Bioactive mineralized small intestinal submucosa acellular matrix/PMMA bone cement for vertebral bone regeneration

Abstract Polymethylmethacrylate (PMMA) bone cement extensively utilized for the treatment of osteoporotic vertebral compression fractures due to its exceptional handleability and mechanical properties. Nevertheless, the clinical application of PMMA bone cement is restricted by its poor bioactivity and excessively high modulus of elasticity. Herein, mineralized small intestinal submucosa (mSIS) was incorporated into PMMA to prepare a partially degradable bone cement (mSIS–PMMA) that provided suitable compressive strength and reduced elastic modulus compared to pure PMMA. The ability of mSIS–PMMA bone cement to promote the attachment, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells was shown through cellular experiments carried out in vitro, and an animal osteoporosis model validated its potential to improve osseointegration. Considering these benefits, mSIS–PMMA bone cement shows promising potential as an injectable biomaterial for orthopedic procedures that require bone augmentation.


Introduction
As the global population ages, osteoporosis is becoming increasingly prevalent [1]. Osteoporosis is a condition of the bones characterized by decreased bone density and compromised bone architecture, leading to a higher susceptibility to bone fractures [2,3]. The most prevalent form of fracture caused by osteoporosis is osteoporotic vertebral compression fracture (OVCF) [4,5], with $1.4 million new cases estimated each year globally [6]. OVCF is primarily characterized by severe low back pain, which can severely limit mobility, significantly diminish the patient's quality of existence and even pose a risk of fatality [7,8]. Percutaneous vertebroplasty and kyphoplasty are common minimally invasive surgical treatments for OVCF [9,10]. In clinical practice, polymethylmethacrylate (PMMA) is the most commonly utilized bone cement [11]; its good handleability and rapid dispersion after injection ensure rapid fracture stabilization and pain relief. Additionally, PMMA bone cement can provide immediate support after implantation, allowing patients to have early mobility [12].
However, PMMA bone cement has several disadvantages. The greater elasticity modulus of PMMA in comparison to the cancellous bone surrounding it can lead to stress shielding, which has the potential to cause secondary fractures in the vertebrae adjacent to the site where PMMA is applied [13]. Moreover, PMMA is biologically inert and nondegradable within the body and does not promote osteocyte growth. Consequently, the adhesion of the bone cement to the adjacent bone interface is weak [14]. Therefore, the bone cement may be displaced at the surgical site [15]. Despite these limitations, PMMA bone cement is an excellent base material, and extensive research has been conducted to reduce its elastic modulus and impart bioactivity to meet the clinical needs of OVCF treatment.
Incorporating active materials into PMMA bone cement can help address these limitations [16]. Porcine small intestinal submucosa (SIS) is a natural substance composed of collagen and various bioactive compounds, such as growth factors that occur naturally in tissues, providing structural support to cells [17][18][19]. These properties, along with its good biocompatibility and 3D microenvironment [20], make SIS an attractive bioactive substance for bone regeneration. SIS-doped PMMA bone cement enhanced cellular growth and bone-forming ability in vitro and improved osseointegration in a vertebral defect animal model. It also had a lower elastic modulus than that of commercially available pure PMMA [21]. Research on SIS scaffolds has demonstrated their capacity to facilitate adherence, growth and differentiation of various cell lineages, such as bone marrow mesenchymal stem cells (BMSCs), osteoblasts and fibroblasts, resulting in accelerated bone regeneration. These findings suggest that SIS scaffolds exhibit both osteoinductive and osteoconductive properties [22]. In addition, SIS can serve as a scaffold for the formation and growth of nano-hydroxyapatite (nHA) under physiological conditions, resulting in the production of mineralized SIS (mSIS) with the potential to promote neo-osteogenesis [23]. This suggests that mSIS can be used to create scaffolds that imitate the composition and structure of natural bone. Enhancing the osteogenic properties of scaffolds can be achieved through the biomineralization process that promotes the adherence of nHA to the surface of collagen scaffolds [24]. Furthermore, compared with conventional PMMA bone cement, nHA-coated PMMA has superior biocompatibility and significantly enhances cell attachment [25].
