to the median value for the quantification cycle of each sample measured in triplicate. E is the amplification efficiency and is obtained from the slope of the log-linear portion of the calibration curve, as:

[0247] (3) E = w[-^slope]

[0248] Chondroqenic Differentiation of hMSCs.

[0249] Alcian Blue staining.

[0250] For the Alcian Blue staining, hMSCs were seeded onto 48-well plates containing glass coverslips with or without HCs@SiO2 at a density of 20 000 hMSCs per well. The well plates were incubated at 37 °C and 5% CO2 for 21 days refreshing the cell medium every second day. hMSCs cultured either in growth media or chondrogenic media were considered. After the 21 days, the cell-seeded wells were fixed using a 4% (v / v) PFA solution at RT for 30 min. The cells were then rinsed in PBS (3x) and first incubated with a pH 2.5 acetic acid solution for 5 min and with an Alcian Blue stain (1 % Alcian Blue in glacial acetic acid solution at pH 2.5) at RT for 30 min. After washing with MQ (3x) to remove the excess stain, the cells were observed by optical microscopy using the inverted optical microscope. Each condition was evaluated in triplicate in at least three independent experiments. The area of hMSCs displaying Alcian Blue signal (mm2) was analyzed using Image J and normalized to the total area of the coverslip (%).

[0251] Chondrogenic gene expression by qRT-PCR.

[0252] The chondrogenic differentiation of hMSCs cultured onto HCs@SiC>2-substrates was assessed by gene expression analysis after 1 , 3, 7, 14 and 21 days of cell culture as previously described in section 2.5.4. 20 000 HObs@SiC>2 per well were evaluated. As controls, hMSCs seeded onto glass coverslips, smooth SiC>2 surface and onto SiC>2- MSs were considered. The SOX9, COL2A1 and ACAN chondrogenic-related genes were considered and the RLINX2 osteogenic-related gene was also considered as a control.

[0253] Statistical Analysis.

[0254] The data was analyzed using one-way ANOVA with a confidence level of 95% (a = 0.05) followed by Tukey's multiple comparison post hoc test (*p < 0.05, **p < 0.01 , *** p < 0.001 and **** p < 0.0001) in an SPSS software (IBM, Armonk, NY, USA).

[0255] Example 3 - Fabrication and Characterization of the Silica Replica-based Substrates

[0256] To demonstrate the versatility of the approach in directing the differentiation of SCs, two different substrates were fabricated. HObs and HCs were chosen as two model cell lines to fabricate the silica replica-based substrates (Figure 1). The silica bioreplication process was conducted by first fixating the HObs and the HCs seeded onto glass substrates followed by a silicification step using TMOS. The resulting HObs / SiO2 and HCs / SiO2 composites were dehydrated using an EtOH gradient and, the silica-based substrates (i.e. , HObs@SiO2 and HCs@SiO2), were obtained after calcination. Figure 2A shows the DIC images of sub-confluent HObs and HCs cultured onto glass substrates and the corresponding silica replicas after silicification and carbonization. The images show how both HObs@SiC>2 and HCs@SiC>2 displayed the same density as their native counterparts. ~68 and 91 % of the total area is covered when 20 000 and 30 000 HObs@SiC>2, respectively, were present in the wells. For the HCs@SiC>2, the area covered was slightly higher with 71 and 95% of the total surface being covered when 20 000 and 30 000 silica replicas, respectively, were present in the wells. The SEM images of the two cell types at the different stages of the fabrication process (i.e., after being fixed, following silicification and upon drying and calcination), indicate that the structural features of the original living cells are well preserved (Figure 2B). The EDS spectra show how, as expected, the fixated HObs and HCs were mainly constituted by carbon and oxygen. After the silicification step, silicon could also be distinguished. Importantly, upon calcination, the carbon peak could not be detected anymore which indicates that the organic matter had been successfully eliminated. The roughness of both types of substrates at a density of 20 000 replicas per well, was also evaluated by AFM (Not shown). The results showed an almost double arithmetic average height (Ra) for HObs@SiC>2-based substrates as compared to the HCs@SiC>2- based ones. The three-dimensional (3D) surface evaluation was given by the kurtosis (Rku) and skewness (RSk) parameters. RkUrevealed values greater than 3 for both types of substrates which indicates a distribution curve with quite some high peaks and low valleys. The RSk oscillated around zero for both SiCh-based substrates thus indicating even amounts of peaks and valleys.

[0257] Example 4 - Evaluation of the osteogenic ability of the Hobs@SiO2-based substrates

[0258] ALP activity

[0259] For the first in vitro investigation, the osteogenic ability of the HObs@SiC>2-based substrates was assessed using SaOS-2 cells. SaOS-2 cells are a type of preosteoblast cell type able to differentiate into osseous tissue. In contrast to other bone- related cell types such as e.g., MG-63, SaOS-2 display a highly mature phenotype close to that of primary osteoblasts. To assess the bone-differentiating ability of the substrates, the ALP activity, which is a strong indicator for early-stage osteogenesis, of pre-seeded SaOS-2 cells was elucidated. Prior to assessing the ALP activity, the biocompatibility of the substrates depending on the amount of HObs@SiC>2 was evaluated. Figure 3Ai shows the nCV readings for SaOS-2 cells incubated with increasing amounts of HObs@SiC>2 (i.e., from 5000, 10 000, 20 000 and 30 000) for up to 7 days. These cell densities correspond to HObs@SiC>2:SaOS-2 ratios of 1 :4, 1 :2, 1 :1 and 1.5:1 , respectively. The results, which have been normalized to the cells seeded onto glass coverslips without the addition of silica replicas, show a slight decrease in nCV (to -85%) for some of the conditions. However, these observed reductions in nCV are not significant and they are not promoted by the HObs@SiC>2, as indicated by the fact that an nCV of -100% was obtained for the highest studied amount of HObs@SiC>2 and for the longest studied time interval (i.e., 30 000 HObs@SiC>2 for 7 days). Following on, the ALP activity was evaluated (Figure 3Aii). On day one, for cells seeded onto glass coverslips, the ALP signal was of only --0.001 pg cells-1min-1while, for the HObs@SiO2-based substrates, a sharp increase in ALP activity was detected for all the studied conditions. At day 1 , the substrate with the lowest amount of HObs@SiO2 (i.e., 5000 HObs@SiO2 which promoted only a -30.8% coverage of the total surface area (data not shown)) resulted already in an ALP signal of -0.07 pg cells'1min-1, which corresponds to a ~70-fold increase as compared to SaOS-2 cells seeded onto glass coverslips. This result indicates that a HObs@SiC>2:SaOS-2 ratio of 1 :4 is already enough to promote a marked increase in ALP activity. The highest ALP activity was obtained for substrates containing 20 000 HObs@SiC>2 (i.e., -0.08 pg cells'1min-1) and any further increase in the number of HObs@SiC>2, did not promote an increment in the ALP signal. Remarkably, these results indicate that only a 46-68% coverage of the total surface by the HObs@SiC>2 was sufficient to achieve the highest ALP activity. Thus, a further coverage of 91% of the total area (as shown for a density of 30 000 HObs@SiC>2) did not result in enhanced ALP activity. Following 3 days of cell culture, the ALP signal was still markedly higher for the HObs@SiC>2-based substrates as compared to the control of glass coverslips. In that case, the glass coverslips rendered an ALP activity of only -0.015 pg cells'1min-1while an ALP signal in the range of -0.040-0.045 pg cells'1min-1, was observed for substrates containing HObs@SiC>2. For the longest incubation times (i.e., 7 days), no significant differences in ALP activity could be detected depending on the type of substrate. This is not surprising since ALP is an early detection marker. Thus, these results show the ability of the silica replicas to promote the osteogenic differentiation of SaOS-2.

