Combining Piezoelectric Stimulation and Extracellular Vesicles for Cartilage Regeneration

Numerous patients experience articular cartilage defects (ACDs), which are characterized by progressive cartilage degradation and often lead to osteoarthritis (OA). Consequently, 44.7% of OA patients sufer from dyskinesia or disability. Current clinical drug treatments ofer limited efectiveness in fully curing the disease. In this study, we propose a collaborative approach that combines physical and biological cues to promote cartilage regeneration. A biodegradable piezoelectric poly (l-lactic acid) (PLLA) nanofber scafold facilitates in situ, battery-free electrical stimulation under natural joint loading, while extracellular vesicles (EVs) serve as communication mediators between cells and promote cell proliferation, migration, and secretion of type II collagen. In this combined approach, EVs attached to PLLA are gradually released by localized piezoelectric electrical stimulation and taken up by chondrocytes. Tis process results in the organization of type II collagen along the PLLA fber surface, ultimately forming cartilage lacunae that facilitate the residence of new chondrocytes. As an outcome, a signifcant round cartilage defect (diameter: 3mm and depth:1mm) in the PLLA/EVs group (rat and knee) was rapidly restored within six weeks. In contrast, individual EVs and PLLA groups demonstrated considerably weaker cartilage regeneration capabilities. Tis research suggests that the synergistic efect of electrome-chanical stimulation and EVs-based biological cues is a crucial intervention method for treating OA.


Introduction
Osteoarthritis (OA) is the leading cause of joint pain symptoms and poses a risk of disability for patients [1][2][3]. Current treatment methods, such as drug therapy, tissue grafts, and synthetic functional scafolds, demonstrate limited efcacy in cartilage regeneration [4,5]. Drug therapy can only alleviate infammation and pain to a certain degree without curing the disease, while tissue grafts are constrained by donor availability and prone to complications such as immune reactions, infection, and donor site morbidity. Synthetic scafolds, or artifcial grafts, have garnered signifcant interest due to their unrestricted supply and customizability for specifc purposes, but they tend to degrade under repeated joint forces. Consequently, there is a need for alternative solutions that promote chondrogenesis and cartilage regeneration. Acknowledging this limitation, we turn to nature for inspiration. Cartilage is known to be sensitive to bioelectric electrical stimulation (ES) [6]. Numerous studies have demonstrated the efectiveness of exogenous ES in promoting cartilage regeneration [7,8]. However, most of these treatments necessitate external energy input and wired connections, which can lead to risks such as infection, reduced efcacy, and device failure. Recent advances in nanotechnology and smart materials enable in situ ES treatments with a high spatial resolution [9,10]. Electromechanical coupling piezoelectric materials have been employed in tissue regeneration engineering but have not been widely accepted for electrically stimulating cartilage cells. Tis is because the implanted piezoelectric scafold can only generate sufcient stimulating potential under substantial deformation, whereas joint mobility in OA patients is typically limited. Extracellular vesicles (EVs) function as crucial biological cues that can facilitate chondrogenesis [11][12][13]. Importantly, based on the principle of electroporation [14], EV cargos can be efectively transferred to receptor cells under the continuous local action of ES, as it increases membrane permeability. In this study, we present a collaborative strategy for stimulating hyaline cartilage regeneration using a cell-free piezoelectric poly (l-lactic acid) (PLLA) nanofber scafold combined with EVs. Although research on EVs-seeded electrospun scafolds for cartilage repair is limited, there has been a successful case of skin tissue repair using electrospun polycaprolactone-EVs scaffolds [15]. In our prior research, the long noncoding RNA DANCR was found to play a crucial role in the chondrogenesis of synovium-derived mesenchymal stem cells (SMSCs) [16]. Studies have suggested that the therapeutic potential of MSCs is derived from their ability to secrete EVs [17,18]. To investigate this, we established a DANCRoverexpressed SMSC cell line (DANCR + SMSCs) and extracted its EVs for examination. Trough six weeks of animal clinical trials, the self-powered biodegradable scaffold with DANCR + SMSC-EVs (DS-EVs) displayed high efciency in promoting rapid cartilage regeneration, eliminating the need for invasive removal procedures typically required for conventional piezoelectric scafolds such as poly (vinylidene difuoride).