We hypothesized that combining mSIS and PMMA could improve PMMA's physical and biological characteristics, yielding a bone cement with a reduced elastic modulus and increased bioactivity compared to those of pure PMMA bone cement. This mSIS-PMMA composite would also maintain the favorable support properties and mechanical flexibility of PMMA. Accordingly, mSIS was prepared using in vitro biomimetic mineralization and used as an active filler to modify PMMA bone cement to produce partially biodegradable mSIS-PMMA bone cement. PMMA bone cement was tested for mechanical and biological properties before and after modification with mSIS. In addition, the mechanism by which mSIS affects osteogenesis was investigated by assessing the gene and protein expression quantities. Finally, the effect of adding mSIS powder to PMMA bone cement on in vivo bone integration was studied in osteoporotic rabbits.

Materials synthesis
Fresh porcine jejunum was mechanically dissociated, defatted, decontaminated, enzymatically digested and lyophilized to obtain dried SIS. Thereafter, the dried SIS was pulverized using a cryo-mill, followed by sifting the resulting powder through a 200-mesh test sieve to obtain powdered SIS. Next, an acidic SIS suspension was prepared by adding deionized water to the SIS powder, followed by the addition of phosphate (7558-80-7; Aladdin, Shanghai, China) and soluble calcium (10043-52-4; Aladdin) solutions at a Ca: P ion ratio of 5:3. Afterward, a sodium hydroxide solution (1310-73-2; Aladdin) was added slowly to neutralize the solution and obtain the mSIS precipitate. After centrifugation and sterile water washing, the mSIS precipitate was collected and lyophilized. The resulting mSIS powder was obtained by grinding through the aforementioned screen. The range of particle sizes in the mSIS powder was then determined by analyzing the particle size distribution using a laserbased method. mSIS was combined with PMMA powder, and liquid MMA was added at a solid to liquid ratio of 2.00:2.27 g/ml. The mSIS-PMMA bone cement was prepared after being fully mixed. Table 1 shows the Mendec Spine V R PMMA bone cement composition before and after modification.

Physical characterization tests
The four stages of the cement curing process (mixing, waiting, working and setting) were evaluated at each mixing weight ratio. This procedure was performed in triplicate to obtain an average value. Fourier-transform infrared (FTIR) analysis was performed on KBr disks of cured PMMA and mSIS-PMMA (mass ratio of mSIS: PMMA ¼ 15:85) bone cement. To confirm the attachment of nHA to the SIS, mSIS (10 mg) morphology was examined by transmission electron microscopy (TEM). Specimen cross sections were imaged using a scanning electron microscope (SEM). Rectangular areas of 400 lm 2 were randomly selected and analyzed for the Ca: P ion ratio, employing energy-dispersive X-ray spectroscopy (EDS).
PMMA and mSIS-PMMA bone cement samples, with varying mSIS: PMMA ratios and dimensions of 6 mm Â 6 mm Â 12 mm, were tested for strength under compression and their ability to withstand deformation using a mechanical tester with a displacement velocity of 20 mm/min. Three specimens of each mixing weight ratio were tested to obtain an average value to ensure measurement accuracy. In addition, three distinct areas of each sample were chosen at random and measured using a water contact angle measurement system to determine the average static contact angle. Combined with previous tests, the proportion suitable for subsequent tests is screened.
The PMMA and mSIS-PMMA specimens (with a mass ratio of mSIS: PMMA ¼ 15:85) were weighed and exposed to a degradation examination by submerging them in simulated body fluid (SBF; G0390, Solarbio, Beijing, China). The samples were extracted from the SBF after 14 and 28 days for subsequent drying and weighing to measure degradation. The degradation rate (DR) was calculated as follows: where m1 and m2 are the masses of the bone cement samples before and after the degradation test, respectively.

