Mechanically Robust, Shape-Memory, and Personalized Silk Fibroin/Magnesium Composite Scaffolds induce in situ Irregular Bone Regeneration

doi:10.21203/rs.3.rs-3639666/v1

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Mechanically Robust, Shape-Memory, and Personalized Silk Fibroin/Magnesium Composite Scaffolds induce in situ Irregular Bone Regeneration

https://doi.org/10.21203/rs.3.rs-3639666/v1

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published 16 May, 2024

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The regeneration of critical-sized bone defects, especially for irregular shapes, remain a clinical challenge. Although various biomaterials were developed to enhance bone regeneration, the limitations on the shape-adaptive capacity, the complexity of clinical operation and the unsatisfied osteogenic bioactivity restricted greatly their clinical application. Herein, we construct a mechanically robust, tailorable and blood-responsive shape memory effect silk fibroin/ Magnesium (SF/MgO) composite scaffold, which can quickly match irregular defects by easily trimming and achieve good interface integration. The SF/MgO composite scaffold exhibits excellent mechanical performance and structure retention during the degradation process, which significantly improved the supporting ability in defective site. In addition, the SF/MgO composite scaffolds can achieve shape recovery very quickly either under water or blood conditions. This scaffold promotes cell proliferation, adhesion, migration of osteoblasts, and osteogenic differentiation of BMSCs in vitro. Subcutaneous implantation results confirmed that with suitable content SF/MgO composite scaffold exhibited good histocompatibility, low foreign body reactions, and as well as significant ectopic mineralization and angiogenesis. Meanwhile, the SF/MgO composite scaffold markedly promote in situ robust bone regeneration of cranial defect. This bioactive shape memory porous scaffolds provides a new design idea for minimally invasive clinical-size irregular bone regeneration.

Health sciences/Health care/Fracture repair

Health sciences/Health care/Quality of life

silk fibroin

magnesium

shape memory scaffold

irregular bone defects

bone regeneration

Regeneration of critical-sized irregular bone defects caused by trauma, tumor, or infection is a major challenge for orthopedic surgeons^1,2. The clinical golden standard to treatment of these bone defects until now is still considered as autologous bone grafting³; however, autografts are associated with several limitations such as postoperative pain, large segmental bone loss and potential donor complications^4,5. The alternatives to clinical bone prostheses, such as titanium alloy, and polyether ether ketone, have excellent mechanical properties^6,7. What is more, their inherent bioinertness and poor biodegradation ability results in poor integration between the grafts and host bone tissue, leading to loosening, detachment and ineffective functional bone regeneration^8–10. Most importantly, most of the existing bone substitutes lack shape adaptability, resulting in insufficient matching for irregular defects^11–12; the bone defects are enlarged into regular ones through open surgery, which increases the difficulty and cost of surgical procedures, and brings severe trauma, pain and severe dysfunction to the patients. Therefore, it is worth looking forward to developing a structurally stable and shape memory porous scaffold that could adapt to the irregular-shaped bone defect to achieve minimally invasive treatment.

The emergence of Enhanced Recovery After Surgery (ERAS) has placed new demands on orthopedic surgeons to minimize surgical trauma using the appropriate bone substitute materials¹³. Calcium phosphate bone cement (CPC) is an popular injectable biomaterial owing to well biocompatibility, osteoconductivity and its fitting ability, which has been used for various micro-invasive patterns (such as vertebroplasty and arthroscopic bone repairing). However, due to imporous structures, slow degradation rate, and low osteoinductivity contribute to limited functional bone regeneration¹⁴. Recently, injectable hydrogels, a kind of minimally invasive materials, have received increasing attention in tissue engineering and regenerative medicine due to their adjustable physicochemical properties in response to the surrounding environment, which can increase patient comfort and shorten recovery time. For example, some photosensitive or heat-sensitive injectable hydrogels have been developed, which had better biocompatibility and bone regeneration capacity^15,16. Shape memory polymers (SMPs), a kind of emerging intelligent biomaterials, have also brought great hope to patients with bone defects. Shape memory polymers have the ability to transform between initial shapes and programmed under various stimuli such as light^17,18, heat¹⁶, electrical or magnetic fields¹⁹, or water²⁰. It can be implanted into bone tissue by minimally invasive method, and “self-adapt” the irregular bone defect shape, so that the scaffold can strengthen the tightness of contact between the material and the surrounding tissue^17,21. However, for biomedical devices, the most physiologically accessible shape-recovery stimuli are temperature and water content. The need for external stimulation of shape memory polymers would inevitably increase the complexity, cost and time of clinical operation. In comparison, it would be very attractive that utilize the blood during the patient's surgery to induce quickly restore the original shape of material. Therefore, a shape memory polymer should be developed to meet the ERAS requirements and benefit for patients, especially for treating irregular bone defects.

