Pre-vascularized callus-like organ as well as preparation method and application thereof

Abstract

The invention belongs to the field of biological materials, and relates to a pre-vascularized callus-like organ for accelerating tissue regeneration as well as a preparation method and application of the pre-vascularized callus-like organ. The pre-vascularized callus organ is a pre-vascularized callus organ which is formed by developing composite hydrogel microspheres loaded with silicate biological ceramics, human bone marrow mesenchymal stem cells and endothelial cells in an organ culture medium and has natural callus functional characteristics. Compared with organoids without silicate ceramics, the pre-vascularized callus organoids under regulation and control of silicate active ceramics show excellent endothelial network forming effect, osteogenic differentiation characteristic and mechanical response characteristic. In addition, the pre-vascularized callus-like organ shows more excellent mineralized matrix deposition capability. Therefore, the pre-vascularized callus-like organ regulated and controlled by the magnesium silicate ceramic can effectively accelerate the regeneration of bone tissues in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to prevascularized callus organoids for tissue regeneration, their preparation methods, and applications. Background Technology

[0002] Organoids are three-dimensional cell clusters formed in vitro from stem cells through self-organization or material-induced tissue formation, possessing similar structure and function to natural tissues. Their ability to self-renew and self-organize, along with their near-natural three-dimensional in vitro culture model, makes organoids a promising platform for replacing animal experiments and screening biomaterials.

[0003] Bone callus is a transitional structure in the fracture healing process. Over time, it gradually mineralizes and hardens, eventually transforming into mature bone tissue. Bone callus plays a crucial role in bone repair and regeneration, helping bones restore integrity and function through multiple processes such as support, vascularization, cell differentiation, and mineralization. Therefore, the development of callus organoids provides a potential strategy for repairing skeletal system injuries. In vitro constructed organoids often lack the vascular network framework, potentially leading to cell necrosis within the organoid; thus, the development of pre-vascularized callus organoids is of great significance. It is worth noting that, based on existing strategies for constructing callus organoids, the composition, structure, and function of these organoids still require further optimization. For example, the culture of bone organoids requires changing multiple culture media and adding large amounts of growth factors, resulting in excessively high culture costs. Furthermore, matrix gels, the gold standard for organoid culture, exhibit significant batch-to-batch variability, while alginate lacks biocompatibility. Therefore, new strategies for organoid culture systems need to be developed to regulate the development and function of pre-vascularized callus organoids. Summary of the Invention

[0004] To address the limitations of existing callus organoids, this invention aims to provide a callus organoid constructed based on silicate bioceramics, its preparation method, and its applications. This invention designs a method to regulate the development and function of callus organoids through inorganic active ceramics. Under the influence of inorganic materials, pre-vascularized callus organoids exhibit osteogenic and vascularization functional characteristics, mechanical properties with a mechanical response, and matrix deposition similar to bone tissue. Therefore, the silicate-regulated callus organoids prepared in this invention provide a novel method for screening biomaterials for organoids and promoting tissue regeneration.

[0005] In a first aspect, the present invention provides a prevascularized callus organoid. The prevascularized callus organoid is a prevascularized callus organoid with the functional characteristics of natural callus, formed by developing composite hydrogel microspheres loaded with silicate bioceramics, human bone marrow mesenchymal stem cells, and endothelial cells in an organoid culture medium.

[0006] Preferably, the composite hydrogel microspheres are composite hydrogel microspheres with a porous structure prepared using microfluidic chip technology.

[0007] Preferably, the endothelial cells are human umbilical vein endothelial cells; the silicate bioceramic is magnesium silicate.

[0008] Preferably, the initial loading ratio of human bone marrow mesenchymal stem cells to endothelial cells is 1:1 to 5:1; more preferably, the total number of bone marrow mesenchymal stem cells and endothelial cells seeded in each composite hydrogel microsphere is 400 to 1000.

[0009] Preferably, the mass concentration of silicate bioceramics in the composite hydrogel microspheres is no higher than 4%.

[0010] Preferably, the silicate bioceramic in the composite hydrogel microspheres has a spherical, rod-shaped, or amorphous shape, and is more preferably nanorod-shaped.

[0011] Preferably, the hydrogel matrix of the composite hydrogel microspheres is GelMA hydrogel; more preferably, the mass fraction of GelMA hydrogel is 4% to 20%.

[0012] Secondly, the present invention provides a method for preparing the aforementioned prevascularized callus organoid. The method includes: mixing silicate bioceramics, human bone marrow mesenchymal stem cells, endothelial cells, and a hydrogel matrix to obtain a cell-loaded composite hydrogel solution; passing the cell-loaded composite hydrogel solution through a microfluidic chip to form uniformly sized composite hydrogel microspheres; curing the (droplet-like) composite hydrogel microspheres through ultraviolet light crosslinking and then culturing them in an organoid culture medium; subsequently changing the medium periodically and continuing culturing to construct the prevascularized callus organoid.

[0013] Preferably, the culture time in organoid culture medium is 1 to 14 days.

[0014] Thirdly, the present invention provides the application of the aforementioned prevascularized callus organoids in the preparation of tissue regeneration materials.

[0015] The present invention has the following beneficial effects:

[0016] The functionalized callus organoids provided by this invention exhibit excellent functional properties, including the formation and maintenance of an ordered endothelial network, osteogenic differentiation, and mechanical response. Simultaneously, the organoids possess the ability to deposit mineralized matrix. Results from rat femoral condyle bone defect repair studies show that prevascularized callus organoids can effectively promote bone regeneration, providing a strong validation model for the use of prevascularized callus organoids in the repair of bone-related defects. Therefore, callus organoids based on silicate bioactive materials offer a novel strategy for regulating important cellular functions and promoting tissue regeneration, with broad application prospects. Attached Figure Description

[0017] Figure 1 The crystal phase and microstructure of magnesium silicate (MS) were characterized. (a) XRD patterns of magnesium silicate with different morphologies. (b) SEM images of magnesium silicate with different morphologies, representing spherical, rod-shaped, and amorphous shapes, respectively. The results show that the prepared spherical magnesium silicate has a diameter of approximately 400 nm and good dispersion; the rod-shaped magnesium silicate has a length of approximately 100 nm and a width of 15 nm.

