In this work, we combined tissue engineering and gene therapy technologies to develop a therapeutic platform for bone regeneration. We have developed photothermal fibrin-based hydrogels that incorporate degradable CuS nanoparticles (CuSNP) which transduce incident near-infrared (NIR) light into heat. A heat-activated and rapamycin-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in the photothermal hydrogels. In the presence of rapamycin, photoinduced mild hyperthermia induced the release of BMP-2 from the NIR responsive cell constructs. Transcriptome analysis of BMP-2 expressing cells showed a signature of induced genes related to stem cell proliferation and angiogenesis. We next generated 4 mm diameter calvarial defects in the left parietal bone of immunocompetent mice. The defects were filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the gene circuit. After one and eight days, rapamycin was administered intraperitoneally followed by irradiation with an NIR laser. Ten weeks after implantation, the animals were euthanized and samples from the bone defect zone were processed for histological analysis using Masson's trichrome staining and for immunohistochemistry analyses using specific CD31 and CD105 antibodies. Samples from mice that were only administered rapamycin or vehicle or that were only NIR-irradiated showed the persistence of fibrous tissue bridging the defect. In animals that were treated with rapamycin, NIR irradiation of implants resulted in the formation of new mineralized tissue with a high degree of vascularization, thus indicating the therapeutic potential of the approach.

Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033).

There is a growing interest in the development of tissue engineering (TE) therapies to repair damaged bone. Among the scaffolds for TE applications, injectable hydrogels have demonstrated great potential as three-dimensional cell cultures in bone TE, owing to their high water content, porous structure that allows cell transplantation and proliferation, similarity to the natural extracellular matrix and ability to match irregular defects. We investigated whether fibrin-based hydrogels capable of transducing near infrared (NIR) energy into heat can be employed to lead bone repair. Hollow gold nanoparticles with a plasmon surface band absorption at ∼750 nm, a NIR wavelength within the so called “tissue optical window”, were used as fillers in injectable fibrin-based hydrogels. These composites were loaded with genetically-modified cells harbouring a heat-activated and rapamycin-dependent gene circuit to regulate transgenic expression of the reporter gene firefly luciferase (fLuc). NIR-responsive cell constructs were injected to fill a 4 mm diameter critical-sized defect (CSD) in the parietal bone of mouse calvaria. NIR-irradiation in the presence of rapamycin triggered a pattern of fLuc activity that faithfully matched the illuminated area of the implanted hydrogel. Having shown that this platform can control the expression of a transgene product, we tested its effectiveness on regulating the secretion of transgenic bone morphogenetic protein 2 (BMP-2) from NIR-responsive hydrogels implanted in CSD. The spatiotemporal pattern of transgenic BMP-2 secretion induced by NIR-irradiation in the presence of rapamycin significantly stimulated bone regeneration from the edge of osteotomy in the CSD practiced, validating the therapeutic approach.

As near-infrared (NIR) photothermal agents, copper sulfide nanoparticles (CuSNP) offer several advantages over plasmonic gold nanoparticles (GNP), the most widely used photothermal nanotransducers in biomedical applications. CuSNP exhibit strong optical absorption at NIR wavelengths (650–1100 nm) and convert it into heat due excitation of electronic transitions or plasmonic photoexcitation. In contrast with GNP, CuSNP are degradable, readily prepared, inexpensive to produce, efficiently cleared from the body and their photothermal efficiency is less sensitive to the dielectric constant of the surrounding medium. We explored the feasibility of CuSNP to function as degradable NIR nanotransducers within fibrin-based cellular scaffolds, paying great attention to the stability and photothermal efficiency of the composite. We tested in vitro and in vivo whether NIR-responsive fibrin hydrogels comprising CuSNP (CuSNP hydrogels) are reliable platforms for triggering transgene expression in cells harboring a gene circuit activatable by heat and dependent of rapamycin. NIR laser irradiation of the CuSNP hydrogels increased local temperature and, in the presence of rapamycin, triggers the gene switch based on the promoter of the highly heat-inducible HSP70B gene (HSPA7). After implantation of such a cell-containing CuSNP hydrogel, transgenic expression can be remotely triggered by NIR-irradiation. Interestingly, we found that CuSNP hydrogels induce remodeling activity in stem cells and stimulate an angiogenic response. In short, CuSNP hydrogels offer compelling features for tissue engineering applications, as fully degradable implants with enhanced integration capacity in host tissues that can provide for remote control in the deployment of therapeutic gene products.

Summary Statement

Parathytorid hormone-related protein (107–111) loaded onto biopolymer-coated nanocrystalline hydroxyapatite (HA_Glu) improves the bone repair in a cavitary defect in rat tibiae.