[0260] As a next step, the osteogenic potential of the HObs@SiC>2-based substrates and of the BMP-2 growth factor (osteogenic media) were compared. Bone morphogenetic proteins (BMPs) play an essential role in bone formation and are involved in the remodeling of fractured bone. Amongst them, BMP-2 is well known to mediate cell proliferation, differentiation and to promote osteogenesis. Although BMPs are FDA- approved for orthopedic surgery and have been extensively used as osteoinductive factors for the regeneration of bone tissue, their widespread use is hampered by important limitations. One of the main drawbacks is that it remains very challenging to administer them at an optimal dose with minimized side effects while also preventing their denaturation.

[0261] The ALP activity of SaOS-2 cells was evaluated again but this time up to 14 days and considering an additional control of cells cultured onto glass coverslips in osteogenic media containing BMP-2. Only substrates containing 10 000 and 20 000 HObs@SiO2 were considered. To rule out any potential detrimental effect onto the seeded cells, the nCV was first evaluated. Figure 3Bi shows no significant decrease in nCV for any of the studied conditions. After 1 day of cell culture, the highest ALP signal was obtained for cells seeded onto HObs@SiC>2-based substrates (Figure 3Bii). The two studied amounts of HObs@SiC>2 (i.e., 10 000 and 20 000) promoted a very similar ALP activity, which was ~8 times higher than that obtained of SaOS-2 cells seeded onto glass coverslips and in the presence of BMP-2 (osteogenic media). In that case, the ALP signal was of only -0.01 pg cells-1min-1. After 3 days of cell culture, the results were different and, while cells cultured in glass coverslips and growth media only still rendered a very low ALP signal (i.e., -0.017 pg cells-1min-1); the cells cultured onto glass coverslips and osteogenic media and onto the HObs@SiC>2-based substrates promoted a very similar ALP activity (i.e., -0.045-0.050 pg cells-1min-1). Interestingly, for SaOS-2 seeded in coverslips and in osteogenic media, the highest ALP activity was observed at day 3 as opposed to the HObs@SiC>2-based substrates which yielded the highest activity at day 1 . This result suggests that the as-prepared silica replica-based substrates are not only able to enhance but also to accelerate the osteogenic differentiation of SaOS-2 when compared to the widely used osteogenic inducers. For the longer incubation times (i.e., 7 and 14 days) the ALP signals remained low, and no marked differences could be observed depending on the type of cell culture medium and the presence of HObs@SiC>2. This is not surprising since ALP is an early osteogenic marker. The ability of the as-prepared HObs@SiC>2-based substrates to direct the osteogenic differentiation of hMSCs was then assessed. In a similar way as was done for the SaOS-2 cells, the biocompatibility of the silica-based substrates towards hMSCs was first evaluated. The nCV readings for hMSCs cultured for up to 21 days onto HObs@SiC>2-based substrates containing increasing amounts of silica replicas (i.e. , from 5000 to 30 000) showed slight decreases (down to -90% in nCV) and increases (up to -110%) in nCV for some of the conditions. However, these fluctuations were not significant. Additionally, the highest nCV readings were observed for hMSCs cultured onto the HObs@SiC>2-based substrates for the longest incubation period (i.e., a nCV of -115% after 21 days of cell culture). This indicates that there was no cytotoxicity even when the hMSCs were cultured onto HObs@SiC>2-based substrates for 21 days. Next, the osteogenic potential of the substrates towards hMSCs was first evaluated by monitoring the ALP activity. Figure 4B shows how, in contrast to the results showed for SaOS-2 cells (see Figure 3), the highest levels of ALP activity are obtained after 14 days of cell culture independently of the studied condition. At day 14, the ALP signal of hMSCs cultured onto glass coverslips and in growth media was only of -0.02 pg cells-1min-1while, as expected, a markedly higher signal was detected for hMSCs cultured in osteogenic media (i.e., 0.038 pg cells-1min-1which is -1.9-fold increase). Importantly, a very similar ALP activity (i.e., of -0.034-0.038 pg cells-1min-1) was also detected for hMSCs cultured onto silica-based substrates containing at least 10 000 HObs@SiC>2 without any addition of soluble osteogenic factors (i.e., in growth media only). The differences in ALP signal for substrates containing 10 000, 20 000 and 30 000 HObs@SiC>2were non-significant. Therefore, these results indicate that the as- prepared HObs@SiC>2-based substrates are as good as the biochemical factors (i.e., BMP-2) in promoting the early-stage osteogenic differentiation of hMSCs. To corroborate these results, ALP staining was conducted following 14 days of cell culture (Figure 5). For cells cultured in glass coverslips, a deep violet color can be observed only for cells cultured in osteogenic medium. In agreement with the ALP activity results, cells cultured onto HObs@SiC>2-based substrates also displayed this deep violet color. The intensity of this violet color was higher when hMSCs had been cultured onto the silica replica-based substrates and also using osteogenic media, indicating an additive effect of the two conditions. Quantitative data were extracted to determine the percentage of cells displaying ALP staining. In agreement with Figure 4B, the least marked ALP staining was observed for hMSCs seeded onto glass coverslips and in growth media since only 0.35% of the area covered by the cells showed a deep violet color. In contrast, 81.53% of the area covered by cells displayed ALP staining when hMSCs were cultured onto glass coverslips but using osteogenic media. Importantly, the same trend as for the ALP activity results was observed since a very similar value of stained cells (of 79.07%), was observed for hMSCs seeded onto the HObs@SiC>2- based substrates and growth media.

[0262] ARS staining.

[0263] During osteogenesis, the cells secrete mineral deposits and, hence, mineralized nodule formation is a typical phenotype to identify osteoblasts. Thus, the mineralization degree was evaluated by detecting the deposition of calcium at different stages of cell culture (i.e. , 1 and 21 days) using ARS staining. No substantial color change was observed for cells seeded onto the glass coverslips for up to 21 days. In contrast, massive mineralization nodules were formed and stained red for hMSCs cultured onto HObs@SiC>2-based substrates containing 20 000 silica replicas (Figure 6). Thus, ARS staining revealed that the substrates containing the silica replicas had also a positive effect on the mineralization of the pre-seeded hMSCs. Table 1 shows the quantitative data. The results show marked ARS staining for cells grown onto the HObs@SiC>2- based substrates as compared to glass coverslips since, 26.8% of all the hMSCs displayed a dark red color, as compared to only 3.05% of all the hMSCs that had been cultured onto the glass coverslips.

[0264] Table 1. Quantification of the percentage of cells displaying Alizarin Red S (ARS) staining for hMSCs cells cultured onto glass coverslips or onto HObs@SiO2-based substrates containing 20 000 silica replicas derived from human osteoblasts (HObs) (i.e., HObs@SiO2) for 1 or 21 days. The cells were cultured in growth medium.

[0265] Gene expression analysis.