[0018] Figure 2 Characterization of composite hydrogel microspheres prepared by microfluidic method. (a) Design and physical image of the microfluidic chip. (b) SEM images of composite hydrogel microspheres containing different concentrations of magnesium silicate. The results show that the microfluidic method can prepare microsphere organoids with uniform size, and the composite hydrogel microspheres at various magnesium silicate concentrations maintain a porous structure.

[0019] Figure 3 Developmental assessment of callus organoids constructed with different concentrations of spherical magnesium silicate. (a) ENG and CD31 staining images of callus organoids constructed with different material concentrations on days 7 and 14. (b) Statistical analysis of mean fluorescence intensity of ENG and CD31 in callus organoids constructed with different material concentrations. The results showed that the 2NS-GM group exhibited a relatively superior endothelial network; furthermore, ENG expression decreased in all groups with prolonged culture time.

[0020] Figure 4 PCR results of BMP2, OPN, and VEGF in callus organoids constructed from different concentrations of spherical magnesium silicate. The results showed that the 2NS-GM group exhibited relatively high expression of BMP2, OPN, and VEGF genes; however, gene expression levels decreased with increasing culture time.

[0021] Figure 5 Evaluation of prevascularization development of callus organoids constructed with different magnesium silicate morphologies. (a) CD31 and PDGFRβ staining images of callus organoids constructed with different material morphologies on days 7 and 14. (b) Statistical analysis of the average PDGFRβ fluorescence area of ​​callus organoids constructed with different material morphologies. The results showed that the 2NT-GM group exhibited a relatively intact endothelial network; furthermore, the endothelial network of each group showed a decreasing trend with prolonged culture time.

[0022] Figure 6 PCR results of KDR, PDGFRβ, and FGF2 in callus organoids constructed from magnesium silicate with different morphologies. The results showed that the 2NT-GM group exhibited relatively high expression of angiogenesis-related genes KDR, PDGFRβ, and FGF2; gene expression levels decreased with increasing culture time.

[0023] Figure 7 PCR results of osteogenic differentiation-related genes in callus organoids constructed from magnesium silicate with different morphologies. The results showed that the 2NT-GM group exhibited relatively high expression of OPN, OGN, BSP, ALP, OCN, and COL1; gene expression levels decreased with increasing culture time.

[0024] Figure 8 The effects of different morphologies of magnesium silicate on the mechanical microenvironment in callus organoids were investigated. (a) Storage modulus of hydrogels loaded with different morphologies of magnesium silicate at 1, 3, and 7 days. (b) Young's modulus of hydrogels loaded with different morphologies of magnesium silicate. The results showed that the 2NT-GM group exhibited the best mechanical properties; the mechanical properties of the hydrogels decreased with increasing culture days.

[0025] Figure 9 The mechanical response of callus organoids constructed with different magnesium silicate morphologies was evaluated. (a) FAK staining images of callus organoids constructed with different material morphologies on day 7. (b) Statistical graph of the average immunofluorescence intensity of FAK protein. (c) YAP staining images of callus organoids constructed with different material morphologies on day 7. (d) Statistical analysis of the relative expression levels of YAP protein in the nucleus and cytoplasm. The results showed that the 2NT-GM group had the best mechanical response and could effectively promote the nuclear transport of YAP protein.

[0026] Figure 10 Histological staining results of callus organoids constructed with magnesium silicate after 7 days of culture. Organoid structures were represented by hematoxylin and eosin (H&E) staining. Periodic acid-Schiff (PAS) staining was used for polysaccharide staining. Sirius red (SR) staining was used for collagen staining. Masson's trichrome staining revealed type I and type II collagen. The results showed that the 7-day-cultured callus organoids exhibited extracellular matrix components of native human bone marrow mesenchymal stem cells, including glycogen and collagen, as well as self-renewal and differentiation characteristics. The 2NT-GM group organoids showed greater deposition of glycogen and collagen fibers than the GM group, indicating that magnesium silicate-treated callus organoids possess optimal mineralization matrix deposition capacity.

[0027] Figure 11To illustrate the in vivo bone repair effect of prevascularized callus organoids. (a) Micro-CT images of the cross-section and longitudinal section of newly formed bone (red); (b) Statistical analysis of the ratio of new bone surface area (BS, bone area) to total defect volume (TV, total volume), the ratio of new bone volume (BV, bone volume) to total defect volume, the number of trabeculae (Tb.N), and the trabecular separation (Tb.Sp) in different groups based on the Micro-CT results. Callus organoids cultured in complete medium were designated BOs / GM; organoids formed in conditioned medium containing 2% nanorod-shaped magnesium silicate were designated BOs / 2NT-GM. The results showed that the BS / TV, BV / TV, and Tb.N of the BOs / 2NT-GM group were significantly higher than those of the other two groups. In particular, the newly formed bone in the BOs / 2NT-GM group was also significantly higher than that in the BOs / GM group, indicating that the prevascularized callus organoids under the action of magnesium silicate have superior in vivo osteogenic activity.

[0028] Figure 12 Images of bone defect regeneration stained with Masson's ointment and Saf. O. The results showed that varying degrees of bone defect areas remained in the Blank, BOs / GM, and BOs / 2NT-GM groups. The Blank group showed only a small amount of matrix deposition in the defect area, while the other two groups showed a small amount of trabecular bone structure. The BOs / 2NT-GM group exhibited the most prominent newly formed trabecular bone structure, indicating that the callus organoid regulated by the nanorod-shaped magnesium silicate material has the best bone regeneration-promoting effect. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and the following embodiments. It should be understood that the drawings and the following embodiments are for illustrative purposes only and are not intended to limit the present invention. The following exemplarily describes prevascularized callus organoids constructed from silicate biomaterials, their preparation methods, and applications.