Isolation and Identifcation of DANCR + SMSC-EVs.
Once DANCR + SMSCs reached 90% confuence (10 cm cell culture dishes), the culture medium was replaced with serum-free medium and incubated continuously. Te cell supernatant was collected three days later. DS-EVs were isolated by diferential centrifugation (Support Information S1) and identifed by Western blot for DS-EVs surface markers (Syntenin-1 and CD81); the size and concentration of DS-EVs were determined by Nanoparticle Tracking Analysis (NTA); and the DS-EVs morphology was examined under a transmission electron microscope (TEM). All identifcation experiments on DS-EVs, including Western blot, NTA, and TEM, were performed in triplicate.

Nanoparticle Tracking
Analysis. Te number and size distribution of DS-EVs were analyzed by nanoparticle tracking using Electrophoresis and Brownian Motion Laser Scattering Microscopy (ZetaView, Germany). Briefy, samples in solution or each particle sample were diluted in PBS, and their relative concentrations were determined according to the dilution factor. Data analysis was conducted with NTA 2.2 software. For each sample, a set of at least four individual movies was acquired, and the count was averaged.

Transmission Electron
Microscopy. DS-EVs were resuspended in 2.5% glutaraldehyde in 0.1 M sodium cacodylate bufer and fxed on formvar carbon-coated grids at room temperature for 30 min, followed by negative staining with 2% uranyl acetate for 1 min. Images were captured using a transmission electron microscope (JEM-2100, JEOL).

Cell Migration and Proliferation Studies.
Te isolated and identifed DS-EVs were resuspended in 50-100 µL PBS, and their concentrations were determined using an Enhanced BCA Protein Assay Kit (P0010, Beyotime, China).
A chondrocyte scratch test was conducted to assess the impact of DS-EVs on isolated chondrocyte migration. Briefy, approximately 1.2 × 10 5 cells (per well) were initially transferred to a 6-well plate and incubated for 24 h until the cells reached nearly 95% confuence. Te prepared DS-EVs/ PBS solution, diluted separately to 0 µg/mL, 20 µg/mL, 40 µg/ mL, and 60 µg/mL concentrations, was added to the six-well plates and continuously observed for 0 h, 6 h, 12 h, and 24 h under a light microscope. Images were recorded with a digital microscope to examine the migration capabilities of chondrocytes.
To assess chondrocyte proliferation, an MTT assay was performed to study the efect of DS-EVs on chondrocyte proliferation. Isolated rat chondrocytes (0.5 × 10 4 cells/well) were transferred to 96-well plates and treated with sodium nitroprusside, followed by a 60 µg/(mL * day) treatment of DS-EVs for 24, 72, and 120 h, after which proliferation was evaluated. MTT reagent (100 µL, 10%) was added to each well for 4 h, and the wells were analyzed using a microplate reader, with optical activity observed at 570 nm.
2.6. Western Blot Assays. Isolated DS-EVs pellets and chondrocytes co-cultured with DS-EVs for varying durations were extracted with RIPA bufer (Santa Cruz) containing a protease inhibitor cocktail (Med ChemExpress) and incubated on ice for 30 min. Protein content of DS-EVs and chondrocyte lysates was quantifed using a BCA kit (Termo Fisher Scientifc), loaded onto SDS-PAGE (10% gels), and transferred to PVDF membranes (Millipore). Te membranes were blocked for 1 h in 3% BSA and incubated with corresponding antibodies, including Syntenin-1 and CD81, overnight at 4°C. Secondary antibodies were IRDye 680RD antirabbit immunoglobulin (Ig)G (H + L) and IRDye 800CW antimouse IgG (H + L) (Invitrogen). Detection was performed with an Odyssey Imaging System (LI-COR, Biosciences).