Silk fibroin (SF) is extracted from cocoons of silkworms, and has demonstrated easy accessibility, good biocompatibility, controllable biodegradation and excellent mechanical properties, leading to its wide application in the fields of tissue engineering, regenerative medicine, drug delivery and medical devices^22–26. However, traditional silk fibroin materials do not have shape memory effect, and the structure will be permanently damaged under external force, resulting in a tight fit with bone defects after implantation. Currently, there have been some reports about SF-based shape memory materials. Kaplan et al. reported a shape memory SF sponge by addition of hygroscopic plasticizers, and tyrosine modifications with hydrophilic sulfonic acid chemistries²⁷. For biomedical devices, implantable shape memory materials must be sufficiently tough and fatigue resistant, exhibit a recovery force strong enough to expand in a confined space. Previously, we have developed cryogelled dual-crosslinked SF scaffolds with robust mechanical, fatigue resistant and water-trigger shape memory performance, as well as good bone marrow mesenchymal stem cells (BMSCs) biocompatibility and histocompatibility, it has been successfully applied to the repair of joint cartilage^28,29. However, bone repair is a complex process that involves many types of cells, signaling molecules, and multiple growth factors. Growth factor can promote bone formation, accelerate bone healing and improve the quality of bone repair. The lack of biochemical cues that trigger cell proliferation and differentiation significantly limits application of SF in these areas. Therefore, in order to improve the biological activity of SF materials, they are generally used in conjunction with other bioactive agents or cells^30,31. However, biomaterials containing cells and growth factors are clinically limited due to ethical and biosafety concerns (e.g., tumorigenesis in BMP-2)³².

Magnesium is an essential element for bone health^33,34 because it involved in the physiological processes of bone tissue formation, bone metabolism and bone mineral crystallization. In the human body, approximately 60% of magnesium is stored in the bone matrix³⁵. Previously, our group have demonstrated that the viability and osteogenic differentiation properties of pre-osteoblasts and mesenchymal stem cells (MSC) could be upregulated when the concentration of magnesium ion (Mg²⁺) at 50 to 100 ppm^36,37. Our group further revealed that the magnesium ions (Mg²⁺) could promote the neuronal calcitonin gene-related polypeptide-a (CGRP) -mediated osteogenic differentiation³⁸. Studies also proved that magnesium could also regulate the cell functions including proliferation, adhesion, and migration, as well as promote neovascularization^36,39. However, the rapid degradation and subsequent hydrogen release of Mg and its alloys in vivo has been an urgent problem for researchers. In contrast, magnesium oxide (MgO), an oxide of magnesium, neither degrades rapidly nor produces hydrogen in vivo⁴⁰. Therefore, it can be used as a source of Mg²⁺ in bone tissue materials. Based on its bioactivity, we designed to introduce MgO particles to dual-crosslinked SF scaffolds and then regulate its osteopromotive function, and retained its shape memory ability. On the other hand, silk fibroin is a negatively charged, while MgO particles are positively charged; electrostatic interactions can occur between them, which further promotes the formation of multiple cross-linked structures in the silk fibroin system. Therefore, with the introduction of MgO particles, the mechanical properties of SF scaffolds can be further increased, and promoted the structural stability of their service process.

Herein, we developed a mechanically robust, water-responsive and personalized silk fibroin/Mg shape memory porous scaffold. Based on clinical adaptability, this scaffold was expected to be versatile, that is, with a uniform porous structure, good excellent mechanical properties, excellent biological performances, and a blood-responsive shape memory effect that enables it to tight-contact with surrounding tissue. After being implanted, Mg ions will be released slowly with the degradation of MgO particles to achieve the repair of critical-sized bone defect (Fig. 1b). Therefore, this shape memory silk fibroin/Mg scaffold is expected to have broad clinical applicability and can guide minimally invasive treatment of critical-sized bone tissue repairing.