[0266] To draw a stronger conclusion on the effect of the HObs@SiC>2-based substrates onto the osteogenic differentiation of hMSCs, the expression of four osteogenic genes (i.e., RUNX2, ALP, COL1A1 and OCN) after 1 , 3, 7, 14 and 21 days of cell culture was quantified by RT-qPCR. As a control to demonstrate the cell-type specificity of our approach, the chondrogenic gene S0X9 was also considered. HObs@SiC>2-based substrates containing 10 000 silica replicas were considered since such an amount already yielded maximum ALP activity (Figure 4B). As controls, apart from hMSCs seeded onto glass coverslips, a smooth SiC>2 substrate and SiCh-MSs of 50 pm in diameter were considered. The aim for choosing the smooth SiC>2 substrate and SiC>2- MSs was to discern the effects of the morphology and topography from the material itself (i.e., smooth SiC>2 substrate, SiCh-MSs, HObs@SiC>2 replicas are constituted by the same material but with different morphology and rugosity). Figures 7A and 7B show how the expression levels of RUNX2 and ALP were upregulated at day 1 and day 3, respectively, while lower expression levels were obtained for the longer incubation times (i.e., 7, 14 and 21 days). This is not surprising since both RUNX2 and ALP are considered as markers for early osteoblast differentiation. For cells cultured in growth media only, a significantly higher upregulation of RUNX2 can be observed for the silica- containing substrates (i.e., smooth SiC>2, SiCh-MSs and HObs@SiC>2) as compared to the glass coverslips. This is shown by a ~2-fold increase in RUNX2 expression at day 3 (Figure 7A). Thus, in the absence of soluble biochemical factors, the material (i.e., silica or glass) has an important effect in directing the fate of hMSCs. This is not surprising since the role of silicon (Si) in bone repair and skeletal development has been acknowledged for many years. In this context, Si-containing biomaterials such as Si-substituted calcium phosphates, Si-based bioglasses and bioceramics have been widely investigated for bone tissue engineering applications.32For cells cultured in osteogenic media, as expected, enhanced upregulation is observed for all the studied groups (i.e., glass coverslips, smooth SiC>2, SiCh-MSs and HObs@SiC>2). However, in this case, not only the material but also the morphology and topology had a profound effect on the early osteogenic differentiation of hMSCs, as can be seen from the rugosity values in terms of Ra, RSk and RkUfor the HObs@SiC>2-based substrates (not shwon). There were no significant differences between the smooth SiC>2 and SiCh-MSs groups. In combination with osteogenic factors, as much as a ~4-fold increase in gene expression could be detected for the HObs@SiC>2 as compared to the glass coverslips. This result suggests a synergistic effect of the biochemical osteogenic inducers and the HObs@SiC>2. The material had also an important effect as shown by the ~2.3-fold increase in gene expression when comparing both smooth SiC>2 and SiCh-MSs to the glass coverslips. Although the effects were not as marked as for RLINX2, ALP expression was also up-regulated in the presence of HObs@SiC>2 (Figure 7B). As an early osteogenic marker, the ALP expression was at its highest value on day 7 for all the four different substrates. Although non-significant, for cells cultured in growth media only, the highest ALP expression was obtained for HObs@SiC>2 substrates, and such an expression was twice as high than the one detected for the glass coverslips. The ALP expression for hMSCs cultured in osteogenic media was higher than the expression observed in growth media for all the studied substrates. In this case, the differences among the substrates were very pronounced. The HObs@SiC>2 promoted the highest ALP expression which was as much as ~3.3-fold higher than the one promoted by the glass coverslips and ~2.7- and ~2.0-fold higher than the one rendered by hMSCs cultured onto smooth SiC>2 and SiCh-MS, respectively. In this case, the material (i.e. , silica versus glass) also had an effect on osteogenic differentiation. Interestingly, there was also a difference depending on the cells being cultured onto flat and smooth SiC>2 or onto SiCh-MSs since SiCh-MSs resulted in a ~1.5-fold higher ALP expression. Thus, all in all, while slight differences depending on the substrate could be observed when using growth media only, when used in combination with biochemical osteogenic inducers, the HObs@SiC>2-based substrates remarkably outperform all the other substrates in the early osteogenic differentiation of hMSCs. This indicates that not only the material, but also the morphology and topography of the substrate has a central effect. Thus, these results highlight that the silica replica-containing substrates stimulate the osteogenic differentiation of hMSCs at an early stage.

[0267] COL1A1 expression appeared later (at day 7) and as expected, the highest expression levels of OCN were detected at day 14, since OCN expression usually accompanies the mineralization process (Figure 7C and Figure 7D). Similar results were obtained for these two genes, with the highest expression levels obtained for hMSCs cultured onto HObs@SiC>2-based substrates and osteogenic media. Regarding the expression of COL1A1 , in the absence of soluble osteogenic inducers, the silica-containing substrates (i.e., smooth SiC>2, SiCh-MSs and HObs@SiC>2) promoted a ~3.75-fold increase in gene expression. In contrast, in the presence of osteogenic media, a marked difference could be detected in the expression levels depending on the silica- based substrate used. The highest expression levels were again observed for the HObs@SiC>2 which were -1.7- and ~1.6-fold higher than the ones promoted by smooth SiC>2, SiC>2-MSs, respectively. Remarkably, when osteogenic media was used, the HObs@SiC>2 replicas promoted a ~3-fold enhanced expression of COL1 A1 as compared to hMSCs cultured onto glass coverslips. Very similar results were observed for the OCN expression. In this case, at day 21 in osteogenic media, HObs@SiC>2 promoted a ~2.4-fold increase in gene expression as compared to glass substrates. When comparing among the different silica-based substrates, up regulation promoted by HObs@SiC>2was -1.88- and -1.74-fold higher than smooth SiC>2, SiCh-MSs, respectively. When osteogenic media was used, HObs@SiC>2 replicas boosted the expression of OCN since, a -2.4-fold higher expression, was detected for the silica replicas as compared to the glass coverslips.

[0268] All in all, the enhanced gene expression for the different studied osteogenic markers suggests that the silica replica-containing scaffolds display significant osteoinductivity. Importantly, to verify the cell type-specific ability of the HObs@SiO2-based substrates, the expression of the chondrogenic SOX9 gene was also evaluated. Figure 7E shows that there was no upregulation for this marker for any of the studied substrates thus highlighting the ability of the as-prepared silica replicas to direct the differentiation of stem cells to pre-defined cell lineages.

[0269] Example 5 - Evaluation of the Chondrogenic Ability of the HCs@SiO2-based Substrates

[0270] To demonstrate the versatility of this approach and, since MSCs are also broadly used for repairing cartilage defects, the potential of the silica replica-based substrates to promote the chondrogenesis of hMSCs was assessed.

[0271] Alcian Blue staining.

[0272] As a first step, the chondrogenic potential of the HCs@SiC>2-based substrates was assessed by conducting Alcian Blue staining for glycosaminoglycan matrix synthesis, which is an important component of the ECM of the cartilage tissue. For that, the hMSCs were cultured onto bare glass coverslips and onto the HCs@SiC>2-based substrates for 21 days and both growth media and chondrogenic media were considered. Macroscopic images of the different substrates were captured, showing positive staining with Alcian Blue for all the treatment groups. A slightly stronger staining was observed for the samples containing the HCs@SiC>2-based substrates as compared to the glass coverslips. These results are corroborated by the presence of a strong Alcian Blue staining for hMSCs cultured onto the HCs@SiC>2-based substrates while a weaker blue staining was detected for cells seeded onto the glass coverslips. Table 2 shows the quantitative data extracted from the stainings (Data not shown). The results showed enhanced Alcian Blue staining for cells grown onto the HCs@SiC>2- based substrates as compared to glass coverslips when growth medium is used since, 15.4% of all the hMSCs, displayed a blue color as compared to only 1.3% of the hMSCs cultured onto the glass coverslips. Likewise, when chondrogenic media is used, 29.88% and 49.46% of the cells displayed a bluish color upon being cultured onto glass coverslips and HCs@SiC>2-based substrates, respectively, for 21 days.

[0273] Table 2. Quantification of the percentage of cells displaying Alcian Blue (AB) staining for hMSCs cells cultured onto glass coverslips or onto HCs@SiO2-based substrates containing 20 000 silica replicas (i.e. , HCs@SiO2) for 21 days. The cells were cultured either in growth media or in chondrogenic media. The silica replicas have been derived from human chondrocytes (HCs).

[0274] Gene expression analysis.