[0030] The prevascularized callus organoids are prevascularized callus organoids with the functional characteristics of natural callus, formed by developing composite hydrogel microspheres loaded with silicate bioceramics, human bone marrow mesenchymal stem cells, and endothelial cells in organoid culture medium.

[0031] In an optional embodiment, the prevascularized callus organoid is an organoid with the functional characteristics of natural callus, formed by developing composite hydrogel spheres (containing human bone marrow mesenchymal stem cells, endothelial cells, and magnesium silicate bioactive ceramics loaded onto a hydrogel matrix using microfluidic technology) in an organoid conditioned culture medium. Specifically, the prevascularized callus organoid is formed under the action of silicate biomaterials. Preferably, the silicate biomaterial is magnesium silicate (MS).

[0032] In other words, the prevascularized callus organoids are prepared using a microfluidic chip. A composite hydrogel loaded with silicate bioceramics, human bone marrow mesenchymal stem cells, and endothelial cells is used to create cell-loaded composite hydrogel microspheres. Through cultivation in organoid culture medium, the development of these cell-loaded composite hydrogel microspheres is regulated under the influence of different concentrations and morphologies of inorganic active ceramic magnesium silicate, thereby forming organoids with the functional characteristics of natural callus. Therefore, the prevascularized callus organoids constructed based on silicate biomaterials in this invention provide a novel method for regulating organoid function and promoting bone tissue regeneration.

[0033] The construction of the prevascularized callus organoid requires the following materials: microfluidic chip, hydrogel matrix, human bone marrow mesenchymal stem cells (HBMSC), endothelial cells (EC), and appropriate amounts of silicate biomaterials.

[0034] The fabrication of microfluidic chips is an existing technology in this field. As an example, the material used for the microfluidic chip is PDMS (polydimethylsiloxane). The microfluidic chip fabrication process includes chip channel design, photolithography, thin film deposition, and bonding. First, based on the droplet formation principle, oil and water phase channels are designed. Then, photosensitive materials are coated onto a silicon wafer using photolithography. Ultraviolet light exposure and development processes are then used to form channel microstructures on the silicon wafer. Next, PDMS prepolymer and curing agent are mixed in a specific ratio, followed by degassing to remove air bubbles. The mixture is then poured into a mold with a silicon wafer attached to the bottom and cured at high temperature to obtain a PDMS film with printed channels. The thickness of the PDMS film can be approximately 3–5 mm. Subsequently, the PDMS is separated from the silicon wafer, and liquid inlets and outlets are formed on the PDMS using drilling and cutting techniques to ensure smooth liquid flow into and out of the chip. A vacuum cleaner is used to clean the PDMS surface and the surface of the adhesive glass substrate, making the surfaces more hydrophilic and thus enhancing the adhesion between the two. The PDMS with the imprinted pathways is then bonded to a glass slide to form a closed microfluidic channel structure. Finally, the chip undergoes functional testing, including performance tests on liquid flow, reaction speed, and mixing effect, to ensure the chip's stability and functionality. The diameter of the microfluidic chip pathways can be, for example, 100–300 μm, preferably 200–300 μm.

[0035] By utilizing microfluidic chips and adjusting the parameters of the microfluidic device, uniformly sized and controllable microsphere organoids can be obtained. The size of the microfluidically prepared composite hydrogel microspheres can be adjusted by regulating the flow rates of the aqueous and oil phases, and is not necessarily related to the microfluidic chip pathway size. As an example, the flow rate of the composite hydrogel phase (aqueous phase) is 2–15 μL / min, and the flow rate of the oil phase is 10–30 μL / min.

[0036] Microfluidically prepared hydrogel microspheres exhibit ultra-high uniformity and precise size control. Microfluidic chips can generate highly uniform microspheres (CV < 5%) by precisely controlling the flow rates of the two phases (continuous phase / dispersed phase), far superior to 3D printing (CV typically > 10%). By adjusting the chip channel size or flow rate, microspheres ranging from 50 to 500 μm can be prepared to meet the needs of different organoids. Microfluidic technology effectively avoids batch variations, is suitable for standardized production, reduces experimental errors, and improves data comparability. Finally, microfluidics can enhance cell viability in organoids and achieve precise cell encapsulation. Microfluidic technology is a gentle cell encapsulation method; its low-shear design protects cell viability (survival rate > 90%), superior to the extrusion pressure of 3D printing (survival rate 70%–90%). Simultaneously, microfluidic mixing ensures uniform cell dispersion within the microspheres, avoiding cell sedimentation or aggregation problems that may occur in 3D printing.

[0037] Inorganic bioactive materials possess excellent stem cell regulatory effects and offer advantages in promoting organoid growth and development. Among them, magnesium silicate bioceramics release bioactive ions that can promote angiogenesis and regulate intercellular interactions. Therefore, under the influence of magnesium silicate bioceramics, the development of callus organoids is expected to be further optimized, including enhancing the maintenance of vascular networks, improving the response to matrix mechanics, promoting the regulation of human bone marrow mesenchymal stem cell differentiation, and enhancing the deposition of organoids in in vitro mineralized matrix, thus further broadening the application scope of callus organoids.

[0038] The sources of magnesium silicate bioceramics are unrestricted. For example, magnesium silicate bioceramics can be synthesized via hydrothermal methods.

[0039] The morphology of the silicate bioceramics in the composite hydrogel microspheres is not limited, and includes, but is not limited to, nanospheres, nanorods, and amorphous forms, preferably nanorods. In some embodiments, magnesium silicate is in the form of nanowires with a diameter of 400–500 nm. In some embodiments, magnesium silicate has a nanorod morphology with a length of approximately 100–200 nm and a width of 10–20 nm. In still other embodiments, magnesium silicate has an irregular amorphous morphology.