Design and characterization of bioactive Silk Fibroin/Magnesium composite scaffold

To fabricate a structurally stable and shape memory porous scaffolds for irregular bone defects, the silk fibroin/Mg composite scaffold was firstly fabricated (Fig. 1a). The detailed experimental details are described in the Supplementary Information and Supplementary Fig. 1. Briefly, the SF/MgO composite scaffolds with various contents of MgO particles (0 wt%, 10 wt%, 30 wt%, relative to the weight of the SF matrix) were prepared via cryogelation at − 10°C. During the process, the phenyl hydroxyl and amino groups of concentrated SF solution could react with the epoxy group of EGDE, and then SF chains also form β-sheet crystalline structure through self-assembly as physical crosslinks. The negative surface charge of the MgO particles enables loading with positively charged silk fibroin molecules by simple charge-charge interaction. The results also demonstrated that the scaffolds have shape memory properties and customization, which could closely match the irregular cranial defect sites (Fig. 1b). For the preparation of SF/MgO composite scaffold, we first extracted silk fibroin according to our previous experimental procedure⁴¹. Then, 10% and 30% MgO particles were added to the blank SF, and a homogeneous mixture solution was prepared using vortices method (Supplementary Fig. 1). As shown in Fig. 2a and Supplementary Fig. 2, 3, the changes before and after the mixed solution gel could be clearly seen. After 24 h of cryogelation, scanning electron microscopy (SEM) images confirmed that the three scaffolds had continuous porous structures with pore sizes of ∼100 µm (Fig. 2a, and Supplementary Fig. 2, 3). Moreover, MgO particles were uniformly distributed in the pore wall and inside the matrix of the SF/MgO scaffolds. Energy-dispersive spectrometry maps and spectra also confirmed the successful incorporation of MgO particles into SF scaffolds.

Due to the existence of MgO particles, the typical peaks of appear at 18.5° and 42.8° in the corresponding XRD patterns of the SF/MgO composite scaffolds while no peak is in that of the SF scaffolds (Fig. 2b). The structure of the newly prepared SF/MgO composite scaffolds and pure SF scaffolds was also characterized by FTIR. The absorption bands show around 3700 cm^–1 corresponding to the characteristic peaks of MgO particles, which indicated the newly prepared SF/MgO composite (Fig. 2c). Secondary structure was analyzed to correlate changes in protein structure with differences in SF scaffolds mechanical and degradation properties with and without MgO particles. In the Amide I band, random coils and α-helix structures were generally found at 1640–1660 cm^− 1, whereas the β-sheet structure was commonly identified at 1620–1637 cm^{− 1 42}. The peaks of three groups at 1621 cm^− 1 indicated that their secondary structure is mainly based on the β-sheet structure. Detailed conformation measure from peak deconvolution in the amide I region provides information of the β-sheet conformation in SF and SF/MgO scaffolds (Supplementary Fig. 4). Figure 2d showed that the three groups contained the same β-sheet conformation (∼44%), which indicate MgO particles did not affect the secondary structure of SF scaffolds. Our preliminary study revealed that the chemical cross-linking in cryogelation may have promoted the formation of more β-sheet conformation⁴¹.

To study whether the addition of MgO particles affects the degradation behavior of SF scaffolds, according to the previous relevant procedures⁴³, SF, SF-1nMgO, and SF-3nMgO were cultured in 2 U/mL protease XIV solution to evaluate the degradation effect (pH = 7.4, 37°C). The macroscopic appearance of the three scaffolds after various times was showed in Fig. 2e. The addition of MgO particles reduced the degradation rate of SF scaffolds. After 4 weeks, the degradation rate of the three scaffolds followed the order: SF-3nMgO > SF-1nMgO > SF (Fig. 2f). This trend implies that the degradation rate of SF-MgO scaffolds can be changed by adjusting the proportion of MgO particles. The results of FITR showed that the secondary structure content is the same among the three groups, which determines the degradation rate of SF scaffolds. However, the degradation rate of the three groups is not different. To further explore the reason, we carried out Zeta Potential test of the three solutions. In the case of pure SF solutions, the zeta potentials were around − 4 mV, while the zeta potential of the SF-1nMgO and SF-3nMgO solutions were around − 7 mV and − 12 mV, separately (Fig. 2g). The lower value of zeta potentials indicates a more stable material system⁴⁴, which is also the reason for the different degradation trends among the three groups. The accumulative Mg²⁺ releasing curve and pH value of the SF-1nMgO and SF-3nMgO scaffolds were measured in simulated body fluid (SBF) for 3 to 28 days to demonstrate the Mg²⁺ releasing profile of the SF/MgO composite (Fig. 2h, i). Compared to the SF-1nMgO scaffolds, SF-3nMgO scaffolds released Mg²⁺ rapidly during the 4 weeks. Since more Mg²⁺ are released in SF-3nMgO scaffolds, the pH value of the corresponding soaking solution is also larger.