[0275] The cartilage differentiation properties of the HCs@SiC>2-based substrates were next evaluated by monitoring by RT-qPCR the expression of four chondrogenic genes following 1 , 3, 7, 14 and 21 days of cell culture. Specifically, S0X9, COL2A1 and ACAN were considered. COL2A1 is a type of collagen specifically expressed in cartilage tissues while ACAN is the main proteoglycan component of the ECM of cartilage tissue. S0X9 is expressed both in chondrogenic progenitor cells and mature chondrocytes, playing a crucial role in chondrogenesis by directing the expression of other specific cartilage markers such as the aforementioned COL2A1 and ACAN. As a control to demonstrate the cell-type specificity of our approach, the osteogenic marker RLINX2 was evaluated. The same amounts of silica replicas as for the osteogenic differentiation were considered (i.e. , 20 000 HCs@SiC>2). To differentiate the effects from the material (i.e., glass versus silica) from the morphology and topology rendered by the silica replicas, a smooth SiC>2 substrate and a coverslip containing SiCh-MSs were also evaluated. Figure 8 shows the gene expression analysis of the hMSCs cultured onto the four different substrates. SOX9 was upregulated at days 14 and 21 but only in the presence of chondrogenic media (Figure 8A). In this case, both the type of material as well as the morphology and topology had an important effect on the chondrogenic differentiation of hMSCs with the strongest effect observed for the HCs@SiC>2-based substrates. For SOX9 no differences in expression were detected in growth media only. In contrast, when chondrogenic media was used, the HCs@SiC>2 promoted a ~2.66-fold increase in gene expression as compared to the glass coverslip. When comparing the different silica-containing substrates, the upregulation of SOX9 was 1.6 times higher for HCs@SiC>2 as compared to smooth SiC>2 and SiCh-MSs. For COL2A1 , which had the highest expression on day 3, the differences among the different substrates were not that obvious (Figure 8B). Similar expression levels were observed in growth media only (i.e., ~2-2.5 relative fold change). The COL2A1 upregulation was much more pronounced when the hMSCs were cultured in chondrogenic media. In that case, a ~1.8-fold increase was observed for HCs@SiC>2 as compared to the glass coverslips. However, the morphology and the topology did not have such a dramatic effect on the chondrogenic differentiation since only a ~1.2-fold increase in upregulation was observed when comparing the HCs@SiC>2 to the smooth SiC>2 and SiCh-MSs. For the case of ACAN expression, the results were slightly different. Upregulation was only detected in chondrogenic media, and the three silica- based substrates had a similar effect since a ~1.8-fold increase was observed when comparing HCs@SiC>2, smooth SiC>2 and SiCh-MSs to the glass coverslips. This indicates that the influence of the morphology and topography onto the late stage chondrogenic differentiation was not that strong.

[0276] Thus, these results confirmed that the differentiation of hMSCs can be regulated by a cell-topography effect. The upregulation of SOX9, COL2A1 and ACAN indicated that silica replicas prepared from live chondrocytes can guide the differentiation of hMSCs towards a chondrogenic lineage. Interestingly, all SiC>2, SiCh-MSs and HCs@SiC>2 replicas promoted a ~0.5-fold increase in RUNX2 expression compared to the glass coverslip. However, there were no marked differences depending on the silica surface employed. This indicated that the material and not that much the topography had an effect on the osteogenic differentiation of hMSCs.

[0277] Example 6 - Conclusions

[0278] In this work, silica replicas of mammalian cells have been generated and used as a biomimetic platform to physically direct the differentiation of stem cells, here hMSCs, into either the osteogenic or the chondrogenic lineage. The silica bioreplication approach is an ingenious way to capture biological structures on silica. With this method, which can essentially be considered as a fossilization process, the organic content of the biological sample is replaced by inorganic substances. The method allows for the replication of the topographical details of the biological surface with a high level of spatial accuracy.

[0279] The silica replication procedure entailed the incubation of mammalian cells (i.e. , HObs and HCs) in TMOS, which results on the specific deposition of silica within the molecularly crowded cell environment. After drying and exposure to extreme temperatures, the nano- to macroscale cellular heterogeneity of the living cells used as templates is captured and preserved. The SEM micrographs of the two types of silica replicas (i.e., HObs@SiC>2 and HCs@SiC>2) demonstrated preservation of the architectural features of the living cells (Figure 2). The EDS spectra confirmed that the replicas were constituted by silica, and that all the organic components had been successfully removed following calcination. The two types of substrates where also characterized by AFM and the Ra, Rku and RSk values were identified. Important differences were observed for the average height parameter with an almost double Rafor the HObs@SiC>2 as compared to the HCs@SiC>2. However, the RkUand RSk values were quite similar for both types of substrates indicating a distribution curve with similar numbers of high peaks and valleys.

[0280] To demonstrate the potential of the as-prepared silica replicas to direct cell differentiation, MSCs, which are the most multipotent adult SCs in our body, have been chosen. Since they can differentiate into multiple cell lineages including osteoblasts, chondrocytes or adipocytes, MSCs have the potential to form fat, bone and cartilage tissues. MSCs are particularly relevant for tissue engineering applications due to their excellent accessibility since they can be extracted with minimally invasive procedures from bone marrow, adipose tissue or from the blood of the umbilical cord. Additionally, MSCs can be obtained from adult tissue and, hence, they are free from ethical concerns as compared to other cell types such as embryonic SCs. By being an autologous cell source, MSCs can also bypass important complications resulting from immune rejection. However, despite all these advantages, the potential application of MSCs in tissue engineering is hampered due to the difficulties to guide their differentiation towards a specific cell lineage when using the traditional biochemical factors. Thus, approaches to guide their differentiation eliminating or at least minimizing the use of soluble chemical compounds are really sought after.

[0281] The above results indicate that the silica replica-containing substrates can promote the differentiation of MSCs towards osteogenic or chondrogenic lineages. Specifically, in the absence of osteogenic soluble factors, HObs@SiC>2-based substrates can remarkably enhance ALP activity for both the pre-osteoblast SaOS-2 cells and hMSCs. Actually, HObs@SiC>2 substrates at a density of 10 000 replicas per well are equally good than the osteogenic media in promoting ALP activity for both studied cell types (Figure 3 and Figure 4). In agreement with the ALP activity results, ALP staining is shown for hMSCs cultured onto HObs@SiC>2-based substrates using growth media only (Figure 5). Finally, enhanced degree of mineralization was observed when HObs@SiC>2 replicas were used as the substrates as shown by ARS staining (Figure 6). The effect of the HObs@SiC>2 replicas on the osteogenic differentiation of hMSCs was less pronounced when gene expression analysis was conducted (Figure 7). In the absence of soluble osteogenic inducers, enhanced expression of the osteogenic markers RLINX2, ALP, COL1A1 and OCN was detected for hMSCs incubated with the HObs@SiC>2-based substrates. However, such differences were non-significant. In contrast, marked and significant enhanced upregulation for the four studied genes was detected when the HObs@SiC>2 replicas were used in combination with osteogenic media, suggesting a potential synergistic effect. Interestingly, when the osteogenic inducers were present, increased gene expression was observed for the smooth SiC>2 and the SiCh-MS used as controls, suggesting an effect from the silica material. Remarkably, no upregulation of the chondrogenic SOX9 markers was observed when the HObs@SiC>2-based substrates were used in growth or osteogenic media which highlights the cell type-specificity of the present approach.

[0282] The differentiation of hMSCs towards the chondrogenic lineage, although to a lesser extent, could also be promoted when silica replicas are used (i.e. , in this case HCs@SiC>2 replicas were considered). This has been demonstrated through Alcian Blue staining and gene expression analysis. Enhanced upregulation for the SOX9, COL2A1 and ACAN was only observed when the HCs@SiC>2-based substrates were used in combination with chondrogenic media. The observed higher gene upregulation when the silica replicas were used to induce the osteogenic differentiation as compared to the chondrogenic differentiation of MSCs, can be attributed to both the mechanical and osteoinductive properties of silica which can further influence cell differentiation to an osteogenic lineage. A very slight, although statistically significant, upregulation of the RLINX2 marker was observed for the silica-based substrates (i.e., smooth SiC>2, SiC>2-MSs and HCs@SiC>2 replicas) as compared to the glass coverslips when growth media was used. However, since there were no marked differences among the different silica-containing substrates, these osteogenic properties are likely to be attributed to the material and not to the topography of the substrates.