[0040] In the composite hydrogel microspheres, the mass concentration of silicate bioceramics is not higher than 4%, preferably 1% to 4%, and more preferably 2%.

[0041] In the composite hydrogel microspheres, the hydrogel matrix can be GelMA (methacrylic anhydride gelatin) hydrogel. The mass fraction of the hydrogel matrix can be 4% to 20%. As an example, the mass fraction of GelMA is 7% to 14%.

[0042] The initial loading ratio of bone marrow mesenchymal stem cells (BMSCs) to endothelial cells is 1:1 to 5:1. For example, the preferred seeding ratio of human BMSCs to endothelial cells is 3:1. The total number of human BMSCs and endothelial cells seeded in each microfluidically prepared composite hydrogel microsphere is 400 to 1000. Preferably, the total number of human BMSCs and endothelial cells seeded in each microfluidically prepared composite hydrogel microsphere is 500. For example, each hydrogel microsphere contains 375 human BMSCs and 125 human umbilical vein endothelial cells.

[0043] The conditioned medium is composed of organoid complete medium. Any organoid complete medium commonly used in the art can be used. As an example, the components of the organoid complete medium may include: Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12) medium and human serum-free endothelial medium (SFM) supplemented with 0.1 mM 2-mercaptoethanol, 1 vol% non-essential amino acids, 1 vol% P / S, 20 ng / mL recombinant human fibroblast growth factor 2 (hFGF-2), recombinant human epidermal growth factor (hEGF), recombinant human platelet-derived growth factor AB (hPDGF-AB), recombinant human tumor suppressor M (hOSM), 40 ng / mL recombinant human insulin-like growth factor 1 (hIGF-1), and 2 vol% chicken embryo extract (CEE). The volume ratio of DMEM / F12 to SFM medium can be 1:2. All supplemented compounds are concentrations in percentage of their total content in the organoid complete medium.

[0044] This invention provides a method for preparing the aforementioned callus organoids. The invention involves mixing silicate bioceramics, human bone marrow mesenchymal stem cells, and endothelial cells with a hydrogel matrix, forming uniform microspheres using a microfluidic chip, and then cross-linking and curing them under ultraviolet light to form microspherical organoids. In an optional embodiment, the preparation method includes: mixing hydrogel, silicate bioceramic particles, human bone marrow mesenchymal stem cells, and human umbilical vein endothelial cells, forming composite hydrogel spheres using a microfluidic chip; subsequently, cross-linking and curing the microspheres under ultraviolet light, then dispersing and seeding them in a six-well plate, adding organoid culture medium, and continuing culture to construct the callus organoid. The following exemplarily illustrates the method for preparing the prevascularized callus organoids.

[0045] Microfluidic chips were fabricated. The fabricated chips were then irradiated with ultraviolet light in a clean bench for 12 hours before use.

[0046] Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were digested with trypsin, centrifuged, and counted. Using a microfluidic chip, these cells were seeded into composite hydrogel microspheres, which were then cross-linked and solidified under UV light. The culture medium was replaced with organoid culture medium, and the cells were cultured in six-well plates for a certain period to construct prevascularized callus organoids. The culture time for the organoids was 7–14 days, preferably 7 days.

[0047] In an optional implementation, individual callus organoid culture units are formed by adding nanospheres of magnesium silicate at different concentrations to the cell-carrying composite hydrogel construct. These units are then cultured for at least 7 days to form callus organoids. The magnesium silicate concentrations are 0%, 1%, 2%, and 4%. The composite hydrogel microspheres with a magnesium silicate concentration of 0% are designated as the GM group. The composite hydrogel microspheres with a magnesium silicate concentration of 1% are designated as the 1NS-GM group. The composite hydrogel microspheres with a magnesium silicate concentration of 2% are designated as the 2NS-GM group. The composite hydrogel microspheres with a magnesium silicate concentration of 4% are designated as the 4NS-GM group.

[0048] In an optional embodiment, the morphology of magnesium silicate is controlled when the concentration of nanospheres within the composite hydrogel microspheres is 2%. On day 0, the microspheres are inoculated into a culture system containing organoid culture medium, and prevascularized callus organoids are constructed by day 7. Callus organoids regulated by nanosphere-shaped magnesium silicate are designated 2NS-GM, while callus organoids regulated by nanorod-shaped and amorphous magnesium silicate are designated 2NT-GM and 2IR-GM, respectively.

[0049] The following uses 2NT-GM as an example to illustrate the preparation method of prevascularized callus organoids. Microfluidic chip preparation: The prepared chip was irradiated with ultraviolet light in a clean bench for 12 hours before use. Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were digested with trypsin, centrifuged, and counted. Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were mixed at a ratio of 3:1, and then mixed with GelMA hydrogel preheated to 37℃ and ultrasonically dispersed in a nanorod-shaped magnesium silicate dispersion. This mixture was connected to a microfluidic device to prepare composite hydrogel microspheres. After UV curing, the composite hydrogel microspheres were collected, dispersed, and seeded in six-well plates, then added to organoid complete culture medium. Cultured for a certain period to construct prevascularized callus organoids.

[0050] This invention provides the application of the aforementioned prevascularized callus organoids in the preparation of tissue regeneration materials. The prevascularized callus organoids of this invention are primarily used to promote tissue regeneration. This invention also provides the use of the aforementioned prevascularized callus organoids in regulating cell behavior and promoting bone repair and regeneration, particularly in the repair of bone defects.

[0051] This invention focuses on the field of bone tissue regeneration, specifically involving the construction of prevascularized callus organoids and their application in bone defect repair. By regulating organoid development through silicate bioceramics, it addresses the problems of lack of vascular network and high culture costs associated with existing callus organoids. This invention introduces magnesium silicate bioceramics (especially nanorod morphology) to regulate organoid development, solving the problem of requiring large amounts of growth factors in existing technologies, and achieving prevascularization, osteogenic differentiation, and mechanoresponsiveness in the organoids. Prevascularized callus organoids regulated by magnesium silicate bioceramics exhibit excellent osteogenic and vascularization functional properties, possess mechanoresponsive mechanical properties, and can deposit a matrix similar to bone tissue, achieving a breakthrough in the functional characteristics of prevascularized callus organoids.