Mechanical properties and shape memory effect of the bioactive Silk Fibroin/Magnesium composite scaffolds

The mechanical properties of the SF/MgO composite scaffolds were systematically investigated. The obtained SF/MgO composite scaffolds could withstand complex mechanical patterns; as the applied loads were released, the composite scaffolds could completely recover to its original shape without structural failure by water adsorption, indicating excellent mechanical properties and perfect shape recovery ability of the SF/MgO composite scaffolds (Fig. 3a). As a bone implant material, its mechanical retention during degradation is essential for the formation of new bone tissue and tight integration with the surrounding bone tissue. The mechanical properties of the SF/MgO composite scaffolds were measured during degradation. The compression stress − strain curves of the scaffolds were illustrated in Fig. 3b1-3b3. Figure 3c displayed results of the compressive modulus of the scaffolds. With increased of MgO particles content, the compression modulus displayed increasing trend from SF scaffolds to SF-3nMgO scaffolds, relative to 60 kPa for SF scaffolds, 110 kPa for SF-1nMgO scaffolds, and 245 kPa for SF-3nMgO scaffolds. After 28 days immersion in phosphate buffered saline (PBS) solution (pH = 7.4, 37°C), the compressive modulus of the SF scaffold and SF/MgO composite scaffolds did not show significant attenuation, which could demonstrate the excellent structure stability of SF/MgO composite scaffolds during degradation. The cyclic compression test illustrated that the SF-1nMgO scaffolds could undergo multiple loading and unloading cycles with fast shape recovery, demonstrating the outstanding shape memory effect and fatigue-resistant ability of the SF-1nMgO scaffolds (Fig. 3d-3e and Supplementary Video 1).

The processability of bone implants during surgery is important for personalized customization of irregularly shaped bone defects. As shown in Fig. 4a, the SF/MgO composite scaffolds can be processed into large blocks that can be trimmed during surgery according to demand. The trimmed SF/MgO composite scaffolds can be implanted into defects with small caliber gaps (Supplementary Video 2). The shape memory property enables the SF/MgO composite scaffolds to instantly restore its original state, and self-adapt to the shape of the defect, achieving a tight-contacting to the bone defect. To reduce the complexity and cost of surgery, the most physiologically accessible shape-recovery stimuli are temperature and water content for biomedical devices. Figure 4b and Supplementary Video 3, 4, 5 show the SF/MgO composite scaffolds can achieve shape recovery very quickly either under water or blood conditions. In addition, because of the recovery effect, the SF/MgO composite scaffolds could lift more than 25% at a 50g weight (Fig. 4c). These results proved that the SF/MgO composite scaffolds had good blood-responsive shape memory effect and supporting ability. Compared with other tissue engineering materials with shape memory performance, SF/MgO composite scaffolds are also basically at the forefront in terms of shape recovery rate and recovery time (Fig. 4d).

In vitro biocompatibility, migration assessment and osteogenic effect of the bioactive Silk Fibroin/Magnesium composite scaffolds

