Demineralized bone matrix (DBM) is widely used as an alternative to autografts for repairing bone defects, but its clinical efficacy is limited by poor retention at the defect site and inadequate osteogenic capacity. Here, we developed a multifunctional carrier system aimed at improving the capacity of DBM by utilizing nanoclay-based self-assembly along with cell-derived exosome mimetics (EMs). EMs were derived from mesenchymal stem cells (MSCs) osteogenically induced in spheroids and were functionalized with bisphosphonates (BP) to assemble a stable gel network with laponite nanoclays via BP-nanoclay edge interactions. The resulting self-assembled nanoclay gel exhibited excellent injectability, moldability, and self-healing properties, even when loaded with a high concentration of DBM, and supported MSC osteogenic differentiation. We further enhanced the potency of DBM-mediated osteogenesis and bone morphogenetic protein (BMP) signaling in the nanoclay gel by incorporating BP-functionalized EMs loaded with miR-200c that can target BMP antagonist noggin. Lastly, we validated the bone regeneration efficacy of DBM-loaded nanoclay gels in comparison to commercially available DBM putties using a mouse calvarial defect model. This approach presents a versatile nanoclay gel carrier platform that overcomes the limitations of current DBM formulations and provides a promising strategy for improved bone repair.

Preparation and characterization of EM-BPs

MSC spheroids were generated via the spontaneous aggregation of MSCs in agarose microwells. The spheroids were dissociated using trypsin–EDTA and sequentially extruded through a polycarbonate membrane with pore sizes of 5 μm and 1 μm (Whatman) using a mini-extruder system (Avanti Polar Lipids). To ensure that the produced EMs have a uniform size and a narrow size distribution, the cell suspension was extruded through each filter for at least 15 to 20 cycles. The concentration of EMs was measured using a Pierce BCA protein assay kit (Thermo Fisher). The particle size, zeta potential, and PDI of EMs were measured using a dynamic light scattering (DLS) instrument (Malvern). To insert DSPE-PEG-BP into the EM membrane or load miRNA in the vesicles, the DSPE-PEG-BP or miRNA was added to the cell suspension prior to extrusion.

Preparation and characterization of LAP/EM-BP nanoclay gel

To prepare the LAP/EM-BP nanoclay gel, 100 μL 8% LAP was mixed with 300 μL EM-BP. The two components self-assembled to stable gel within a few minutes, and the gelation time varied with the different concentrations of EM-BP incorporated. To incorporate DBM into the nanoclay gels, the DBM particles were added to the LAP/EM-BP nanoclay gel and uniformly mixed using a spatula to form a composite gel. The compressive properties of LAP/EM-BP nanoclay gels with and without DBM were tested using a UniVert kN Mechanical Test System (CellScale) equipped with a 20 N load cell.

Bioactivity of LAP/EM-BP nanoclay gel

MSCs were encapsulated in the gels and cultured in growth medium (DMEM with 10% FBS and 1% P/S) for 1, 4, and 7 days. Cell viability at each time point was evaluated using a live/dead assay (Invitrogen). Cell proliferation was quantified using a 10% (v/v) AlamarBlue assay. To evaluate osteogenic differentiation, MSCs encapsulated in the gels were cultured in osteogenic differentiation medium. For qRT-PCR experiments, total RNA was extracted using the RNeasy Mini Kit (Qiagen) and RNA concentration was measured with a NanoDrop spectrophotometer. Complementary DNA (cDNA) synthesis was conducted using a SuperScript III First-Strand Synthesis Kit (Invitrogen), followed by gene amplification on a LightCycler 480 system (Roche). To evaluate ALP activity, cells in gels were fixed in 10% formalin and incubated with an ALP staining solution. To quantify ALP activity, cells were lysed and then centrifuged to remove debris. The resulting supernatant was incubated with ALP substrate solution and the absorbance was measured at 405 nm. Mineralization was evaluated using ARS staining. ARS-stained cells were dispersed in 10% (v/v) acetic acid, and absorbance was measured at 405 nm. The osteogenic effect of nanoclay gel loaded with DBM and noggin-targeting miRNA was further investigated by Western blotting. Cells loaded in gel were lysed to extract total protein. Lysates were separated via SDS-PAGE and transferred onto PVDF membranes. Membranes were incubated with primary antibodies specific to noggin, pSMAD1/5, or GAPDH (Santa Cruz Biotechnology, USA), followed by HRP-conjugated secondary antibodies. Protein bands were visualized using SuperSignal West Pico chemiluminescent substrate and imaged with a ChemiDoc™ Touch system (Bio-Rad, USA). Band intensities were quantified using AIWBwell software (Servicebio, China) to assess relative protein expression levels.

Bone regeneration capacity of nanoclay gels

The in vivo bone regenerative effect of nanoclay gels incorporating DBM and noggin-targeting miRNA was assessed using a calvarial defect model in mice. After eight weeks of surgery, all the animals were sacrificed to collect the calvarial samples. The calvarial samples were first fixed utilizing 4% formaldehyde for two days. The fixed samples were then scanned with a micro-CT instrument (SkyScan, Bruker). A 0.25 mm filter and 20 μm resolution were selected during the scanning. Three-dimensional reconstruction was conducted using NRecon, DataViewer, and Dragonfly. The relative area of new bone formation was calculated using the reconstructed images via ImageJ. The other microstructural parameters were obtained using CTAn software (Bruker).

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