[0052] In summary, the callus organoids of this invention are prepared using microfluidic technology to create hydrogel microspheres loaded with silicate bioceramics, human bone marrow mesenchymal stem cells, and human umbilical vein endothelial cells. These microspheres are then cultured in vitro to develop into organoids with functional properties similar to natural callus. The pre-vascularized callus organoids prepared in this invention exhibit significantly enhanced development and possess the ability to promote osteogenic differentiation of stem cells and deposition of bone-related matrix. The pre-vascularized callus organoids possess excellent functional characteristics of callus organoids, such as osteogenic differentiation, angiogenic differentiation, and mechanotransformation. In vivo femoral condyle repair results show that the pre-vascularized callus organoids can effectively promote the repair of femoral condyle defects in rats, providing strong evidence for the use of callus organoids in the repair of skeletal system defects.

[0053] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0054] Preparation and characterization of magnesium silicate

[0055] References for the preparation method of spherical magnesium silicate: (3D multicellular micropatterning biomaterials for hair regeneration and vascularization, Mater. Horiz., 2023, 10, 3773-3784). References for the preparation method of rod-shaped magnesium silicate: (Superhydrophobic and self-healing dual-function coatings based on mercaptabenzimidazole inhibitor-loaded magnesium silicate nanotubes for corrosion protection of AZ31B magnesium alloys, Chem. Eng. J., 2021, 404127106).

[0056] For the characterization of the crystal phase and microstructure of magnesium silicate, please refer to [link to relevant documentation]. Figure 1 The microstructure of magnesium silicate with different morphologies was characterized by SEM images. The results showed that the prepared hollow spherical magnesium silicate had a diameter of approximately 400 nm and good dispersion; the nanorod magnesium silicate had a length of approximately 100 nm and a width of 15 nm. The crystal phase was characterized by XRD patterns of magnesium silicate with different morphologies. XRD analysis of the synthesized spherical and amorphous magnesium silicate showed that their diffraction patterns corresponded to Mg3Si4O. 10 (OH)2 (PDF No. 19-0770) has low crystallinity; XRD analysis of the synthesized nanorod-shaped magnesium silicate showed that its diffraction pattern corresponds to Mg3Si2O5(OH)4 (PDF No. 52-1562), which has high crystallinity.

[0057] Fabrication and characterization of microfluidic chips

[0058] The fabrication method of the microfluidic chip is described in the reference (Generation of cell-laden hydrogel microspheres using 3D printing-enabled microfluidics, J. Mater. Res., 2018, 33(14): 2012-2018). The channel diameter of the fabricated PDMS microfluidic chip is 200 μm.

[0059] Preparation of complete organoid culture medium

[0060] The specific components of the complete organoid culture medium were: DMEM / F12 medium and human endothelial serum-free medium (SFM) supplemented with 0.1 mM 2-mercaptoethanol, 1% non-essential amino acids, 1% P / S, 20 ng / mL recombinant human fibroblast growth factor 2 (hFGF-2), recombinant human epidermal growth factor (hEGF), recombinant human platelet-derived growth factor AB (hPDGF-AB), recombinant human tumor suppressor M (hOSM), 40 ng / mL recombinant human insulin-like growth factor 1 (hIGF-1), and 2% chicken embryo extract (CEE). The volume ratio of DMEM / F12 to SFM medium was 1:2. The concentrations of all supplemented compounds were their percentages in the complete organoid culture medium.

[0061] Example 1

[0062] The microfluidic chip was sterilized by UV irradiation. A 14 wt% GelMA hydrogel solution was prepared and sterilized by passing it through a 0.22 μm bacterial filter membrane and preheating to 37°C for later use. Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were digested with trypsin, centrifuged, counted, and mixed at a 3:1 ratio. GelMA hydrogel, PBS, and cell suspension were mixed at a 2:1:1 volume ratio. The microfluidic device was connected to control the diameter of each composite hydrogel microsphere to 250 μm, encapsulating 375 human bone marrow mesenchymal stem cells and 125 human umbilical vein endothelial cells. The microspheres were collected and cross-linked with UV light. The composite hydrogel microspheres were then uniformly dispersed and seeded in six-well plates, and then replaced with fresh organoid complete culture medium, and cultured for 7–14 days. The organoids prepared in Example 1 did not contain nanosphere magnesium silicate ceramics and were labeled as the GM group.

[0063] Example 2

[0064] The microfluidic chip was sterilized by UV irradiation. A 14 wt% GelMA hydrogel solution was prepared and sterilized by passing it through a 0.22 μm bacterial filter membrane and preheating to 37°C for later use. Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were digested with trypsin, centrifuged, counted, and mixed at a 3:1 ratio. GelMA hydrogel, PBS suspension of nanosphere magnesium silicate, and cell suspension were mixed at a 2:1:1 volume ratio. The microfluidic device was connected to control the diameter of each composite hydrogel microsphere to 250 μm, encapsulating 375 human bone marrow mesenchymal stem cells and 125 human umbilical vein endothelial cells. The microspheres were collected and UV cross-linked, and the composite hydrogel microspheres were uniformly dispersed and seeded in six-well plates. The culture medium was then replaced with fresh organoid complete culture medium, and cultured for 7–14 days to form callus organoids. The organoids prepared in Example 2 were labeled 1NS-GM, and the concentration of nanosphere magnesium silicate ceramics in the organoids was 1%.