[0283] All in all, these results reveal that the micro- and nanoscale topographical cues of the silica replicas can effectively promote the differentiation of SCs. Cells in general have the ability to sense the topographical features in their environment and transmit this information to their nucleus as biochemical signals. As such, the topographic cues can strongly influence the polarity of the attached cells by contact guidance through cytoskeletal and nucleo-skeletal rearrangement. These changes that take place within the subcellular structures are correlated with modifications in the gene expression profile which, subsequently, affects their differentiation into a particular lineage. The interspacing between the different topographical features is also an important parameter that needs to be taken into consideration.

[0284] The silica replicas of intact tissues or isolated cell types as presented in this work are non-toxic (silicon dioxide is considered as GRAS (generally regarded as safe) under FDA regulations), biodegradable and can potentially be prepared at high quantities and low cost. Hence, these silica replicas may find applications for in vitro cell culture vessels to reduce the use of growth factors thus minimizing the costs and risks of side effects. Furthermore, due to the mechanical properties and osteoinductive properties of silica, the as-prepared replicas could be embedded within scaffolds for bone tissue engineering applications.

[0285] We have presented a biomimetic substrate to induce the differentiation of SCs based on silica replicas of mammalian cells using a “live-cell” template strategy. Specifically, substrates containing silica particles replicating the morphology and topography of HObs and HCs have been able to direct the differentiation of hMSCs towards the osteogenic and chondrogenic lineages, respectively. RT-qPCR results have shown how MSCs cultured onto substrates containing silica replicas fabricated from mature HObs showed enhanced upregulation of specific osteogenic markers (i.e., RLINX2, ALP, COL1A1 and OCN). Importantly, for all the genes studied, the highest expression levels were obtained for hMSCs incubated onto the silica-based substrates and in the presence of osteogenic media. This fact indicates a potential synergistic effect. Similar results were obtained for MSCs cultured onto silica replica-based substrates that had been derived from mature HCs, which demonstrates the versatility of this approach. Similarly, the RT-qPCR data shows the highest gene expression of specific chondrogenic genes (i.e., SOX9, COL2A1 and ACAN) for MSCs cultured onto the silica replicas as compared to glass coverslips. Also, for chondrogenesis, for all the genes studied, the highest expression levels were detected for the combination of the silica replicas and the chondrogenic media.

[0286] All in all, the above data demonstrate that silica replicas can be successfully employed as substrates to physically regulate the fate of stem cells. At a minimum, such an approach can reduce the usage of biochemical soluble compounds (i.e., growth factors) which have important limitations such as off-target effects, or challenges in obtaining optimal release kinetics. Additionally, the as-prepared substrates could reduce the high costs of differentiation since they can be autoclavable and reusable.

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[0292] Nicolas, J.; Magli, S.; Rabbachin, L.; Sampaolesi, S.; Nicotra, F.; Russo, L. 3D Extracellular Matrix Mimics: Fundamental Concepts and Role of Materials Chemistry to Influence Stem Cell Fate. Biomacromolecules 2020, 21 (6), 1968-1994. DOI: 10.1021 / acs.biomac.0c00045.

[0293] Wang, W.; Cui, H.; Zhang, P.; Meng, J.; Zhang, F.; Wang, S. Efficient Capture of Cancer Cells by Their Replicated Surfaces Reveals Multiscale Topographic Interactions Coupled with Molecular Recognition. ACS Appl. Mater. Interfaces. 2017, 9 (12), 10537-10543. DOI: 10.1021 / acsami.7b01147.

[0294] Items

[0295] 1. A method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with the immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type.

[0296] 2. A method of obtaining cells of a specific cell lineage of interest and / or of a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type, and obtaining cells of said specific cell lineage and / or of said specific cell type.

[0297] 3. The method according to any one of the preceding items, wherein the immature cells are stem cells.

[0298] 4. The method according to any one of the preceding items, wherein the immature cells are progenitor cells.

[0299] 5. The method according to any one of the preceding items, wherein the immature cells are partially-differentiated cells.

[0300] 6. The method according to any one of the preceding items wherein the immature cells are progenitor cells or precursor cells of the specific cell lineage or cell type of interest.

[0301] 7. The method according to any one of the preceding items wherein the differentiated cells are partially-differentiated cells.

[0302] 8. The method according to any one of the preceding items wherein the differentiated cells are terminally-differentiated cells.

[0303] 9. The method according to any one of items 3 to 8, wherein the stem cells are pluripotent stem cells, such as wherein the stem cells are mesenchymal stem cells, such as wherein the stem cells are induced pluripotent stem cells, such as wherein the stem cells are embryonic stem cells, such as wherein the stem cells are hematopoietic stem cells, such as wherein the stem cells are neural stem cells, such as wherein the stem cells are epidermal stem cells, such as wherein the stem cells are intestinal stem cells, such as wherein the stem cells are hair follicle stem cells, such as wherein the stem cells are muscle stem cells, such as wherein the stem cells are cardiac stem cells, such as wherein the stem cells are liver stem cells, or such as wherein the stem cells are dental stem cells. The method according to any one of the preceding items, wherein said specific cell lineage or cell type is a mammalian cell lineage or cell type. The method according to any one of the preceding items wherein said specific cell lineage or cell type is a human cell lineage or cell type. The method according to any one of the preceding items, wherein said specific cell lineage is selected from the group consisting of: epithelial lineage, neural lineage, hematopoietic lineage, muscle lineage, connective tissue lineage, adipocyte lineage, osteogenic lineage, germ cell lineage, endothelial lineage, pancreatic lineage, hepatic lineage, epidermal lineage, intestinal lineage. The method according to any one of the preceding items, wherein said specific cell lineage is osteogenic. The method according to any one of the preceding items, wherein said specific cell lineage is chondrogenic. The method according to any one of the preceding items, wherein said specific cell type is selecting from the group consisting of: osteoblasts, osteocytes and osteoclasts. The method according to any one of the preceding items, wherein said specific cell type is chondrocytes. 17. The method according to any one of the preceding items, further comprising fixing and / or plating said cells of the specific cell lineage or specific cell type prior to silicification, preferably wherein said cells have not reached confluence.

[0304] 18. The method according to any one of the preceding items, wherein step i) comprises or consists of: i) Incubating said cells of said specific cell lineage or specific cell type at a temperature between 25 °C and 55 °C, such as between 30°C and 50°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours in the presence of a silicic acid compound, preferably selected from the group of orthosilicic acid, tetramethoxysilane, tetraethoxysilane and their derivatives such as methyltriethoxysilane, methyltrimethoxysilane, or a mixture thereof, and ii) Optionally, contacting said cells of said specific cell lineage or specific cell type with a compound selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably wherein the compound is ethanol, preferably for a duration of at least 10 minutes; optionally, the compound is first added as a 50% (v / v) solution, then as a 100% (v / v) solution.

[0305] 19. The method according to item 18, wherein the incubation temperature in step i) is at least 25 °C, such as at least 30 °C, such as at least 35 °C, such as at least 40 °C.

[0306] 20. The method according to item 18, wherein the incubation temperature in step i) is at most 55 °C, such as at most 50 °C, such as at most 45 °C, such as at most 40 °C.