To verify the effects of SF/MgO scaffold on morphology, cell viability, proliferation and migration, the MC3T3-E1 pre-osteoblasts were cultured with the extract of the SF, SF-1nMgO, SF-3nMgO scaffolds while the control group had no treatment. F-actin cytoskeleton and Live/Dead staining fluorescence images showed that the cells incubated with various samples displayed well-spreading morphology and high viability (Fig. 5a, b). The CCK-8 assay indicated the cells proliferated more along with time in all groups. The optical density (OD) values of the SF-3nMgO group were significantly higher than that of the SF-1nMgO and SF group at day 3,5 and 7 culture, which indicates the release of Mg²⁺ in promoting cell adhesion and growth, and there was no significant difference among SF-3nMgO and control groups in terms of the OD values (Fig. 5d). The ability of the scaffolds extracts to promote cell migration was studied by cell scratch. As shown in Fig. 5c, the results suggested that the migration ability of MC3T3-E1 cells was enhanced after treatment of SF-1nMgO and SF-3nMgO extracts compared with SF and control groups. Quantitative analysis of the cell scratch assay showed that the scratch healing rate of SF-1nMgO and SF-3nMgO groups reached 85.14 ± 4.50% and 95.68 ± 2.82%, respectively, indicating the SF-3nMgO extracts exhibited the strongest migration-promoting effect (Fig. 5e).

Alkaline phosphatase (ALP) and quantification on day 3,7 and 14 were firstly performed to assess the osteogenic differentiation performance of the extraction to the BMSCs (Fig. 6a, b). At 3 days, the purple ALP-staining colony of the was darker with the increasing content of the Mg, and there was no significant difference between control and SF group. After 7 days and 14 days incubation, the stained colony of the all groups was darker and SF-3nMgO group still exhibited the most intense staining followed by SF-1nMgO, SF and control groups. The ALP activity assay suggested that threefold value is found on SF-3nMgO group in comparison to the control group. The mineralization property of BMSCs incubation with various extraction were further assessed by Alizarin red S (ARS) staining (Fig. 6c, d). After 14 and 21 days of culturing, compared with the control group and the pure SF group, the calcium nodules were significantly improved with the increasing of Mg particles. The quantitative analysis of alizarin red showed that mineralized nodules in SF-3nMgO is around 3-fold higher values than on the control and SF groups. Furthermore, the gene expression of the Runx2, OCN, OPN and COL Ⅰ of the BMSCs after cultured for 7 days and 14 days were determined by qPCR. Generally, all the gene expression levels were gradually up-regulated over time in addition to the control group, and the related expression level in the SF-3nMgO group was significantly higher than those in the SF and SF-1nMgO groups (Fig. 6e-h). Subsequently, Western Blot analysis was conducted to detect the proteins levels of the Runx2, OCN, OPN and COL Ⅰ after 7 days of incubation. The expression levels of all genes in the SF/MgO groups were significantly increased, and the SF-3nMgO group presented the highest levels of gene expression (Fig. 6i, j). These results indicated the osteopromotive effects of the SF/MgO scaffold.

In vivo histological and ectopic osteogenesis assessment of the bioactive Silk Fibroin/Magnesium composite scaffolds

The foreign-body reaction (FBR) caused by the implantation of scaffolds seriously affects tissue-biomaterial integration and tissue repair⁴⁵. Generally, in the later stage of FBR, macrophages fuse into foreign-body giant cells (FBGCs) because of biomaterials are incapable of degrading, and the formation of fibrous capsule at the interface of material and tissue resulting in the significant reduction of biomaterial-tissue integration. To identify the host response to scaffolds, SF, SF-1nMgO and SF-3nMgO scaffolds were implanted subcutaneously into SD rats for 4 weeks (Fig. 7a). H&E and Masson’s trichrome staining were performed to analyze host cell infiltration, the formation of foreign-body giant cells (FBGCs) and collagenous fibrotic capsule. After 1 week of implantation, the host cells had already infiltrated across the tissue-scaffolds interface into the interior of scaffolds (Supplementary Fig. 5). At 4 weeks after implantation, the SF and SF-3nMgO scaffolds exhibited less host cell infiltration, in contrast, cell infiltration was significantly improved in the SF-1nMgO scaffolds (Fig. 7b, c). In addition, the SF and SF-1nMgO group exhibited similar FBGCs and collagenous fibrotic capsule formation, which significantly decreased compared with that of the SF-3nMgO group (Fig. 7b, d, e, f). These results indicate that SF-1nMgO scaffold can effectively mitigate FBR and accelerate the integration of tissue and scaffold.