[0065] Example 3

[0066] The microfluidic chip was sterilized by UV irradiation. A 14 wt% GelMA hydrogel solution was prepared and sterilized by passing it through a 0.22 μm bacterial filter membrane and preheating to 37°C for later use. Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were digested with trypsin, centrifuged, counted, and mixed at a 3:1 ratio. GelMA hydrogel, PBS suspension of nanosphere magnesium silicate, and cell suspension were mixed at a 2:1:1 volume ratio. The microfluidic device was connected, and the diameter of each composite hydrogel microsphere was controlled to be 250 μm, encapsulating 375 human bone marrow mesenchymal stem cells and 125 human umbilical vein endothelial cells. The microspheres were collected and cross-linked with UV light. The composite hydrogel microspheres were then uniformly dispersed and seeded in six-well plates, and then replaced with fresh organoid complete culture medium. The culture was continued for 7–14 days to form callus organoids. The organoids prepared in Example 3 were labeled 2NS-GM, and the concentration of nanosphere magnesium silicate ceramics in the organoids was 2%.

[0067] Example 4

[0068] The microfluidic chip was sterilized by UV irradiation. A 14 wt% GelMA hydrogel solution was prepared and sterilized by passing it through a 0.22 μm bacterial filter membrane and preheating to 37°C for later use. Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were digested with trypsin, centrifuged, counted, and mixed at a 3:1 ratio. GelMA hydrogel, PBS suspension of nanosphere magnesium silicate, and cell suspension were mixed at a 2:1:1 volume ratio. The microfluidic device was connected to control the diameter of each composite hydrogel microsphere to 250 μm, encapsulating 375 human bone marrow mesenchymal stem cells and 125 human umbilical vein endothelial cells. The microspheres were collected and UV cross-linked, and the composite hydrogel microspheres were uniformly dispersed and seeded in six-well plates. The culture medium was then replaced with fresh organoid complete culture medium, and cultured for 7–14 days to form callus organoids. The organoids prepared in Example 4 were labeled 4NS-GM, and the concentration of nanosphere magnesium silicate ceramics in the organoids was 4%.

[0069] See the schematic diagram, physical image, and SEM image of the composite hydrogel for the microfluidic chip. Figure 2 The magnesium silicate hydrogel microspheres containing different concentrations, prepared using microfluidic chips, exhibited uniform size and maintained a porous structure.

[0070] The developmental effects of the prepared organoids were analyzed, and the results are shown in [reference needed]. Figure 3-4 .

[0071] The callus organoids controlled with 2% magnesium silicate by mass were used as the optimal experimental setup for subsequent evaluation.

[0072] Example 5

[0073] The microfluidic chip was sterilized by UV irradiation. A 14 wt% GelMA hydrogel solution was prepared and sterilized by passing it through a 0.22 μm bacterial filter membrane, preheated to 37°C for later use. Human bone marrow mesenchymal stem cells and human umbilical vein endothelial cells were digested with trypsin, centrifuged, counted, and mixed at a 3:1 ratio. GelMA hydrogel, magnesium silicate in PBS suspension, and cell suspension were mixed at a 2:1:1 volume ratio. The magnesium silicate morphology included amorphous, nanospheres, and nanorods. A microfluidic device was connected to control the diameter of each composite hydrogel microsphere to 250 μm, encapsulating 375 human bone marrow mesenchymal stem cells and 125 human umbilical vein endothelial cells. The microspheres were collected and cross-linked with UV light. The composite hydrogel microspheres were then uniformly dispersed and seeded in six-well plates, followed by replacement with fresh organoid complete culture medium, and cultured for 7–14 days to form callus organoids. The concentration of the nanosphere magnesium silicate ceramics in the organoids was 2%.

[0074] The prevascularization development effect of the prepared organoids was analyzed, and the results are shown in [reference needed]. Figure 5-6 .

[0075] The osteogenic differentiation characteristics of the prepared callus organoids were investigated, and the results are shown in [reference needed]. Figure 7 .

[0076] The mechanical response characteristics of the prepared callus organoids were investigated, and the results are shown in [reference needed]. Figure 8-9 .

[0077] The callus organoid regulated by nanorod-shaped magnesium silicate was used as the optimal experimental model for subsequent evaluation.

[0078] The deposition capacity of the prepared callus organoids for mineralization matrix was investigated, and the results are shown in [reference needed]. Figure 10 .

[0079] The study investigated how the prepared callus organoids promoted the in vivo repair of femoral condyle defects. The results are shown in [link to study]. Figure 11-12 .

[0080] Development of prevascularized callus organoids under the influence of different concentrations of magnesium silicate

[0081] Inorganic bioactive materials hold immense application potential in the field of organoids. Studies have shown that bioactive ions released by silicate materials, such as silicon and magnesium ions, have a positive impact on angiogenesis. Furthermore, silicate materials with different elemental compositions exhibit varying effects in promoting organoid development. Therefore, considering the roles of different silicate material components in maintaining the endothelial network of callus organoids and other potential influences, magnesium silicate was introduced into the culture system of callus organoids to promote their development into prevascularized callus organoids.

[0082] Examples 1-4 verified the effect of different magnesium silicate concentrations. First, immunofluorescence images of organoids at different time points under different magnesium silicate concentrations were captured. The four groups of organoids cultured for 7 and 14 days were then stained with two proteins using mesenchymal markers (Endoglin, ENG) and endothelial markers (CD31). Figure 3 a). Furthermore, the mean fluorescence intensity of ENG and CD31 was statistically analyzed. On day 7, ENG expression in the 2NS-GM group was higher than that in the GM, 1NS-GM, and 4NS-GM groups. CD31 expression in the 2NS-GM and 4NS-GM groups was significantly higher than in the other two groups, and the endothelial network was clearly visible. The expression was more significant in the 2NS-GM group. However, on day 14, ENG expression decreased significantly, indicating a reduced proportion of stem cells in the initial differentiation stage. The CD31 network also showed a significant decrease (…). Figure 3 b). Secondly, the expression of genes related to osteogenic and angiogenic differentiation in organoids was characterized. On day 7, the expression levels of BMP2, OPN, and VEGF were all highest in the 2NS-GM group, while on day 14, the expression levels of all three genes were lower than on day 7. Figure 4 In the subsequent construction of callus organoids, the material concentration of the 2NS-GM group was selected as the optimal condition for organoid construction.