[0307] 21. The method according to item 18, wherein the incubation temperature in step i) is in a range of 25 °C to 55 °C, such as in a range of 30 °C to 50 °C, such as in a range of 35 °C to 45 °C. 22. The method according to item 18, wherein the incubation duration in step i) is at least 7 hours, such as at least 8 hours, such as at least 9 hours, such as at least 10 hours, such as at least 11 hours, such as at least 12 hours.

[0308] 23. The method according to item 18, wherein the incubation duration in step i) is at most 17 hours, such as at most 16 hours, such as at most 15 hours, such as at most 14 hours, such as at most 13 hours, such as at most 12 hours.

[0309] 24. The method according to item 18, wherein the incubation duration in step i) is in a range of 7 to 17 hours, such as in a range of 8 to 16 hours, such as in a range of 9 to 15 hours, such as in a range of 10 to 14 hours, such as in a range of 11 to 13 hours.

[0310] 25. The method according to item 18, wherein the orthosilicic acid used in step i) is tetramethoxysilane, preferably a solution of tetramethoxysilane.

[0311] 26. The method according to item 25, wherein the tetramethoxysilane solution has a tetramethoxysilane concentration of at least 70 mM, such as at least 80 mM, such as at least 90 mM, such as at least 100 mM.

[0312] 27. The method according to item 25, wherein the tetramethoxysilane solution has a tetramethoxysilane concentration of at most 130 mM, such as at most 120 mM, such as at most 110 mM, such as at most 100 mM.

[0313] 28. The method according to item 25, wherein the tetramethoxysilane solution has a tetramethoxysilane concentration of 100 mM.

[0314] 29. The method according to item 25, wherein the tetramethoxysilane solution has a pH of at least 1 , such as at least 1 .2, such as at least 1 .5, such as at least 1.8, such as at least 2, such as at least 2.2, such as at least 2.5, such as at least 2.8, such as at least 3.

[0315] 30. The method according to item 25, wherein the tetramethoxysilane solution has a pH of at most 5, such as at most 4.8, such as at most 4.5, such as at most 4.2, such as at most 4, such as at most 3.8, such as at most 3.5, such as at most 3.2, such as at most 3.

[0316] 31. The method according to item 25, wherein the tetramethoxysilane solution has a pH of 3.

[0317] 32. The method according to any one of the preceding items, further comprising a step of drying said cells of said specific lineage or said specific cell type before step ii), such as at a temperature between 30°C and 50°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours.

[0318] 33. The method according to item 32, wherein the temperature of the drying step is at least 25 °C, such as at least 30 °C, such as at least 35 °C, such as at least 40 °C.

[0319] 34. The method according to item 32, wherein the temperature of the drying step is at most 55 °C, such as at most 50 °C, such as at most 45 °C, such as at most 40 °C.

[0320] 35. The method according to item 32, wherein the temperature of the drying step is in a range of 25 °C to 55 °C, such as in a range of 30 °C to 50 °C, such as in a range of 35 °C to 45 °C.

[0321] 36. The method according to item 32, wherein the duration of the drying step is at least 7 hours, such as at least 8 hours, such as at least 9 hours, such as at least 10 hours, such as at least 11 hours, such as at least 12 hours.

[0322] 37. The method according to item 32, wherein the duration of the drying step is at most 17 hours, such as at most 16 hours, such as at most 15 hours, such as at most 14 hours, such as at most 13 hours, such as at most 12 hours.

[0323] 38. The method according to item 32, wherein the duration of the drying step is in a range of 7 to 17 hours, such as in a range of 8 to 16 hours, such as in a range of 9 to 15 hours, such as in a range of 10 to 14 hours, such as in a range of 11 to 13 hours. The method according to any one of the preceding items, further comprising a step of dehydrating said silicified cells between step i) and ii), wherein said dehydration step comprises or consists of contacting said scaffold with a compound gradient, wherein the compound is selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably wherein the compound is ethanol, preferably for a duration of at least 10 minutes. The method according to item 39, wherein the dehydration step further comprises a step of drying said silicified cells, such as at a temperature between 30°C and 50°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours. The method according to item 40, wherein the temperature of the drying step is at least 25 °C, such as at least 30 °C, such as at least 35 °C, such as at least 40 °C. The method according to item 40, wherein the temperature of the drying step is at most 55 °C, such as at most 50 °C, such as at most 45 °C, such as at most 40 °C. The method according to item 40, wherein the temperature of the drying step is in a range of 25 °C to 55 °C, such as in a range of 30 °C to 50 °C, such as in a range of 35 °C to 45 °C. The method according to item 40, wherein the duration of the drying step is at least 7 hours, such as at least 8 hours, such as at least 9 hours, such as at least 10 hours, such as at least 11 hours, such as at least 12 hours. The method according to item 40, wherein the duration of the drying step is at most 17 hours, such as at most 16 hours, such as at most 15 hours, such as at most 14 hours, such as at most 13 hours, such as at most 12 hours. The method according to item 40, wherein the duration of the drying step is in a range of 7 to 17 hours, such as in a range of 8 to 16 hours, such as in a range of 9 to 15 hours, such as in a range of 10 to 14 hours, such as in a range of 11 to 13 hours. The method according to any one of the preceding items, wherein step ii) comprises or consists of pyrolyzing said silicified cells at a high temperature in an inert atmosphere. The method according to any one of the preceding items, wherein step ii) comprises or consists of pyrolyzing said silicified cells at a temperature of at least 400°C, such as at least 450°C, such as at least 500°C, such as at least 525°C, such as at least 550°C in an inert atmosphere. The method according to any one of the preceding items, wherein step ii) comprises or consists of pyrolyzing said silicified cells at a temperature of 400 to 700°C, such as 450 to 650°C, such as 500 to 600°C, such as 525 to 575°C in an inert atmosphere. The method according to any one of the preceding items, wherein the calcination of step ii) is performed for at least 1 hour, such as at least 2 hours, such as at least 3 hours. The method according to any one of the preceding items, wherein the calcination of step ii) is performed for 1 to 5 hours, such as 2 to 4 hours, such as 2.5 to 3.5 hours. The method according to any one of the preceding items, wherein the scaffold of steps ii) and / or iii) comprises or consists of at most 50000 silicified cells / cm2, such as at most 40000 silicified cells / cm2, such as at most 30000 silicified cells / cm2, such as at most 25000 silicified cells / cm2, such as at most 20000 silicified cells / cm2cm2, such as at most 15000 silicified cells / cm2, such as at most 10000 silicified cells / cm2, such as at most 5000 silicified cells / cm2. The method according to any one of the preceding items, wherein the scaffold of steps ii) and / or iii) comprises or consists of 5000 to 50000 silicified cells / cm2, such as 10000 to 40000 silicified cells / cm2, such as 15000 to 30000 silicified cells / cm2, such as 20000 to 25000 silicified cells / cm2.

[0324] 54. The method according to any one of the preceding items, wherein the culture medium comprises differentiation factors.

[0325] 55. The method according to any one of the preceding items, wherein the culture medium does not comprise differentiation factors.

[0326] 56. The method according to any one of the preceding items, wherein the scaffold is contacted with the immature cells for at least 1 day, such as at least 2 days, such as at least 3 days, such as at least 5 days, such as at least 7 days, such as at least 10 days, such as at least 14 days, such as at least 21 days.

[0327] 57. The method according to any one of the preceding items, further comprising a step comprising or consisting of measuring the increased expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in said differentiated cells.

[0328] 58. The method according to any one of the preceding items, wherein relative fold change in gene expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in said differentiated cells is at least 0.25, such as at least 0.5, such as at least 0,75 such as at least 1 , such as at least 1 , such as at least 1.8, such as at least 2, such as at least 2.5, wherein the relative fold change is calculated by reference to the gene expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in cells differentiated on glass coverslips.