Quantitative
Real-Time PCR. Cells and ground animal tissue were washed with PBS twice before RNA purifcation. RNA was purifed using TRIZOL following the manufacturer's protocol. Te concentration and purity were determined using a NanoDrop 2000 spectrophotometer (Termo Fisher). cDNA was synthesized using an RT2 First Strand Kit (Qiagen). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted using GoTaq qPCR Master Mix (Promega) on an ABI 7900 (Applied Bioscience). Relative gene expression was calculated using the 2 −ΔΔ CT method, with glyceraldehyde-3phosphate dehydrogenase (GAPDH) serving as an internal control (see Table 1).

Fabrication and Characterizations of PLLA/DS-EVs
Scafold. PLLA nanofbers (NFs) were fabricated through electrospinning using a #21 nozzle. For additional information on the electrospinning process, please refer to Supporting Information S2. Subsequently, the asreceived PLLA NFs mat was immersed in the DS-EVs suspension. Te morphology and structural distribution were examined using SEM (Supra 55, Carl Zeiss) and TEM (JEM-2100, JEOL). Piezoelectric response measurements of individual NFs were carried out using PFM (Multimode 8, Bruker) in contact mode. For further information on PFM characterizations, consult Supporting Information S3.

Detection of the DS-EVs Retention Rate of PLLA/DS-EVs.
Straightforward modifcations to the electrospinning conditions can produce PLLA fbers with minimal piezoelectric efects. In this study, 4000-rpm PLLA nanofber was used to fabricate piezoelectric PLLA, while 300-rpm PLLA served as a nonpiezoelectric control.
Both types of prepared PLLA/DS-EVs (thickness: 1 mm; volume: 7.07 mm 3 ; loaded with 300 µg DS-EVs) were placed in a sterile cell culture dish and soaked with 1 mL sterile PBS. Ten, 1 mL of PBS was collected every 24 h. Te material's surface was repeatedly washed and squeezed 10 times in 1 mL of freshly added PBS, and the sterile PBS was continuously collected for 10 days. An NTA particle size analyzer was employed to measure the DS-EVs concentration in the sterile PBS collected daily and to calculate the adsorption rate, adsorption amount per unit volume, and retention rate of PLLA on DS-EVs.
Preprepared DS-EVs stained with DiR dye were used to assess the in vivo EVs retention rate. Two randomly selected SD rats were used, with one receiving a DS-EVs DiR injection and the other receiving a PLLA/DS-EVs DiR implant. Te fuorescence of DS-EVs was analyzed for two weeks using an LB983 in vivo imaging system (Berthold Technologies).

Animal Models.
Fifty-seven adult male SPF-grade SD rats, weighing between 280 and 310 g, were utilized in this study. Tree rats were employed for primary chondrocyte extraction and other in vivo experiments. Te remaining rats were randomly assigned to the MOCK group (blank control), Sham group (Sham surgery), ACD group (defect diameter: 3 mm; depth: 1 mm), PLLA implanted group, DS-EVs injection group, and PLLA/DS-EVs implanted group. For further information on the animal models, please refer to Support Informations S4 and S5.
Te rats were provided by Shanghai SIPPR-BK Laboratory Animal Co. Ltd., and the quality of the experimental rats was verifed by the Shanghai Laboratory Animal Quality Supervision and Inspection Station. Te use and breeding of experimental animals were approved by the Department of Comparative Medicine, Jinling Hospital, Nanjing University School of Medicine (Approval No. 2020JLHKJLWDWLS-001).