[0083] Development of prevascularized callus organoids under the influence of magnesium silicate with different morphologies

[0084] In the above analysis, stable network structures were found in organoids containing different concentrations of nanosphere-shaped magnesium silicate, indicating that the constructed three-dimensional composite hydrogel organoids can partially mimic the vascular niche in the natural callus environment and display the structural characteristics of callus. The 2NS-GM group showed a relatively superior endothelial network, which plays an important role in the function of the organoids. In addition, the 2NS-GM group showed relatively superior osteogenic differentiation characteristics. Therefore, in subsequent studies, composite hydrogel microspheres with a magnesium silicate concentration of 2% were selected as the optimal construction strategy for callus organoids.

[0085] The development of callus organoids was regulated using three different morphologies of magnesium silicate. The control group (GM group) consisted of callus organoids cultured with hydrogel microspheres without magnesium silicate; the 2NS-GM group consisted of callus organoids regulated with 2% concentration of spherical magnesium silicate; the 2IR-GM group consisted of callus organoids regulated with 2% concentration of amorphous magnesium silicate; and the 2NT-GM group consisted of callus organoids regulated with 2% concentration of rod-shaped magnesium silicate.

[0086] Images of CD31 and perivascular cell marker protein (PDGFRβ) staining, along with corresponding average fluorescence intensity statistics, showed that the addition of magnesium silicate further maintained the endothelial network of callus organoids. On day 7, compared to the control group, all three morphologies of magnesium silicate enhanced CD31 expression, indicating a certain promoting effect on organoid prevascularization. Furthermore, the endothelial network in each group showed a decreasing trend with prolonged culture time. Overall, nanorod-shaped magnesium silicate was the most effective in maintaining the endothelial network. Figure 5 PCR results for KDR, PDGFRβ, and FGF2 showed that the 2NT-GM group exhibited relatively high expression of angiogenesis-related genes KDR, PDGFRβ, and FGF2; however, gene expression levels decreased with increasing culture time. Figure 6 ).

[0087] Meanwhile, the effects of different magnesium silicate morphologies on the osteogenic differentiation characteristics of callus organoids were investigated. PCR results of osteogenic differentiation-related genes in callus organoids constructed from magnesium silicate with different morphologies are shown below. Figure 7 As shown in the figure, the 2NT-GM group exhibited relatively high expression levels of OPN, OGN, BSP, ALP, OCN, and COL1; however, gene expression levels decreased with increasing culture time. This indicates that nanorod-shaped magnesium silicate has a relatively superior effect in promoting the development of callus organoids.

[0088] Investigation of the mechanical response characteristics of magnesium silicate with different morphologies to prevascularized callus organoids

[0089] Bone, as a tissue highly sensitive to mechanical forces, has its growth and development closely related to the external mechanical environment. In particular, the stiffness of the matrix plays a crucial role in the differentiation of bone marrow mesenchymal stem cells. Studies have found that when the matrix stiffness is high, stem cells tend to differentiate into osteoblasts and chondrocytes, while in a softer matrix, stem cells preferentially differentiate into adipocytes. This phenomenon reflects the cell's response to the mechanical environment and the regulation of its differentiation potential. Example 5 also explored the biomechanical characteristics of callus organoids and their relationship with the stiffness of the culture matrix.

[0090] First, the mechanical properties of composite hydrogel matrices with different morphologies of magnesium silicate systems were characterized. Figure 8Storage modulus reflects a material's ability to store elastic energy, while Young's modulus measures its stiffness. These mechanical property parameters effectively reflect the influence of different culture substrates on the mechanical behavior of hydrogels. Results showed that on day 1, there was no significant difference in storage modulus among the component-based composite hydrogels. However, as the number of culture days increased, the 2NT-GM group exhibited a higher storage modulus. Furthermore, the 2NT-GM group had a higher elastic modulus compared to the other three groups. This indicates that the 2NT-GM group exhibited the best mechanical properties; the mechanical properties of the hydrogel decreased with increasing culture days.

[0091] Subsequently, immunofluorescence staining was performed on two key proteins in the biomechanical response pathway of callus organoids. The results showed that the expression level of activated focal adhesion kinase (FAK) was higher in the 2NT-GM group than in the other three groups. Figure 9 (ab). This indicates that cells in the 2NT-GM group showed a more significant mechanical response under high-intensity matrix conditions, promoting FAK protein expression. Furthermore, we quantitatively analyzed the distribution of YAP protein in the nucleus and cytoplasm of the four organoid groups (ab). Figure 9 The results showed that significant nuclear translocation of YAP protein occurred in the 2NT-GM group, indicating that the nuclear translocation process of YAP protein was more pronounced in this group than in other groups. Nuclear translocation is a key step in the function of YAP protein, which can promote the expression of osteogenic differentiation-related genes, thereby driving osteogenic differentiation of organoids.

[0092] The above experimental results are consistent with the previous mechanical strength test results of the composite hydrogel, further demonstrating that magnesium silicate with different morphologies participates in regulating the mechanical response characteristics of organoids and the osteogenic differentiation process by altering the strength of the hydrogel matrix. The 2NT-GM group showed better organoid mechanical response. Using nanorod-shaped magnesium silicate composite hydrogels as a matrix can effectively enhance the mechanical properties of organoids while promoting their osteogenic differentiation characteristics, providing strong theoretical support and technical guarantee for bone repair and regeneration.

[0093] An investigation into the ability of mineralized matrix deposition in prevascularized callus organoids

[0094] In the above analysis, the stable network structure found in spheres with different magnesium silicate morphologies indicates that the constructed three-dimensional composite hydrogel spheres can partially simulate the vascular niche in the natural callus environment, displaying the structural characteristics of callus. The 2NS-GM group showed a relatively superior endothelial network, which plays an important role in the function of the organoid. In addition, the 2NS-GM group showed relatively superior osteogenic differentiation and mechanoresponsive functional properties. Therefore, in subsequent studies, nanorod-shaped magnesium silicate composite hydrogel spheres were selected as the optimal construction strategy for callus organoids.