[0329] 59. The method according to any one of the preceding items, wherein the increased expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in said differentiated cells is measured after 1 day, such as after more than one day, such as after 2 days, such as after 3 days, such as after 4 days, such as after 5 days, such as after 10 days, such as after 14 days after contacting the scaffold with the immature cells. 60. The method according to any one of the preceding items, further comprising a step comprising or consisting of measuring the absence of increased expression and / or activity of one or more marker associated with a cell lineage or cell type unrelated to the specific cell lineage or specific cell type of interest.

[0330] 61. The method according to item 60, wherein the specific cell lineage is osteogenic, such as wherein the specific cell type is osteoblasts, and the marker is selected from alkaline phosphatase (ALP), RLINX2, COL1A1 and OCN.

[0331] 62. The method according to item 60, wherein the specific cell lineage is chondrogenic, such as wherein the specific cell type is chondrocytes, and the marker is SOX9.

[0332] 63. The method according to any one of the preceding items, further comprising a step comprising or consisting of determining enzymatic activity associated with said specific cell lineage or said specific cell type, preferably ALP activity.

[0333] 64. The method according to any one of items 60 to 63, wherein the expression and / or activity of the one or more marker is performed by a cell lineage or cell type-specific staining of the differentiated cells.

[0334] 65. The method according to item 64, wherein the cell lineage is osteogenic, such as wherein the specific cell type is osteoblasts, and wherein the staining is selected from the group consisting of: Alkaline phosphatase staining (ALP) and Alizarin red staining (ARS) staining.

[0335] 66. The method according to item 64, wherein the cell lineage is chondrogenic, such as wherein the specific cell type is chondrocytes, and wherein the staining is Alcian blue (AB) staining.

[0336] 67. The method according to any one of the preceding items, wherein the cells of a specific cell lineage or of a specific cell type are provided as a monolayer cell culture. 68. The method according to any one of the preceding items, wherein the cells of a specific cell lineage or of a specific cell type are not a tissue.

[0337] 69. The method according to any one of the preceding items, further comprising a step of conjugating the cells of said specific cell lineage or specific cell type to a purification moiety.

[0338] 70. The method according to item 69, wherein the purification moiety is magnetic, preferably magnetic beads.

[0339] 71. The method according to any one of items 69 to 70, wherein the step of conjugating is performed during or preferably prior to the step i) of silicification.

[0340] 72. Use of a scaffold comprising or consisting of silicified cells as a substrate to regulate the fate of immature and / or to differentiate immature cells into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined in any one of the preceding items, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type of interest are as defined in any one of the preceding items.

[0341] 73. A scaffold comprising or consisting of silicified cells, wherein the scaffold promotes the differentiation of immature into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined in any one of the preceding items, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type are as defined in any one of the preceding items.

[0342] 74. The scaffold according to item 73, wherein the scaffold is reusable.

[0343] 75. The scaffold according to any one of items 73 to 74, wherein the scaffold is sterilizable, preferably wherein the scaffold is autoclavable. 76. The scaffold according to any one of items 73 to 75, wherein the silicified cells have a roughness as measured by atomic force microscopy characterized by a kurtosis value of in a range of 0.1 to 5.9, such as in a range of 0.5 to 5.5, such in a range of 1 to 5, such as in a range of 1.5 to 4.5, such as in a range of 2 to 4.

[0344] 77. The scaffold according to any one of items 73 to 75, wherein the silicified cells have a roughness as measured by atomic force microscopy characterized by a skewness value of in a range of -3 to 3, such as in a range of -2.5 to 2.5, such as in a range of -2 to 2, such as in a range of -1.5 to 1.5, such as in a range of - 1 to 1 , such as in a range of -0.5 to 0.5.

[0345] 78. A scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold is obtained by a method comprising the steps of: i) Silicificating cells of a specific cell lineage or a specific cell type to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold, preferably wherein the scaffold, the silicified cells and the specific cell lineage or specific cell type are as defined in any of the preceding items.

[0346] 79. The scaffold according to item 78, wherein the scaffold is obtained by the method according to any of items 1 to 71.

[0347] 80. The scaffold according to any one of items 78 to 79, wherein the method further comprises fixing and / or plating the cells of a specific lineage or specific cell type prior to silicification.

[0348] 81. The scaffold according to any one of items 78 to 80, wherein step i) further comprises or consists of a step of contacting said cells with a silicic acid compound selected from the group of orthosilicic acid, tetramethoxysilane, tetraethoxysilane, or a mixture thereof is used for silicification, preferably wherein the silic acid tetramethoxysilane is used for silicification. The scaffold according to any one of items 78 to 81 , wherein the method further comprises a step of dehydrating said silicified cells between step i) and ii), wherein the dehydration comprises or consists of contacting said silicified cells with a compound selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably ethanol. The scaffold according to item 82, wherein the method further comprises drying said silicified cells overnight prior to step ii), preferably between steps i) and ii). The scaffold according to any one of the items 78 to 83, wherein step ii) further comprises pyrolyzing said silicified cells at a high temperature in an inert atmosphere. The scaffold according to any one of the items 78 to 84, wherein step ii) further comprises pyrolyzing said silicified cells at a temperature of at least 450°C, such as at least 500°C, such as at least 525°C, such as at least 550°C, in an inert atmosphere. The scaffold according to any one of the items 78 to 85, wherein step ii) further comprises pyrolyzing said silicified cells at a temperature of 400 to 700°C, such as 450 to 650°C, such as 500 to 600°C, such as 525 to 575°C, in an inert atmosphere. The scaffold according to any one of the items 78 to 86, wherein step ii) further comprises calcinating the silicified cells for at least 1 hour, such as at least 2 hours, such as at least 3 hours. The scaffold according to any one of the items 78 to 87, wherein step ii) further comprises calcinating for 1 to 5 hours, such as 2 to 4 hours, such as 2.5 to 3.5 hours. The scaffold according to any one of items 78 to 88, wherein the scaffold comprises or consists of a monolayer of silicified cells. 90. The scaffold according to any one of items 78 to 89, wherein the scaffold comprises or consists of 0.1x104cells / cm2, such as 0.5x104cells / cm2, such as 1x104 cells / cm2, such as 2x104cells / cm2, such as 5x104cells / cm2

[0349] 91 . The scaffold according to any one of items 78 to 90, wherein the calcinated cells do not comprise organic matter as measured by the absence of carbon detection in any suitable spectroscopy technique, such as Energy-dispersive X- ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS) and / or secondary ion mass spectroscopy (SIMS).

[0350] 92. The scaffold according to any one of items 73 to 91 , wherein the scaffold is biocompatible.

[0351] 93. The scaffold according to any one of items 73 to 92 for use in a method of repairing tissue defect in a mammal.

[0352] 94. The scaffold according to any one of items 73 to 93, wherein the scaffold is a bone engineering scaffold.

[0353] 95. A population of differentiated cells of a specific cell lineage or a specific cell type of interest, obtained by the method of any one of items 1 to 44.

[0354] 96. Use of a scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold, the silicified cells and the specific cell lineage or the specific cell type of interest are as defined in any one of the preceding items.

[0355] 97. The use according to item 96, wherein the scaffold is used for bone tissue engineering applications.

[0356] 98. The use according to item 96 to 97, wherein the scaffold is implanted into a body.

[0357] 99. The use according to item 96 to 98, wherein the scaffold is embedded in a collagen matrix. 100. An implant comprising the scaffold according to any one of items 73 to 94.

[0358] 101. A scaffold for use in bone engineering, wherein the scaffold is as described in any one of items 73 to 94.

[0359] 102. A kit-of-parts comprising: i) The scaffold as defined in any one of the preceding items comprising silicified cells of a specific lineage or specific cell type of interest; ii) Immature cells, preferably stem cells, even more preferably as defined in any one of the preceding items; iii) Optionally, instructions for use.

