A method for preparing a mineralized nanofiber scaffold composite poly-l-glutamic acid-based injectable macroporous hydrogel

Abstract

This invention relates to a method for preparing a mineralized fiber scaffold composite injectable macroporous hydrogel that mimics natural bone emulsion (ECM). The method proposes preparing a PBLG fiber scaffold using electrospinning, gas foaming, and in-situ mineralization. Subsequently, the mineralized fiber scaffold composite injectable macroporous hydrogel is constructed through negative pressure perfusion, mechanical fragmentation, and gel fragmentation and reorganization. The mineralized fiber scaffold composite injectable macroporous hydrogel prepared by this invention can adapt to irregular bone defect areas, providing temporary support after implantation and offering excellent osteogenic induction and angiogenic activity for subsequent bone defect repair. The macroporous structure formed by microgel reorganization also supports the ingrowth of new tissue. This material has promising applications in bone defect repair and can also be extended to other medical fields such as cartilage defect repair and drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of bone tissue engineering scaffold materials technology, and particularly relates to a mineralized fiber scaffold composite injectable macroporous hydrogel and its preparation method. Background Technology

[0002] Treatment of complex bone defects with irregular shapes or located at soft tissue interfaces (such as cartilage-bone, tendon-bone, and ligament-bone) remains a clinical challenge. Autologous bone grafting remains the "gold standard" for treating such injuries. However, its clinical application is severely limited due to the limited availability of donors, high morbidity at donor sites, and difficulty in adapting to irregular defects. Injectable hydrogels, implanted minimally invasively, avoid the high trauma of surgical procedures, possess good biocompatibility, and adaptability to filling complex-shaped defects, providing a promising new approach for bone defect repair and bone tissue regeneration (Liu M, Zeng X, Ma C, et al. Bone Res. 2017, 5, 17014.).

[0003] Natural bone matrix is ​​a three-dimensional mineralized nanofiber network structure formed by interwoven collagen and nano-hydroxyapatite (nHA). Three-dimensional mineralized collagen nanofibers are the basic structural units of the bone matrix and form the nanostructural basis for its excellent biological and mechanical properties (Koons GL, Diba M, Mikos AG. Nat. Rev. Mater. 2020, 5, 584-603.). Constructing biomaterials that mimic the structure and function of natural bone matrix can effectively expand its application in bone tissue engineering. In recent years, layered electrospun fiber membranes have been transformed into three-dimensional fiber scaffolds through gas foaming (Chen SX, Wang HJ, McCarthy A. Nano Lett. 2019, 19, 2059-2065.). However, these fiber scaffolds cannot be injected. Based on the biomimetic mineralization of three-dimensional nanofiber scaffolds, how to introduce mineralized nanofiber scaffolds into injectable hydrogel systems for bone repair, mimicking the micro-nano topology of natural bone matrix, and enhancing hydrogel strength while promoting cell adhesion and osteogenic differentiation, has become an urgent problem to be solved.

[0004] To promote cell penetration and ingrowth, angiogenesis, and nutrient transport, injectable hydrogels for bone repair should possess interconnected macroporous structures to facilitate osteogenic and vascularization (Swanson WB, Omi M, Zhang Z, et al. Biomaterials 2021, 272, 120769.). However, the nanoscale or submicron-scale pore structure between the polymer networks within traditional fibrous composite hydrogels limits cell penetration and ingrowth. Current methods for preparing porous hydrogels mainly include porogen leaching, freeze-drying, or 3D printing, but these methods are not suitable for injectable hydrogel systems (Daly AC, Riley L, Segura T, et al. Nat. Rev. Mater. 2020, 5, 20-43.). Microgel assembly is an effective method for preparing injectable macroporous hydrogels. The interconnected macroporous structure formed after microgel stacking and assembly can support cell proliferation and migration, providing sufficient space for bone tissue ingrowth. Currently, the main techniques for preparing microgels include emulsification, microfluidics, photolithography, and mechanical fragmentation. Compared with other methods, mechanical fragmentation has the advantages of high speed, simplicity, and large production capacity. This method is suitable for fiber scaffold composite hydrogels. By breaking down the bulk fiber scaffold composite hydrogel into microgels, it can be endowed with injectable properties. After injection, microgels can be assembled to obtain microfiber scaffold composite hydrogels with macroporous structures.

[0005] Poly-L-glutamic acid (PLGA) and its derivative, poly-L-glutamic acid benzyl ester (PBLG), possess protein-like secondary structures that can mimic the collagen components in the natural bone matrix. However, no reports have been published on mineralized nanofiber scaffolds composited with poly-L-glutamic acid-based injectable macroporous hydrogels for bone defect repair. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies and solve the problems of traditional fiber scaffold composite hydrogels being non-injectable, lacking macroporous structure, and lacking osteogenic and angiogenic activity, this invention provides a method for preparing a mineralized nanofiber scaffold composite PLGA-based injectable macroporous hydrogel from a biomimetic perspective.