Biological characterization tests
To conduct cell culture and proliferation tests, the surfaces of the different bone cements (10 mm diameter, 1 mm thickness, n ¼ 5) were seeded with mouse BMSCs (MUBMX-01001; OriCell, Guangzhou, China) with a consistent initial cell seeding density of 6.5 Â 10 3 cells/cm 2 and allowed to incubate in Dulbecco's modified Eagle medium (MUXMX-90011; OriCell) for 7 days. Cell proliferation was assessed by adding a CCK-8 working solution (HY-K0301; MedChemExpress, Princeton, NJ, USA) to the culture medium on Days 1, 3, 5 and 7. Optical density measurement at 450 nm was performed using a microplate reader on the supernatant after a 2-hour dark incubation of the samples. Following 7 days of incubation in a proliferation medium, cell viability on the BMSC-loaded bone cement disks was evaluated using calcein/PI (C2015S; Beyotime, Beijing, China). Stained cells were imaged and counted under a laser microscope. To examine BMSC morphology, the specimens underwent sequential dehydration using increasing ethanol strengths from 30% to 100%. The specimens were soaked in tert-butanol (T119717; Aladdin) overnight at 4 C and freeze-dried for SEM observation.
BMSCs were subjected to 21 days of bone-forming differentiation by culturing them in a medium that promotes this type of differentiation. Quantitative assays for alkaline phosphatase (ALP) were performed by lysing differentiated cells with lysis buffer (P0013J; Beyotime). After adding the working and reaction termination solutions to the lysed differentiated cells, a microplate reader was used to detect the ALP enzymatic reaction signal at a wavelength of 405 nm. The differentiated BMSCs were immobilized using 4% paraformaldehyde and subsequently incubated with 1 ml of alizarin red staining solution (C0148S; Beyotime) for 5 min. Thereafter, the stained Ca on the bone cement was imaged using a scanner.
Reverse transcription polymerase chain reaction (RT-qPCR) was used to investigate the gene expression of differentiated BMSCs. Briefly, the cells were subjected to RNA extraction using a commercial kit (CW0584; CoWin Biosciences, Beijing, China). A FastQuant RT kit (AG11707; Accurate Biology, Changsha, China) was utilized to reverse transcribe RNA to cDNA. Subsequently, RT-qPCR was conducted using 5 ng of cDNA per reaction, with the forward and reverse primers specified in Supplementary Table S1, following the SYBR Green method.
In the rabbit osteoporosis model, adult female New Zealand white rabbits aged 5 months (Cixi Yanling Otter Farm, China; weight 3.02 þ 0.25 kg, n ¼ 10) were utilized following the approved protocol of the Animal Experimentation Committee of Ningbo University (11473). Dual-energy X-rays were used to evaluate the lumbar vertebrae bone mineral density of the rabbits 1 week before the surgery. After anesthesia, bilateral ovariectomies were performed on the rabbits. Intramuscular injections of dexamethasone sodium phosphate were administered at a dosage of 1.8 mg/day for eight consecutive weeks from the second week after osteoporosis surgery, and bone density was periodically determined during this period.
Osteoporotic female rabbits were divided into two groups for percutaneous vertebroplasty surgery: one group received pure PMMA bone cement, while the other group received mSIS-PMMA bone cement. The L3 spinous process was penetrated using a 14G bone biopsy needle to a depth of 2-3 mm, after which 0.2 ml of bone cement was injected.
Six weeks after percutaneous vertebroplasty surgery, the vertebral bodies injected with bone cement were excised. Subsequently, micro-CT scans were performed on all specimens. For data analysis, 3D CT reconstructions were performed for each specimen under the same conditions. A region of interest measuring 1.5 mm Â 1.5 mm, located at roughly the same position in each specimen, was chosen for assessing the 3D microarchitecture of the trabecular bone, encompassing the trabecular number (Tb.N) and volumetric ratio (BV/TV) of trabeculae. The effectiveness of the cement's regenerative capacity was evaluated via histological scoring (Supplementary Table S2) at the bone-implant interfaces. This allowed for the assessment of tissue response and bone ingrowth resulting from the cement [26].
To prepare for histological staining and imaging, the vertebral specimens of the rabbits were dehydrated, encased in MMA and sliced into 10-lm-thick sections. Four slices containing bone cement were randomly selected from each specimen and subjected to methylene blue and magenta and Masson staining, followed by observation using a slice scanner.

Statistical analyses
Values are expressed as the mean 6 standard deviation (SD) of a minimum of three procedural replicates. Statistical analysis was executed utilizing SPSS package 28.0.1.1 (SPSS Inc., Chicago, IL, USA). Normally distributed variables were evaluated using an independent-sample t-test or one-way analysis of variance (ANOVA). Non-normally distributed variables were evaluated using nonparametric methods combined with appropriate post hoc tests for the least significant difference. Statistical significance was inferred for differences at a threshold of P < 0.05.