[0095] Bone callus organoids without magnesium silicate ceramics are named BOs / GM; organoids formed under the control of 2% concentration of nanorod-shaped magnesium silicate are named BOs / 2NT-GM. That is, bone callus organoids controlled by nanorod-shaped magnesium silicate are used as the optimal experimental material for subsequent evaluation.

[0096] Subsequently, histological staining was performed on the callus organoids to assess their ability to deposit mineralized matrix. Figure 10 Histological staining results of two different callus organoids cultured for 7 days are shown. Hematoxylin and eosin (H&E) staining revealed that most cells grew on the periphery of the structure and were distributed in a three-dimensional network, forming a well-defined luminal structure. PAS and SR staining results indicated that the organoids contained abundant collagen and polysaccharides, such as glycosaminoglycans, which are known extracellular matrix components of human bone marrow mesenchymal stem cell resident sites in natural bone marrow. In addition, Masson's trichrome staining results showed a large distribution of collagen fibers in the organoids. In summary, the 7-day cultured callus organoids exhibited extracellular matrix components of human bone marrow mesenchymal stem cells from natural callus, including collagen fibers, glycogen, and collagen.

[0097] Magnesium silicate regulates the prevascularized callus organoids to promote bone tissue regeneration in vivo.

[0098] Given the excellent in vitro biological activity of callus organoids, a rat femoral condyle defect model was further constructed to demonstrate the performance of prevascularized callus organoids in tissue regeneration. Specifically, callus organoids cultured for 7 days under the action of magnesium nanorods or organoids without magnesium silicate were implanted into the femoral condyle defect. Six weeks after repair, tissue samples were collected for biological evaluation. Analysis was performed based on Micro-CT reconstruction results. Compared with other groups, the BOs / 2NT-GM group showed more newly formed bone in the bone defect area (red, Figure 11 a). Furthermore, statistical analysis showed that the BOs / 2NT-GM group had significantly higher ratios of BS (new bone surface area) to TV (total defect volume), BV (bone volume) to TV (total volume), and trabecular bone number (Tb.N) than the other two groups. Figure 11 b). Stain the regenerated bone defect area with masson and safranin-fast green. Figure 12 The results showed that the Blank group, BOs / GM group and BOs / 2NT-GM group still had bone defect areas of varying degrees. The Blank group had only a small amount of matrix deposition in the defect area, while the other two groups showed a small amount of trabecular bone structure. Among them, the BOs / 2NT-GM group had the most obvious new bone trabecular bone structure, indicating that the callus organoid regulated by the nanorod magnesium silicate material has the best bone regeneration effect.

[0099] In summary, the purpose of this invention is to prepare a prevascularized callus organoid for bone defect repair. The prevascularized callus organoid is formed under the action of the inorganic bioactive material magnesium silicate. The developed callus organoid exhibits ideal in vitro bioactivity, including excellent osteogenic differentiation, vascularization, and mechanotransformation properties, and contains bone-related matrix deposition. The addition of magnesium silicate further enhances the performance of the constructed prevascularized callus organoid, significantly promoting the repair of femoral condyle bone defects in rats. This invention provides an innovative paradigm for using callus organoids to repair skeletal system defects.

Claims

1. A prevascularized callus organoid, characterized in that, The prevascularized callus organoids are prevascularized callus organoids with the functional characteristics of natural callus, formed by developing composite hydrogel microspheres loaded with silicate bioceramics, human bone marrow mesenchymal stem cells, and endothelial cells in organoid culture medium.

2. The prevascularized callus organoid according to claim 1, characterized in that, The composite hydrogel microspheres have a porous structure; preferably, the composite hydrogel microspheres are composite hydrogel microspheres with a porous structure prepared using a microfluidic chip.

3. The prevascularized callus organoid according to claim 1 or 2, characterized in that, The endothelial cells are human umbilical vein endothelial cells; the silicate bioceramic is magnesium silicate.

4. The prevascularized callus organoid according to any one of claims 1 to 3, characterized in that, The initial loading ratio of human bone marrow mesenchymal stem cells to endothelial cells is 1:1 to 5:1; preferably, the total number of bone marrow mesenchymal stem cells and endothelial cells seeded in each composite hydrogel microsphere is 400 to 1000.

5. The prevascularized callus organoid according to any one of claims 1 to 4, characterized in that, The mass concentration of silicate bioceramics in the composite hydrogel microspheres is no higher than 4%.

6. The prevascularized callus organoid according to any one of claims 1 to 5, characterized in that, The silicate bioceramics in the composite hydrogel microspheres have spherical, rod-shaped, or amorphous shapes, preferably nanorod-shaped.

7. The prevascularized callus organoid according to any one of claims 1 to 6, characterized in that, The hydrogel matrix of the composite hydrogel microspheres is GelMA hydrogel; preferably, the mass fraction of GelMA hydrogel is 4% to 20%.

8. The method for preparing prevascularized callus organoids according to any one of claims 1 to 7, characterized in that, The preparation method includes: mixing silicate bioceramics, human bone marrow mesenchymal stem cells, endothelial cells and hydrogel matrix to obtain a cell-loaded composite hydrogel solution; passing the cell-loaded composite hydrogel solution through a microfluidic chip to form composite hydrogel microspheres of uniform size; after cross-linking and solidification, the composite hydrogel microspheres are cultured in organoid culture medium; and then the medium is changed regularly to continue culturing, thereby constructing a prevascularized callus organoid.

9. The preparation method according to claim 8, characterized in that, The culture time in organoid culture medium is 1 to 14 days.

10. The use of a prevascularized callus organoid according to any one of claims 1 to 7 in the preparation of tissue regeneration materials.

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