[0007] This invention proposes to form a mineralized fiber scaffold composite hydrogel in situ by perfusing a poly-L-glutamic acid / maltodextrin (PLGA / MDex) precursor into a poly-L-glutamic acid benzyl ester (PBLG) mineralized fiber scaffold. A "fragmentation and recombination" strategy is employed, utilizing dynamic Schiff base bonds between microgels obtained after mechanical fragmentation / chelation of copper-doped nano-hydroxyapatite (n-CuHA)-bisphosphonic acid groups (BP) to construct a mineralized nanofiber scaffold composite PLGA-based injectable macroporous hydrogel. This approach imparts injectability to the fiber scaffold composite hydrogel while providing the macroporous structure necessary for bone repair and excellent osteogenic and angiogenic activity.

[0008] Furthermore, the present invention also provides a method for preparing a mineralized nanofiber scaffold composite PLGA-based injectable macroporous hydrogel, comprising the following preparation steps: (1) A mineralized PBLG nanofiber scaffold with surface-deposited n-CuHA was prepared by electrospinning, foaming expansion and in-situ co-precipitation mineralization. (2) Using bisphosphonic acid-hydrazide PLGA (PLGA-BP-ADH) and aldehyde-modified maltodextrin (MDex-CHO) as precursors, the mineralized PBLG nanofiber scaffold was immersed in the precursor solution. The solution was filled with the fiber scaffold by drawing negative pressure with a vacuum pump. After standing for a period of time, it was taken out to obtain the mineralized nanofiber scaffold composite PLGA-based hydrogel. (3) After freezing the mineralized nanofiber scaffold composite PLGA-based hydrogel, it was placed in anhydrous ethanol and mechanically broken down using an emulsifier. Subsequently, the mineralized nanofiber scaffold composite PLGA-based microgel was obtained by centrifugation and PBS replacement.

[0009] (4) PBLG nanofiber scaffold@PLGA-MDex injectable macroporous hydrogel was constructed by recombining mineralized fiber scaffold with PLGA-based microgel.

[0010] The weight-average molecular weight of PBLG is 30~120kDa, the molecular weight of PLGA is 80~120kDa, the ratio of -NH2:-CHO in the precursor solution is 1:2~2:1, the solid content in the precursor solution is 8%~12%, the emulsifier speed is set to 10000~15000rpm, and the emulsifier crushing time is set to 2~20min.

[0011] Compared with the prior art, the beneficial effects of the present invention are: (1) The mineralized nanofiber scaffold composite PLGA-based injectable macroporous hydrogel of the present invention has both a three-dimensional mineralized nanofiber scaffold and a biomimetic structure with interconnected macropores, effectively solving the problems of traditional injectable hydrogels lacking three-dimensional micro-nano topology and insufficient internal pore connectivity.

[0012] (2) The mineralized nanofiber scaffold composite PLGA-based injectable macroporous hydrogel involved in this invention combines multiple factors to synergistically promote osteogenic and angiogenic processes, and is expected to better exert the biological activity of the material.

[0013] (3) The mineralized nanofiber scaffold composite PLGA-based injectable macroporous hydrogel involved in this invention has good interfacial bonding and multiple cross-linking structure, which can effectively enhance the structural stability of the composite hydrogel and accelerate microgel recombination. Attached Figure Description

[0014] Figure 1This is a schematic diagram illustrating the preparation and principle of the mineralized nanofiber scaffold composite PLGA-based injectable macroporous hydrogel involved in this invention. Detailed Implementation

[0015] The technical solution of the present invention will be described in detail below with reference to specific embodiments. It should be noted that the following embodiments are merely preferred implementation examples of the present invention, intended to help understand the core ideas and implementation methods of the present invention, and do not constitute a limitation on the scope of protection. Based on the basic principles of the present invention, all technical solutions obtained by those skilled in the art through conventional experimental methods or reasonable technical extensions, based on a full understanding of the contents of this disclosure, fall within the protection scope of the claims of the present invention.

[0016] Example 1 Poly(L-glutamic acid benzyl ester) (PBLG, 80 kDa) mineralized nanofiber scaffolds (n-CuHA / PBLG-BP) were prepared by electrospinning, sodium borohydride gas foaming and in-situ mineralization.

[0017] The poly-L-glutamic acid (150kDa) / maltodextrin (PLGA-BP-ADH / MDex-CHO) hydrogel precursor (-NH2:-CHO ratio of 1:1, solid content of 10%) was injected into the interior of the n-CuHA / PBLG-BP mineralized fiber scaffold by negative pressure injection to form a mineralized fiber scaffold composite hydrogel in situ.

[0018] The blocky mineralized fiber scaffold composite hydrogel was frozen at -20°C and then transferred to anhydrous ethanol. Mechanical disruption was performed using an emulsifier at 12,000 rpm for 10 minutes. The mineralized fiber scaffold composite microgel was collected by centrifugation at 8,000 rpm for 10 minutes. The ethanol in the microgel was replaced with PBS using a solvent displacement method.

[0019] Mineralized fiber scaffold composite microgel was injected into a specific mold and allowed to stand for 30 minutes to obtain mineralized PBLG nanofiber scaffold@PLGA-MDex injectable macroporous hydrogel.