mSIS-PMMA cement composition and properties
The mSIS powder had an average diameter of 163.1 mm, with particle size frequency analysis indicating that 10%, 50% (median diameter) and 90% of the particles had diameters of 14.76, 97.44 and 404.9 mm, respectively (Fig. 1A). The nanomorphology of the mSIS observed by TEM is shown in Fig. 1B. The mSIS fibers had an approximate diameter of 100 nm, and their surface comprised a randomly dispersed crystalline material. In terms of operability, mSIS-PMMA bone cement showed similar curing behavior to PMMA; its curing time gradually decreased with increasing mSIS fraction up to an mSIS/PMMA powder ratio of 15:85 (Fig. 1C). mSIS-PMMA bone cement (mSIS: PMMA mass ratio ¼ 15:85) FTIR spectra (Fig. 1D) confirmed organic (SIS) and inorganic (nHA) phases via characteristic peaks: HPO 2À 4 (876 cm À1 ), HPO 3À 4 (1100 cm À1 ), amide II (C-N stretching, 1640 cm À1 ) and amide A (N-H stretching, 3445 cm À1 ). The EDS elemental analysis results presented in Fig. 1E demonstrate a uniform dispersion of calcium (Ca) and phosphorus (P) throughout the mSIS-PMMA bone cement, with a ion ratio of Ca to P of 1.52. SEM images of the crosssectional morphology (Fig. 1F) showed that PMMA had a dense and smooth cross-section, while the mSIS-PMMA contained randomly distributed fibrous mSIS. Before reaching the yield point, the stress-strain curves of all materials exhibited comparable linear elastic behavior, as shown in Fig. 2A   The water contact angle was 113 6 3 for PMMA, typical for a hydrophobic polymeric material. The water contact angle of mSIS-PMMA bone cement decreased with increasing mSIS content to 85 6 2 and 92 6 3 for the 15:85 and 15:100 ratios, respectively (Fig. 2D). Due to its hydrophilicity and mechanical characteristics, 15:85 mSIS-PMMA bone cement was chosen for further biological analyses. Figure 2E depicts the changes observed in PMMA and mSIS-PMMA bone cements (mass ratio of mSIS: PMMA ¼ 15:85) after they were left to soak in SBF for 2 and 4 weeks. There was almost no observed mass change of PMMA in SBF. The mean DR of mSIS-PMMA on Days 14 and 28 were 3.13% and 4.45%, respectively.

Impact of mSIS-PMMA bone cement on BMSCs
The calcein AM/PI staining of BMSCs cultured for 7 days on PMMA or mSIS-PMMA bone cements (Fig. 3A) revealed that mSIS-PMMA had a higher number of viable cells (green) compared with that of PMMA. However, the number of dead cells (red) did not significantly differ, suggesting that mSIS-PMMA enhanced cell attachment and promoted cell viability. After 3 days of culture, CCK-8 tests showed significantly higher optical density values for cells on mSIS-PMMA compared with those on PMMA (Fig. 3B). This indicates that mSIS-PMMA promoted cell proliferation to a greater extent than PMMA. Immunofluorescence staining revealed that after 1 day of culture, BMSCs showed similar attachment on all samples, but mSIS-PMMA supported cells with more and longer cellular pseudopods than those on PMMA. The cell densities on the surface of mSIS-PMMA were considerably higher than those on PMMA at Days 5 and 7 of culture, as shown in Fig. 3C. SEM imaging of the BMSC morphology on Day 7 also showed that compared with PMMA, mSIS-PMMA had more cells on its surface; additionally, the cells were better-attached, with expanded cell morphology and extended pseudopods (Fig. 3D). This confirmed that mSIS-PMMA promoted BMSC attachment and proliferation better than PMMA. Quantitative and statistical analyses were performed on randomly selected cells on the bone cements using ImageJ software (NIH, Bethesda, MD, USA). BMSCs covered 1.3 times larger surface area on mSIS-PMMA than PMMA (Fig. 3E).