Pathological Staining and Immunohistochemistry.
Femur histological sections were decalcifed with EDTA, fxed with paraformaldehyde, and embedded in parafn. Rat femur sections were then cut into 4 µm slices. Hematoxylin-Eosin (H&E) staining, toluidine blue staining, and safranin O staining were performed on bone sections following the manufacturer's instructions. In addition, COL2 antibody (Proteintech 1 : 200) and MMP13 antibody (Abcam 1 : 200) were diluted according to the recommended ratios, and immunohistochemistry was conducted on femoral sections.

Biomechanics Analysis.
Te experiment was divided into three groups: Sham (n � 3), control (n � 3), and PDS-EVs treatment (n � 3). Rat knee joint specimens were prepared as fat surfaces using a wire saw. An MML Nano Indenter was used to perform biomechanics analysis for each group. Te maximum loading force was set at 20 mN, with the loading position situated at the deep layer of cartilage and a loading time of 15 s. A cylindrical indenter from Hysitron, USA, with a bottom diameter of 10 µm, was selected for this experiment.
2.14. Statistical Analysis. Te software GraphPad 8.0.1 was utilized for graphical illustration and statistical analysis. Te outcomes are presented as means ± standard deviation (SD) derived from three autonomous experiments unless indicated otherwise. Te divergence within treatment groups was assessed through the Student's t-test or one-way analysis of variance (ANOVA) along with Bonferroni's multiple comparisons test.

Construction of Bionic Scafold and Its Mechanism in
Cartilage Regeneration. Te physiological structure of the distal femur knee joint can be divided into the cartilage layer, calcifed layer, and subchondral bone layer. Te cartilage layer is characterized by collagen fbers that form grid-like cartilage lacunae. Within these lacunae, articular chondrocytes can reside and secrete glycosaminoglycans and other essential components of the cartilage matrix [19,20]. Research has demonstrated that periodic mechanical stimulation can activate the calcium ion channel Cav1.2 in chondrocytes and enhance the expression of type II collagen [21,22]. Te PLLA scafold utilized in this study exhibited positive piezoelectricity and could convert mechanical energy into bioelectrical energy (Support Information, S2B-S2D) [23,24]. Te mammalian knee cartilage experiences body weight and mechanical compression during static or dynamic processes. Terefore, the PLLA scafold is a suitable option for cartilage tissue engineering (Figures 1(a) and 1(b)).
Extracellular vesicles contain genetic information and perform most of the functions of the originating cell. Tese vesicles primarily facilitate cell communication through paracrine, proximal secretion, and endocrine mechanisms. Tis phenomenon is frequently observed in MSCs [17,18,[25][26][27][28][29]. Our previous research demonstrated that the lncRNA DANCR can regulate chondrogenic diferentiation and proliferation [16]. Furthermore, it was found that DANCR may have a role in inducing chondrogenesis [30]. In addition, in this study, we made an initial discovery that DANCR may have the capacity to upregulate the synthesis of COL2A1. Subsequently, we generated DANCR + SMSCs and isolated their extracellular vesicles for further investigation. We hypothesized that DS-EVs could elicit comparable or analogous biological efects to those of DANCR + SMSCs.
In this study, we proposed a therapeutic approach for repairing ACD that involved combining PLLA with DS-EVs to form EV-seeded electrospun scafolds. Te PLLA NFs could serve as a bridge to promote the migration of normal chondrocytes toward the center of cartilage defects. Furthermore, during the process of cell migration, the piezoelectric stimulation produced by daily knee joint activities may promote chondrocyte uptake of DS-EVs, which are slowly released from PLLA/DS-EVs [31]. Ultimately, this uptake of DS-EVs may enhance the ability of chondrocytes to synthesize type II collagen, thereby promoting cartilage repair (Figure 1(c)) (speculation). Notably, PLLA fber scafolds possess a grid-like structure similar to collagen II, making it a suitable biomimetic material. Moreover, PLLA does not produce harmful substances during the degradation process and exhibits excellent tissue compatibility [32,33].