[0020] Example 2 The molecular weight of PLGA in PLGA-BP-ADH in step (2) of Example 1 was adjusted to 8kDa, while the rest remained the same as in Example 1.

[0021] Example 3 The molar ratio of -NH2:-CHO in the precursor solution of step (2) of Example 1 was adjusted to 1:2, while the rest remained the same as in Example 1.

[0022] Example 4 The solid content of PLGA-BP-ADH / MDex-CHO in the precursor solution of step (2) of Example 1 was adjusted to 12%, while the rest remained the same as in Example 1.

[0023] Example 5 The mechanical crushing rate of the emulsifier in step (3) of Example 1 was adjusted to 15000 rpm, while the rest remained the same as in Example 1.

[0024] Example 6 The mechanical crushing time of the emulsifier in step (3) of Example 1 was adjusted to 5 minutes, while the rest remained the same as in Example 1.

[0025] Comparative Example 1 PLGA-BP-ADH / MDex-CHO was infused into the interior of a mineralized PBLG fiber scaffold using a negative pressure method to obtain a mineralized PBLG fiber scaffold@PLGA-MDex hydrogel.

[0026] Performance testing The mineralized PBLG nanofiber scaffolds@PLGA-MDex hydrogels obtained in the examples and comparative examples were subjected to performance testing, and the test results are as follows: Pore ​​size, porosity, and swelling ratio of mineralized PBLG nanofiber scaffold@PLGA-MDex hydrogel: The microstructure of the mineralized PBLG nanofiber scaffold@PLGA-MDex hydrogel was observed by scanning electron microscopy, and its pore size was measured. Porosity was measured by ethanol displacement method, and swelling ratio was measured by gravimetric method. Specific results are shown in Table 1.

[0027] Table 1. Pore size, porosity, and swelling ratio of mineralized PBLG nanofiber scaffolds@PLGA-MDex hydrogels 2. Rheological and compressive properties: The rheological and compressive properties of a 1cm cubic mineralized PBLG nanofiber scaffold@PLGA-MDex hydrogel were tested using a rotational rheometer and a dynamic thermomechanical analyzer to obtain the material's storage modulus and compressive strength. Specific results are shown in Table 2.

[0028] Table 2 Mechanical Properties of Mineralized Fiber Scaffold Composite Hydrogels The above results indicate that a PLGA-based mineralized fiber scaffold composite injectable macroporous hydrogel that mimics natural ECM can be obtained by using negative pressure infusion, mechanical crushing, and fragmented gel recombination.

[0029] It should be specifically noted that the preferred embodiments disclosed in this specification are merely exemplary examples illustrating the technical solution of the present invention and do not constitute a limitation on the scope of patent protection. Any person skilled in the art, upon fully understanding the technical concept of the present invention, may make equivalent substitutions, reorganize technical features, or adjust process parameters in the above embodiments. Such technical modifications, as long as they do not depart from the substantive technical features specified in the claims, shall be considered within the legal protection scope of the patent right of the present invention. The legal protection boundary of the present invention shall be subject to the content of the claims published by the State Intellectual Property Office. Any non-substantive changes based on the technical solution of the present invention shall not override the statutory protection effect of the patent right.

Claims

1. A method for preparing a mineralized fiber scaffold composite injectable porous hydrogel that mimics natural bone matrix, comprising the following steps: (1) A Cu mineralization layer was prepared sequentially by electrospinning, gas foaming, and in-situ mineralization. 2+ Mineralized poly(L-glutamic acid benzyl ester) fiber scaffold with macroporous structure doped with hydroxyapatite (n-CuHA) (n-CuHA / PBLG). (2) The hydrogel precursors, bisphosphonate-hydrazide-modified poly-L-glutamic acid (PLGA-BP-ADH) and aldehyde-modified maltodextrin (MDex-CHO), are injected into the interior of the n-MeHA / PBLG mineralized fiber scaffold by drawing negative pressure to form a mineralized fiber scaffold composite hydrogel in situ. (3) A “crush-and-reassemble” strategy was adopted, in which the mineralized fiber scaffold composite hydrogel was mechanically crushed by an emulsifier to obtain microgels. Reassembly was achieved through dynamic Schiff base bonding / n-CuHA-bisphosphonic acid (BP) chelation between microgels to construct a mineralized fiber scaffold composite PLGA-based injectable macroporous hydrogel.

2. The method according to claim 1, characterized in that, In step (2), the mass fraction of PLGA-BP-ADH / MDex-CHO in the hydrogel precursor solution is 8%~12%.

3. The method according to claim 1, characterized in that, In step (3), the speed of the emulsifier is 6000~10000 rpm.

4. The method according to claim 1, characterized in that, In step (3), the emulsifier crushing time is 2~20 min.

5. The method according to claims 1 to 4, preparing a mineralized fiber scaffold composite injectable porous hydrogel.

6. The mineralized fiber scaffold composite injectable porous hydrogel described in claim 5 can be applied to medical fields such as tissue repair, hemostasis, and drug delivery.

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