Osteogenic differentiation of BMSCs on the bone cements
Osteogenically differentiated BMSCs were stained to reveal their cytoskeletal structure. Compared with the control, staining revealed higher cell density on mSIS-PMMA bone cement, more pronounced local cluster growth, more obvious fusion, and more extensive pseudopod extension and spreading (Fig. 4A-C). ALP activity was utilized as an indicator to evaluate the osteogenic differentiation potential of mSIS-PMMA bone cement. mSIS-PMMA manifested a significant upsurge in ALP activity as compared to the control (Fig. 4D). Thus, mSIS enhances BMSC osteogenic differentiation and stimulates increased ALP expression. In addition, the cells were stained with alizarin red to assess matrix mineralization (Ca nodules formed at the later stages of osteoblastic differentiation). The differentiation of BMSCs into osteoblasts on mSIS-PMMA bone cement was facilitated, as indicated by the occurrence of more irregular areas that were stained dark red in comparison to the control (Fig. 4E). The impact of mSIS on osteogenic gene and protein expression was also investigated. RT-qPCR showed that BMSCs on mSIS-PMMA had significantly higher levels of ALP, OCN, BMP2, Runx2 and Col-1 genes than those on PMMA. ALP and Runx2 levels were nearly tripled, while OCN levels were upregulated almost 7-fold (Fig. 4F). Protein blot analysis revealed a significant increase in ALP and OCN protein expression in differentiated BMSCs cultured on mSIS-PMMA compared with the control (Fig. 4G), suggesting that mSIS promoted osteogenic differentiation of BMSCs at both gene and protein levels.

Effect of mSIS-PMMA cement on bone regrowth in animals
Mean bone mineral density in rabbits decreased by 23.03% after 8 weeks of dexamethasone sodium phosphate injection. Bone cements were injected into the L3 vertebrae of rabbits, and intraoperative observations were made (Fig. 5A). 3D-CT images showed mSIS-PMMA bone cement with irregular microporous structures due to mSIS degradation. In contrast, the exterior of the PMMA bone cement appeared smooth and intact (Fig. 5B), implying that the utilization of mSIS-PMMA enhances the area of interaction with the adjacent bone tissue upon partial degradation in vivo. Based on the morphometric evaluation, the mSIS-PMMA group exhibited a elevated percentage of peripheral bone volume (BV/TV ¼ 19.14%, P ¼ 0.001, n ¼ 5) and greater peripheral trabeculae thickness (Tb.N ¼ 0.73 mm À1 , P ¼ 0.001, n ¼ 5) compared with those of the control (BV/TV ¼ 13.07%, Tb.N ¼ 0.68 mm À1 , n ¼ 5).
The bone healing efficacy was evaluated by staining sections of the treated vertebrae using methylene blue and magenta (Fig. 5C). A negligible number of inflammatory cells was observed for both groups. The mSIS-PMMA group showed a significant enhancement in bone repair performance relative to the control group. Observation of the mSIS-PMMA group revealed new bone growth, characterized by an incomplete boundary with previously segmented bone tissues beginning to fuse and the emergence of a nascent bone structure. Nevertheless, the PMMA bone cement interface displayed a fine layer of fibrous tissue on its surface, and almost no new bone growth was observed. By employing Masson trichrome staining (Fig. 5D), it was observed that the mSIS-PMMA bone cement exhibited an uneven demarcation, characterized by a green hue resulting from the infiltration of a substantial quantity of cartilage collagen. Collagen within the bone and calcified cartilage were stained with distinct colors. In contrast, the control exhibited a continuous, regular boundary with no new bone inside. The study demonstrated that the degradation of mSIS-PMMA within the physiological milieu facilitated the infiltration of fresh host bone, thereby augmenting osseointegration. Moreover, histological assessment showed that the mSIS-PMMA group obtained substantially elevated scores in comparison to the PMMA group (Fig. 5E-H).

Discussion
PMMA bone cement is widely used for minimally invasive OVCF surgery, with over 40 brands on the market. However, all brands of PMMA bone cement used in OVCF treatment have a high elastic modulus, lack bioactivity and are nondegradable in vivo. Incorporating active substances into PMMA bone cement can mitigate these shortcomings to a certain extent. As the active material degrades, it generates a porous structure and osteogenic microenvironment that enhance cell attachment and proliferation. This, in turn, augments the bonding surface between bone and bone cement while concurrently decreasing the elastic modulus [27]. As the main component of the active material mSIS, SIS has suitable biocompatibility, degradability and mechanical properties for a bone cement additive and contains various growth factors. It can facilitate the growth of new bone tissue while reducing the modulus of elasticity. Moreover, nHA, a constituent of mSIS, exhibits osteoinductive properties and enhances the interaction between osteoblasts and the nearby microenvironment. This eventually leads to the promotion of fresh bone formation [28]. On this basis, mSIS was incorporated into the polymer powder of PMMA bone cement to produce a modified bone cement.