Extraction of DS-EVs and Validation of Its Efect on
Cartilage Regeneration. Traditional tissue engineering technology involves seed cells, scafolds, cytokines, and the microenvironment. However, maintaining the viability of seed cells in vitro poses a signifcant challenge, hindering its clinical applications. As a result, research on decellularized scafolds has garnered signifcant attention among scientists [34,35].
Tis study aimed to investigate the biological function of DS-EVs. Our transmission electron microscopy results demonstrated that the isolated DS-EVs had a classical phospholipid layer spherical structure with a diameter of approximately 120-130 nm (Figure 2(a); Supporting Information, S6). NTA analysis further confrmed that the diameter of DS-EVs was approximately 130.6 nm (Figure 2(b); Supporting Information, S6). Western blot analysis revealed that the isolated DS-EVs expressed CD81 and Syntenin-1 (Figure 2(c)). To verify the successful construction of SMSCs overexpressing DANCR, we performed qPCR (Figure 2(d)). Subsequently, we labeled the isolated DS-EVs with Dil dye and co-cultured them with articular chondrocytes. Under a laser confocal microscope, we observed that the DS-EVs could penetrate the articular chondrocytes and aggregate in the cellular matrix after 6 h (Figure 2(e)). Te outcomes of the cell scratch assay and MTT assay indicated that DS-EVs (60 µg/mL) could enhance the migration and proliferation of chondrocytes (Figures 2(f ) and 2(g)). Furthermore, continuous treatment of articular chondrocytes with 60 µg/mL DS-EVs for fve days resulted in increased secretion of collagen type II by the cells (Figures 3(h) and 3(i)). Tese experimental fndings not only suggest that DS-EVs have the potential to repair cartilage but also provide a theoretical basis for utilizing extracellular vesicles tissue engineering technology to address the innate shortcomings of traditional tissue engineering techniques.