[0360] 103. The kit of parts according to item 102, further comprising a cell culture medium comprising differentiation factors inducing differentiation into said specific lineage or said specific cell type.

Claims

Claims1. A method of differentiating immature cells to a specific cell lineage of interest and / or to a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with the immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type.

2. A method of obtaining cells of a specific cell lineage of interest and / or of a specific cell type, said method comprising: i) Silicificating cells of said specific cell lineage to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold; iii) Contacting the scaffold with immature cells; iv) Culturing the immature cells in contact with the scaffold in a culture medium, thereby inducing differentiation of the immature cells into differentiated cells of said specific cell lineage and / or of said specific cell type, and obtaining cells of said specific cell lineage and / or of said specific cell type.

3. The method according to any one of the preceding claims, wherein the immature cells are stem cells, such as pluripotent stem cells, such as mesenchymal stem cells, such as induced pluripotent stem cells, such as embryonic stem cells, such as hematopoietic stem cells, such as neural stem cells, such as epidermal stem cells, such as intestinal stem cells, such as hair follicle stem cells, such as muscle stem cells, such as cardiac stem cells, such as liver stem cells, or such as dental stem cells.

4. The method according to any one of the preceding claims, wherein the immature cells are progenitor cells, such as progenitor cells or precursor cells of the specific cell lineage or cell type of interest.

5. The method according to any one of the preceding claims, wherein said specific cell lineage or cell type is a mammalian cell lineage or cell type, such as osteogenic or chondrogenic.

6. The method according to any one of the preceding claims, wherein said specific cell lineage is epithelial lineage, neural lineage, hematopoietic lineage, muscle lineage, connective tissue lineage, adipocyte lineage, osteogenic lineage, germ cell lineage, endothelial lineage, pancreatic lineage, hepatic lineage, epidermal lineage or intestinal lineage.

7. The method according to any one of the preceding claims, wherein said specific cell lineage is osteogenic.

8. The method according to any one of the preceding claims, wherein said specific cell lineage is chondrogenic.

9. The method according to any one of the preceding claims, wherein said specific cell type is osteoblasts, osteocytes or osteoclasts, preferably osteoblasts.

10. The method according to any one of the claims 1 to 8, wherein said specific cell type is chondrocytes.

11. The method according to any one of the preceding claims, further comprising fixing and / or plating said cells of the specific cell lineage or specific cell type prior to silicification, preferably wherein said cells have not reached confluence.

12. The method according to any one of the preceding claims, wherein step i) of silicificating cells comprises or consists of: i) Incubating said cells of said specific cell lineage or specific cell type at a temperature between 30°C and 50°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours in the presence of a silicic acid compound, preferably selected from the group of orthosilicic acid, tetramethoxysilane, tetraethoxysilane and their derivatives such as methyltriethoxysilane, methyltrimethoxysilane, or a mixture thereof, andii) Optionally, contacting said cells of said specific cell lineage or specific cell type with a compound selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably wherein the compound is ethanol, preferably for a duration of at least 10 minutes; optionally, the compound is first added as a 50% (v / v) solution, then as a 100% (v / v) solution.

13. The method according to any one of the preceding claims, further comprising a step of drying said cells of said specific lineage or said specific cell type before step ii), such as at a temperature between 30°C and 50°C, such as 40 °C, preferably for a duration of 7 to 17 hours, such as 12 hours.

14. The method according to any one of the preceding claims, further comprising a step of dehydrating said silicified cells between step i) of silicificating cells and step ii) of calcinating a plurality of the silicified cells, wherein said dehydration step comprises or consists of contacting said scaffold with a compound gradient, wherein the compound is selected from the group of ethanol, isopropyl alcohol, acetone and dioxane, preferably wherein the compound is ethanol, preferably for a duration of at least 10 minutes.

15. The method according to any one of the preceding claims, wherein step ii) of calcinating a plurality of the silicified cells comprises or consists of pyrolyzing said silicified cells at a high temperature in an inert atmosphere.

16. The method according to any one of the preceding claims, wherein the scaffold of steps ii) and / or iii) comprises or consists of at most 50000 silicified cells / cm2, such as at most 40000 silicified cells / cm2, such as at most 30000 silicified cells / cm2, such as at most 25000 silicified cells / cm2, such as at most 20000 silicified cells / cm2cm2, such as at most 15000 silicified cells / cm2, such as at most 10000 silicified cells / cm2, such as at most 5000 silicified cells / cm2.

17. The method according to any one of the preceding claims, wherein the culture medium comprises differentiation factors.

18. The method according to any one of the preceding claims, wherein the culture medium does not comprise differentiation factors.

19. The method according to any one of the preceding claims, further comprising a step comprising or consisting of measuring the increased expression and / or activity of one or more marker associated with said specific cell lineage or said cell type in said differentiated cells.

20. The method according to claim 19, wherein the specific cell lineage is osteogenic and the marker is alkaline phosphatase (ALP), RLINX2, COL1A1 or OCN, preferably the specific cell type is osteoblasts.

21. The method according to claim 19, wherein the specific cell lineage is chondrogenic and the marker is SOX9, preferably the specific cell type is chondrocytes.

22. The method according to any one of the preceding claims, further comprising a step comprising or consisting of determining enzymatic activity associated with said specific cell lineage or said specific cell type, preferably ALP activity.

23. The method according to any one of the preceding claims, wherein the cells of a specific cell lineage or of a specific cell type are provided as a monolayer cell culture.

24. The method according to any one of the preceding claims, further comprising a step of conjugating the cells of said specific cell lineage or specific cell type to a purification moiety.

25. The method according to claim 24, wherein the purification moiety is magnetic, preferably magnetic beads.

26. Use of a scaffold comprising or consisting of silicified cells as a substrate to regulate the fate of immature and / or to differentiate immature cells into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells asdefined in any one of the preceding claims, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type of interest are as defined in any one of the preceding claims.

27. A scaffold comprising or consisting of silicified cells, wherein the scaffold promotes the differentiation of immature into differentiated cells of a specific cell lineage or a specific cell type of interest, preferably wherein the scaffold comprises or consists of silicified cells as defined in any one of the preceding claims, and / or wherein the scaffold, the immature cells, the differentiated cells and / or the specific cell lineage or the specific cell type are as defined in any one of the preceding claims.

28. A scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold is obtained by a method comprising the steps of: i) Silicificating cells of a specific cell lineage or a specific cell type to obtain silicified cells; ii) Calcinating a plurality of the silicified cells to obtain a scaffold, preferably wherein the scaffold, the silicified cells and the specific cell lineage or specific cell type are as defined in any of the preceding claims.

29. The scaffold according to any one of claims 27 to 28, for use in a method of repairing tissue defect in a mammal.

30. The scaffold according to any one of claims 27 to 29, wherein the scaffold is biocompatible.31 . The scaffold according to any one of claims 27 to 30, wherein the scaffold is a bone engineering scaffold.

32. A population of differentiated cells of a specific cell lineage or a specific cell type of interest, obtained by the method of any one of claims 1 to 25.

33. Use of a scaffold comprising or consisting of silicified cells of a specific cell lineage or a specific cell type of interest, wherein the scaffold, the silicified cellsand the specific cell lineage or the specific cell type of interest are as defined in any one of the preceding claims.

34. An implant comprising the scaffold according to any one of claims 27 to 31.

35. A scaffold for use in bone engineering, wherein the scaffold is as described in any one of claims 27 to 31.

36. A kit-of-parts comprising: i) The scaffold as defined in any one of the preceding claims comprising silicified cells of a specific lineage or specific cell type of interest; ii) Immature cells, preferably stem cells, even more preferably as defined in any one of the preceding claims; iii) Optionally, instructions for use.

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