One important factor influencing clinical procedures is the required time for bone cement to harden. Curing times that are too long or too short are undesirable in clinical practice; excessively long curing times increase the risk of bone cement leakage, while excessively short ones reduce the handleability of bone cement. Although mSIS is insoluble in liquid MMA, it absorbs some liquid MMA during mixing, shortening curing time. However, the inclusion of a specific proportion of mSIS has an insignificant impact on the handling characteristics of the cement.
The higher elastic modulus of PMMA bone cement relative to cancellous bone results in stress shielding, which modifies stress transfer [29]. This can lead to bone tissue resorption [30], increased osteoclast activity and osteoblast apoptosis [31], which may increase the likelihood of fractures occurring in the surrounding vertebral body. Studies have shown that bone cements with low elastic moduli can effectively reduce stress levels in the neighboring bone [32]. Adding porogens to bone cement is a common method for reducing the elastic modulus. Aqueous sodium hyaluronate and hydroxypropylmethyl cellulose have been added to PMMA powder to produce porous bone cement. This technique can significantly decrease the elastic modulus of bone cement. For example, by incorporating 50 vol.% hydroxyapatite, the elastic modulus of Vertecem V R bone cement was diminished from 1840 to 50 MPa, while the addition of 50 vol.% hydroxypropylmethylcellulose decreased the elastic modulus of Beracryl V R bone cement from 2800 to 120 MPa [33,34]. In addition, Stryker V R PMMA bone cement was modified with castor oil (5, 10 or 15 vol.%) [35]. Increasing the volume fraction of castor oil resulted in a lower modulus of elasticity and lower polymerization temperature but a longer curing time.
In the treatment of OVCF, bone cement plays crucial roles, such as restoring vertebral height, stabilizing the vertebral body, and providing mechanical support to the fractured vertebra. An ideal bone cement must have sufficient compressive strength and an appropriate elastic modulus to provide adequate support and reduce stress shielding [36]. Although incorporating active materials may decrease the elastic modulus of PMMA bone cement, it could potentially result in inadequate compressive strength, which is a concern. If the compressive strength is too low, the bone cement cannot support the fractured vertebral body when stress is applied at the cement-bone interface. For instance, the addition of isotonic saline to PMMA bone cement (Vertecem Vþ Cement Kit; DePuy Synthes) induced a significant reduction in its modulus of elasticity, which decreased from 3409 6 312 to 1131 6 127 MPa. This was primarily attributed to an increase in microporosity. However, the maximum compressive strength of the cement decreased from 86 6 4 to 33 6 3 MPa [37]. Lewin et al. [38] replaced some of the MMA monomers with linoleic acid and observed that the elastic modulus of V-Steady V R PMMA bone cement decreased from 3360 6 277 to 462 6 78 MPa after 24 h of curing, while the compressive strength decreased to 12 6 1 MPa. Although these studies have made progress in reducing the modulus of elasticity of bone cements, the inherently low compressive strength of the resultant bone cements makes them unsuitable for orthopedic surgery. Compared with the bone cements from these studies, mSIS-PMMA exhibited a reduced modulus of elasticity with reasonably high compressive strength. In this study, the inclusion of mSIS into PMMA bone cement decreased the elastic modulus from 2572 6 4.8 to 263.7 6 3.7 MPa at a ratio of 15:85. This material exhibits an elastic modulus range that is similar to the trabecular architecture present in human bones. In addition, the mSIS-PMMA (mass ratio of mSIS: PMMA ¼ 15:85) bone cement still had a compressive strength of more than 70 MPa, sufficient to provide support to the affected vertebral body. The low elastic modulus of mSIS-PMMA relative to that of conventional PMMA bone cement, combined with adequate mechanical strength and handleability, can reduce postoperative complications resulting from stress shielding between the bone cement and cancellous bone.