The subcutaneous implant model in SD rats was performed to examine the ectopic osteogenesis and angiogenesis potential of scaffolds. Immunohistochemical staining images of osteogenesis (OCN) markers of different groups at 1, 2 and 4 weeks after the surgery are shown in (Fig. 8a). The different degrees of positive expression of OCN in all experimental groups was observed at 2 and 4 weeks. At 2 weeks, we found that SF-1nMgO and SF-3nMgO showed increased intensity compared with SF group (Fig. 8b). At 4 weeks, the positive expression of OCN of SF-1nMgO of scaffolds increases significantly compared to other groups (Fig. 8c) Immunofluorescence images of angiogenesis (CD31) markers showed that compared with the “SF” and the “SF-3nMgO” groups, the number of CD31-positive cells representing neovascular endothelial cells was significantly improved on the SF-1nMgO group (Fig. 8d, e, f) at 2 and 4 weeks. These results indicated that SF-1nMgO scaffold was beneficial to the osteogenesis and angiogenesis.

In vivo Osteogenic Effects of the bioactive Silk Fibroin/Magnesium composite scaffolds in a Cranial Defect Model

Encouraged by the above results, we used the rat critical-sized calvarial defect model to characterize the therapeutic performance of the SF/MgO scaffold in vivo (Fig. 9a), and the SF-1nMgO scaffold was selected due to its excellent histocompatibility and capacity for osteogenesis and angiogenesis. We prepared three groups: blank group (only defects), control group (defects filled by the SF scaffold) and experimental group (defects filled by the SF-1nMgO scaffold). Micro-CT analysis was firstly used to assess the bone regeneration at 4 and 8 weeks after surgery, The 3D reconstruction image as shown in Fig. 9b. Compared to the blank group and the SF scaffold group, the SF-1nMgO group presented more newly regenerated bone at each time point, especially at 8 weeks. Moreover, for quantitative analysis, the SF-1nMg group demonstrated the enhanced bone regeneration with highest bone mineral density (BMD) and bone tissue volume/total tissue volume (BV/TV) values, the BMD and BV/TV of the SF-1nMg scaffold group were respectively ~ 0.25 and 27.2% at 8 weeks while those in the SF scaffold group were 0.19 and 24%, and in the blank group were only 0.08 and 13% (Fig. 9c, d). To further analyze the generated bone tissue within the defect area, H&E and Masson’s trichrome staining were performed after 4- and 8-weeks implantation. The results of the H&E staining in Fig. 9e indicated the SF-1nMgO group presented the maximum new bone tissues on each time point compared to other groups explained by known osteogenetic effects of Mg²⁺. The new bone tissues grew into the pore of the scaffold and integrated with the scaffold. Conversely, the control groups were mostly covered by abundant blue fiber connective tissues, and the SF group presented some fibrous tissues with a small amount of new bone tissue. We further performed the Masson’s trichrome staining to assess the bone regeneration (Fig. 9f). More purple staining indicating the mature bone could be found in the SF-1nMg group, while other groups showed more blue stained immature woven bone or fibrous tissues. The immunofluorescence staining of CD31 was further conducted to identify the effects of scaffolds on neovascularization (Fig. 10a, a1). The SF-1nMgO group showed more CD31 positive cells within the defect site and represented 4.1 and 3.4 folds of blood vessel numbers compared to the control groups at 4- and 8-weeks post-operation (Fig. 10f). In addition, immunohistochemical staining images of osteogenesis-related protein expression BMP-2, Runx-2, OCN, and VEGF presented similar results (Fig. 10b-e, b1-e1, g1-g4). The results showed that the highest expression level of osteogenesis-related protein appeared in SF-1nMgO group, while the SF group presented weaker expression, as well as the limited expression in the control group. These results demonstrated the greater potential of the SF-1nMgO scaffold to facilitate the effective bone regeneration and provide promising approach for the clinical treatment of irregular bone defect.

The repair of irregular bone defects has always been a difficult problem for clinicians. And, higher requirements are also put forward for clinical surgery in order to achieve more minimally invasive treatment. Currently, most of the existing bone substitutes lack shape adaptability, poor intraoperative reprocessability and insufficient osteogenic activities, resulting in insufficient matching with bone defect shape, complexity of clinical operation and poor bone regeneration. In order to improve the biological activity bone implants, there were often used in conjunction with growth factors and cells, while the unpredictable clinical risks of exogenous cells and growth factors restrict their clinical use. Therefore, the need for shape memory, self-adaptive, and bioactive bone implants in situ to achieve minimally invasive bone regeneration has been raised.