Synergistic Efects between Piezoelectric Stimulation and EVs Together Enhances the Regeneration of Cartilage.
Electrospinning was used to prepare the PLLA NFs scafolds (Figure 4(a)), and subsequently, PLLA/DS-EVs were prepared using a specifc process (Supporting Information, S2). Te piezoelectric activity of individual PLLA NFs was investigated by piezoresponse force microscopy (PFM), a well-established method for analyzing the local piezoelectric properties of nanostructures [36] (Figures 4(b)-4(f)). Te PFM amplitude (Figure 4(b)) and PFM phase (Figure 4(c)) showed a classical butterfy-shaped curve, indicating the hysteresis loops of the PLLA membrane and confrming the good piezoelectric properties of electrospun PLLA NFs, which is a typical feature of piezoelectric materials [37]. Confocal laser scanning microscope images showed the distribution of DS-EVs on the surface of PLLA NFs through electrostatic adsorption (Figure 4(g)). Furthermore, the physiological voltage generated by rats during the knee joint (Figure 4(h)). Te movement was measured and found to be within the range of voltage changes     Journal of Tissue Engineering and Regenerative Medicine produced by PLLA NFs (Supporting Information, S2). To assess the in vivo retention rate of PLLA/DS-EVs, we implanted them into rat knee joints and tracked DiR-fuorescently labeled DS-EVs using small animal imaging technology. Te implanted group showed detectable fuorescent signals on day 14, whereas the injected group's signal disappeared by day 3 (Figure 4(i)). Tis fnding indicated that DS-EVs adsorbed on PLLA NFs were slowly released in the in vivo environment compared to direct injection. Next, we analyzed the DS-EVs released from nonpiezoelectric and piezoelectric PLLA collected in vitro for 10 consecutive days using an NTA particle size analyzer to obtain the in vitro retention rate of DS-EVs. Te piezoelectric PLLA had a better retention rate of DS-EVs than nonpiezoelectric PLLA, with 40% of total DS-EVs still retained after 7 days (Figure 4(j)). According to the grading method of the International Cartilage Repair Society (ICRS), injuries with a diameter greater than 3 mm are categorized as articular cartilage defects (ACD). Most of these injuries cannot be fully repaired and are instead flled with fbrocartilage. When the diameter of the ACD exceeds 6 mm, it not only becomes irreparable but also results in further damage to the surrounding bone wall and articular cartilage. Tis creates a larger defect cavity, which in turn causes the surrounding cartilage to shift and the articular cartilage to collapse. Ultimately, this leads to knee osteoarthritis [38]. By preventing the onset of osteoarthritis during the ACD stage, the incidence of OA can be greatly reduced. Terefore, we chose ACD models (diameter: 3 mm, depth: 1 mm) for this study.
Te results of HE staining, toluidine blue staining, and safranin O fast green staining indicated that the PLLA/DS-EVs group exhibited a substantial treatment efect. Te HE results revealed that the PLLA scafolds were not completely degraded in the PLLA/DS-EVs group, which aligns with the reported degradation rate of PLLA [39]. In the PLLA/DS-EVs group, a distinct tidal line was formed, and there were more newly formed chondrocytes residing in the cartilage lacunae. Te outcomes of immunohistochemistry demonstrated a higher amount of newly formed COL2A1 in the PLLA/DS-EVs group, while the expression of MMP13 was signifcantly upregulated in the PLLA group, forming macrophages. Te results of immunohistochemistry were consistent with the qPCR outcomes. In addition, based on the CT scanning and 3D reconstruction results, the PLLA/DS-EVs group exhibited a signifcant repair efect (Figure 3(a); Supporting Information, S7). To understand the changes in the cartilage microenvironment during the repair of articular cartilage, we analyzed the regenerated cartilage tissue around the defects using qPCR and discovered a substantial change in the COL2A1 and MMP13 indexes (Figures 3(b) and 3(c)).
We conducted nanoindentation experiments on various rat knee joint specimens, and the application of PLLA/ DS-EVs resulted in favorable biomechanical properties. Specifcally, among the groups, only the ACD, Sham, and PLLA/DS-EVs groups had relatively complete cartilage layers after six weeks of ACD model construction, so only these groups were examined (Figure 3(d)). Our results indicated that the PLLA/DS-EVs group exhibited signifcantly better elastic modulus and hardness compared to the ACD group (Figures 3(e) and 3(f )).
However, we observed that the treatment efect of PLLA alone was not as efective as anticipated. We further validated the expression levels of MMP13 and COL2A1 via qRT-PCR and immunohistochemistry, which demonstrated that the degradation rate of collagen II was higher than its synthesis rate during the repair of ACD using PLLA scafolds alone. Tis underscores the importance of the biological function of DANCR + SMSC-EVs in the repair process. It is noteworthy that we did not delve into the specifc mechanism of DS-EVs in this study. Based on our prior studies and some of our experimental results, we suggest that DS-EVs carry specifc LncRNA from SMSCs, which can regulate cell proliferation and stimulate collagen II production. However, this conclusion requires further experimental evidence in the future.

Conclusion
Overall, our fndings suggest that the combination of PLLA/ DS-EVs could enhance the migration and proliferation of normal chondrocytes towards the injury site in the ACD and modulate the expression of COL2A1 and MMP13. Moreover, the impact of piezoelectric stimulation facilitated by the PLLA/DS-EVs scafold could potentially improve the efciency of cargo delivery from DS-EVs to regenerated chondrocytes. Eventually, regenerated chondrocytes could secrete type II collagen, which aligns along the PLLA surface, generating cartilage lacunae that facilitate chondrocyte adhesion.

Data Availability
Te data that support the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
Tis work has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Tis study was approved by the Department of Comparative Medicine, Jinling Hospital, Nanjing University, School of Medicine (Approval No. 2020JLHKJLWDWLS-001).

Conflicts of Interest
Te authors declare that there are no conficts of interest.