As a biologically inert material, PMMA bone cement does not promote cell proliferation and adhesion. To improve the bioactivity of bone cement, active materials have been introduced to PMMA. For instance, incorporating mineralized collagen into Mendec Spine V R PMMA bone cement enhanced its biocompatibility, and the resulting modified bone cement stimulated the attachment, growth and bone-forming ability of osteoblasts [26]. Another study modified the same PMMA bone cement by replacing a portion of the bone cement powder with tricalcium silicate. In vitro cell culture tests showed that the bone cement's proproliferative effect tended to increase with higher tricalcium silicate content [39]. Biodegradable bone cement was obtained by incorporating Mg microspheres into Alfa Aesar V R PMMA bone cement. In vitro cellular experiments showed improved cell proliferation when exposed to the modified bone cement surface [40]. Although the above studies have achieved higher biological activity, their modification of biological activity and mechanical properties is unbalanced. In contrast, the mSIS-PMMA reported in the present study exhibits simultaneous improvements in biological activity and mechanical properties, representing a more promising bone cement for clinical applications.
The initial stage of the biological response to implanted biomaterials in vivo is cell attachment, which is greatly influenced by the physicochemical properties of the implant surface [41]. Our in vitro cellular experiments demonstrated improved cell growth with extended morphology and intercellular connections forming a mesh-like structure following exposure to mSIS-PMMA bone cement. A hydrophilic surface is more conducive to cell attachment and growth compared with a hydrophobic surface [42], implying that the hydrophilic surface of mSIS-PMMA bone cement may enhance cell attachment. Moreover, cells cultured on mSIS-PMMA bone cement exhibited stronger osteogenic differentiation, as indicated by increased osteogenic gene expression. These findings suggest that mSIS may enhance osteogenic activity, which is critical for new bone formation. This may be because of the ability of SIS to activate the BMP signaling pathway [22] and the ability of nHA, the main degradation product of mSIS in the physiological environment, to bind to proteins such as ALP and OCN in osteoblasts, thus promoting osteogenic cell differentiation [43]. The in vitro cellular experiments we conducted suggest that mSIS-PMMA bone cement possesses both osteoconductive and osteoinductive properties, which could potentially promote the integration of the bone cement into the affected spinal segment.
Conventional PMMA bone cement does not integrate with the bone tissue after implantation into a fractured vertebral body [44]. This is because PMMA cannot be degraded into small molecules in vivo, and the dense internal structure does not provide space for new bone ingrowth [45]. Instead, PMMA bone cement may be encapsulated by fibrous tissue. Furthermore, the low attachment strength between the bone cement and surrounding bone [46] can cause slight movement of the cement [47], resulting in the formation of small particles or even dislodgment of the cement [48]. In contrast, bioactive bone cement has been shown to have excellent osseointegrative properties [49]. Biologically active bone cements can react with human body fluids in the early stage of transplantation to generate nHA at the bone-tissue interface and form organic bonds. Subsequently, nHA induces osteoblast matrix mineralization to generate new bone. Synostosis between the new bone and bone cement improves interfacial integration [50]. The experimental results demonstrate that the mSIS in mSIS-PMMA bone cement gradually degrades in the physiological environment, forming a microporous structure capable of supporting cell ingrowth. Moreover, the excellent biocompatibility of mSIS-PMMA bone cement and the local ionic microenvironment with high P and Ca levels due to the degradation of mSIS can promote cell attachment and proliferation, recruit osteoblasts, and facilitate cell migration. These effects jointly promote the integration of bone tissue and bone cement within the vertebral bone, making it safer for clinical use.
Overall, the partially biodegradable mSIS-PMMA bone cement prepared here has good handleability and provides excellent support, while its elastic modulus closely resembles that of human cancellous bone in contrast to that of conventional bone cement. Additionally, the biocompatible, bioactive and biodegradable characteristics of mSIS-PMMA bone cement can promote the formation of fresh bone tissue, while simultaneously providing structural support to the affected vertebra. Incorporating mSIS into PMMA bone cement holds promising clinical implications for percutaneous kyphoplasty and percutaneous vertebroplasty procedures.

Supplementary data
Supplementary data are available at Regenerative Biomaterials online.