In this study, the optimized of SF/MgO composite scaffolds with the interconnected pore structure, excellent mechanical stability and liquid-trigger shape memory properties was fabricated, which provided mechanical support for tight-contact with surrounding tissue, and enabled the inner growth of new bone and vessels. In comparison with the pure dual-crosslinked SF scaffolds, the mechanical performance of the multi-crosslinking SF/MgO composite material increases significantly after the incorporation of the nano-MgO. The main reasons are the covalent cross-linking between the epoxy group of EGDE and the phenyl hydroxyl group and amino group of silk fibroin, the physical cross-linking of β-sheets formed within or between silk fibroin molecules, and the electrostatic interaction between silk fibroin and MgO particles, which form a multiple cross-linking network structure^46,47. Noticeably, despite the improvement in mechanical properties, the SF/MgO composite material still retains its unique shape memory properties and customization, which significantly improves clinical operability and flexibility. The material successfully overcame the malleability and brittleness of calcium phosphate ceramics scaffolds, which brought difficulties to clinical implantation. Moreover, for bone implants, the mechanical retention during material degradation directly affects the integration ability of the material-tissue interface¹⁸. The SF/MgO scaffolds maintained its excellent mechanical retention of the degradation process under high nano-MgO content (30 wt%), which improves the interfacial integration with the tissues.

Studies have shown that magnesium cation plays an important role in the regulation of most cellular (pre-osteoblasts, BMSCs) functions, such as protein synthesis, activation and bone formation^48,49. To avoid the generation of hydrogen of magnesium metal biomaterials during rapid corrosion in an aqueous environment, the magnesium oxide (MgO) was widely used in conjunction with other materials, which act as the source of magnesium element⁵⁰. Our group previous have also proved that at appropriate dosage range of Mg²⁺ level could promote cell proliferation, viability, adhesion, migration and osteogenic differentiation of osteoblasts^37,51. In vitro cell studies prove that the Mg-contained SF/MgO composite scaffolds (SF-1nMgO and SF-3nMgO) not only significantly enhances attachment of MC3T3-E1 pre-osteoblasts (Fig. 5(a)), but also raises the cell proliferation, viability and migration. The results indicated that the SF/MgO composite scaffolds have good cell biocompatibility. The ALP staining, ALP activity, ARS staining, ARS mineralization, mRNA expression level, and protein expression level were carried out to assess the osteogenic differentiation performances of the SF and SF/MgO scaffolds to the BMSCs. Though the analysis of in vitro osteogenic effect of the SF and SF/MgO composite scaffolds, the composite scaffolds could promote effectively the osteogenic differentiation of BMSCs.

For most implanted biomaterials, the integration of the material and the surrounding tissue is highly dependent on the degradation behavior and structural stability of the materials. Studies showed that rapid degradation of materials may lead to loss of mechanical support and eventually incomplete tissue formation within materials. In contrast, slow degradation of the material could cause severe foreign body reaction, that is, the formation of thick collagen fibrotic capsule around the materials, greatly reducing the interface integration of the materials and the host tissues. In this study, in vitro physicochemical properties studies showed that with the increase of MgO particle content, the structure of SF/MgO composite scaffolds tended to be stable, and the degradation rate also decreased significantly. Therefore, it is very necessary to evaluate the histocompatibility of SF/MgO composite scaffolds with different magnesium content in vivo. The subcutaneous implantation results confirmed that the collagenous fibrous capsule thickness of the SF-1nMgO and SF-3nMgO scaffolds increased with the prolongation of implantation time. However, it can be seen that the structure of fibrous capsule changes from dense to loose, showing a tendency of ablation, which is very beneficial for the infiltration of host cells and the tissue-biomaterial integration. In terms of the number of cell infiltration, the number of SF-1nMgO scaffolds was significantly more than that of SF-3nMgO scaffolds. In addition, the subcutaneous ectopic osteogenesis ability of SF and SF/MgO composite scaffolds was confirmed. With the degradation of SF/MgO composite scaffolds accompanied by the gradual precipitation of Mg²⁺, which enriched interfacial tissue microenvironment. After 4 weeks implantation, immunohistochemistry and immunofluorescence results confirmed that the SF-1nMgO scaffolds showed better Mg²⁺-induced osteogenesis and angiogenesis ability. The qualitative and quantitative results of subcutaneous implantation revealed that the SF-1nMgO scaffolds have a high potential to stimulate bone repair for the repair of critical-sized bone defect.

The long-term cell-free SF/MgO bioactive scaffolds implantation on rat cranial defects confirmed the tissue integration and bone regeneration of the implanted scaffolds. After 8 weeks implantation, the Micro-CT, histological staining, immunohistochemistry and immunofluorescence analysis suggest that SF-1nMgO bioactive scaffolds than those in blank group and the SF group showed not only a great amount of new bone tissue in the bone defect, but also significantly increased the number of blood vessels during the regeneration process, which might be caused by the bioactive effects of released Mg²⁺ from the tight-contacting scaffold. The results of osteogenesis in vivo were consistent with those in vitro and subcutaneous implantations. Additionally, the SF-1nMgO scaffolds has proved to have a good ability to contact tightly the defective site and enhanced biomechanical properties for robust bone regeneration. After 4- and 8- weeks implantation, higher osteo-related expression of BMP-2, Runx2, and OCN in SF/MgO scaffolds was confirmed. The higher positive expression level of VEGF in SF/MgO scaffolds demonstrated a greater potential for facilitating vascularization. Taken together, the excellent structural preservation from the multi-crosslinking network structure, the tight-contacting ability from blood-responsive shape memory effect, and the osteogenic activities of the MgO particles guarantee the SF/MgO scaffolds with robust bone regeneration for the critical-sized bone tissue defects.

In summary, the shape memory porous scaffolds made of multi-crosslinking SF/MgO bioactive composites with tailorable, blood-responsive shape memory effect, tight-contacting abilities and osteopromotive were developed in this work for repair of irregular bone defects. First of all, the SF/MgO composites scaffolds had a uniform porous structure through the cryogelation. In addition, the mechanical performance of the SF/MgO composite scaffolds increases significantly after the incorporation of the nano-MgO, those still retains its unique blood-responsive shape memory properties and customization. The SF/MgO composite scaffolds maintained its excellent mechanical retention of the degradation process led to the supporting ability in defective site. Furthermore, the SF/MgO composite scaffold was proved to support the cell proliferation, viability, adhesion, migration of osteoblasts, and osteogenic differentiation of BMSCs in vitro cell experiments. Subcutaneous implantation results confirmed that SF-1nMgO scaffolds exhibited good histocompatibility and subcutaneous ectopic osteogenesis ability. More importantly, the cranial defects studies demonstrated the SF scaffolds showed limited new bone regeneration, while SF/MgO composite scaffold took advantages of its stable and appropriate magnesium ionic microenvironment to realize the robust bone regeneration. We anticipate that these bioactive shape memory porous scaffolds have great clinical application potential in minimally invasive clinical-size irregular bone regeneration.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the corresponding author.

Disclosure statement

The authors declare no competing interests, financial or otherwise.

Acknowledgements

This work was financially supported by National Natural Science Foundation of China（32201114, 82002303），the Guangdong Basic and Applied Basic Research Foundation (2022A1515220079, 2022A1515011536, 2021A1515220093, 2021A1515220086), Scientific Research Foundation of Peking University Shenzhen hospital (KYQD2022141), Beijing Municipal Health Commission (Grant Number: BMHC-2021-6, BJRITO-RDP-2023), Shenzhen Science and Technology Innovation Committee Project (SGDX20220530111405038, GJHZ20210705142543019).

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There is NO Competing Interest.

Supplementary.docx
Supplementary
SupplementaryVideo1.mp4
Shape memory effect and fatigue-resistant ability of the SF-1nMgO scaffolds
SupplementaryVideo2.mp4
The trimmed SF/MgO composite scaffolds for small caliber defects implantation
SupplementaryVideo3.mp4
Performances and shape memory effect of SF/MgO scaffolds
SupplementaryVideo4.mp4
Shape memory effect of SF/MgO scaffolds triggered by water
SupplementaryVideo5.mp4
Shape memory effect of SF/MgO scaffolds triggered by blood

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Mechanically Robust, Shape-Memory, and Personalized Silk Fibroin/Magnesium Composite Scaffolds induce in situ Irregular Bone Regeneration

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Design and characterization of bioactive Silk Fibroin/Magnesium composite scaffold

Mechanical properties and shape memory effect of the bioactive Silk Fibroin/Magnesium composite scaffolds

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