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Abstract: Exosomes derived from mesenchymal stem cells are an active area of research due to their therapeutic potential in treating osteoporosis. To further harness their therapeutic performance in modulating bone resorption, equipped exosomes with osteoclast‐targeting moieties on their surface as well as chemokine receptor antagonists blocking osteoclast recruitment. Phosphatidylserine (PS), a membrane lipid exerting immunosuppressive and phagocytic signals, is incorporated in the membrane of exosome mimetics (EMs) to achieve a marked affinity for osteoclast precursors and potential anti‐resorptive effects. This is also aimed to tackle a CXCL9‐CXCR3 ligand‐receptor axis, a critical signaling axis in regulating osteoclast precursor recruitment and differentiation at bone resorption sites, by encapsulating a chemical antagonist of CXCR3, AMG487, in the PS‐incorporated EMs (PS‐EMs). The osteoclast‐targeting PS‐EMs loaded with AMG487 effectively protected against bone loss in an ovariectomized mouse model. These findings demonstrate the great promise of PS‐EMs as anti‐resorptive nanotherapies for alleviating osteoporosis.

The bone morphogenetic protein (BMP) signaling pathway plays a crucial role in bone development and regeneration. While BMP-2 is widely used as an alternative to autograft, its clinical application has raised concerns about adverse side effects and deteriorated bone quality. Therefore, there is a need to develop more sophisticated approaches to regulate BMP signaling and promote bone regeneration. Here, we present a novel complementary strategy that targets both BMP antagonist noggin and agonist Trb3 to enhance bone defect repair without the application of exogenous BMP-2. In vitro studies showed that overexpression of Trb3 with simultaneous noggin suppression significantly promotes osteogenic differentiation of mesenchymal stem cells. This was accompanied by increased BMP/Smad signaling. We also developed sterosome nanocarriers, a non-phospholipid liposomal system, to achieve non-viral mediated noggin suppression and Trb3 overexpression. The gene-loaded sterosomes were integrated onto an apatite-coated polymer scaffold for in vivo calvarial defect implantation, resulting in robust bone healing compared to BMP-2 treatments. Our work provides a promising alternative for high-quality bone formation by regulating expression of BMP agonists and antagonists.

Extracellular vesicles have received a great interest as safe biocarriers in biomedical engineering. There is a need to develop more efficient delivery strategies to improve localized therapeutic efficacy and minimize off-target adverse effects. Here, exosome mimetics (EMs) are reported for bone targeting involving the introduction of hydroxyapatite-binding moieties through bioorthogonal functionalization. Bone-binding ability of the engineered EMs is verified with hydroxyapatite-coated scaffolds and an ex vivo bone-binding assay. The EM-bound construct provided a biocompatible substrate for cell adhesion, proliferation, and osteogenic differentiation. Particularly, the incorporation of Smoothened agonist (SAG) into EMs greatly increased the osteogenic capacity through the activation of hedgehog signaling. Furthermore, the scaffold integrated with EM/SAG significantly improved in vivo reossification. Lastly, biodistribution studies confirmed the accumulation of systemically administered EMs in bone tissue. This facile engineering strategy could be a versatile tool to promote bone regeneration, offering a promising nanomedicine approach to the sophisticated treatment of bone diseases.

Aberrant lineage commitment of mesenchymal stem cells (MSCs) in marrow contributes to abnormal bone formation due to reduced osteogenic and increased adipogenic potency. While several major transcriptional factors associated with lineage differentiation have been found during the last few decades, the molecular switch for MSC fate determination and its role in skeletal regeneration remains largely unknown, limiting creation of effective therapeutic approaches. Tribbles homolog 3 (Trb3), a member of tribbles family pseudokinases, is known to exert diverse roles in cellular differentiation. Here, we investigated the reciprocal role of Trb3 in the regulation of osteogenic and adipogenic differentiation of MSCs in the context of bone formation, and examined the mechanisms by which Trb3 controls the adipo-osteogenic balance. Trb3 promoted osteoblastic commitment of MSCs at the expense of adipocyte differentiation. Mechanistically, Trb3 regulated cell-fate choice of MSCs through BMP/Smad and Wnt/β-catenin signals. Importantly, in vivo local delivery of Trb3 using a novel gelatin-conjugated caffeic acid-coated apatite/PLGA (GelCA-PLGA) scaffold stimulated robust bone regeneration and inhibited fat-filled cyst formation in rodent non-healing mandibular defect models. These findings demonstrate Trb3-based therapeutic strategies that favor osteoblastogenesis over adipogenesis for improved skeletal regeneration and future treatment of bone-loss disease. The distinctive approach implementing a scaffold-mediated local gene transfer may further broaden the translational use of targeting specific therapeutic gene related to lineage commitment for clinical bone treatment.

Although bone morphogenetic protein-2 (BMP2) has demonstrated extraordinary potential in bone formation, its clinical applications require supraphysiological milligram-level doses that increase postoperative inflammation and inappropriate adipogenesis, resulting in well-documented life-threatening cervical swelling and cyst-like bone formation. Recent promising alternative biomolecular strategies are toward promoting pro-osteogenic activity of BMP2 while simultaneously suppressing its adverse effects. Here, we demonstrated that small molecular phenamil synergized osteogenesis and bone formation with BMP2 in a rat critical size mandibular defect model. Moreover, we successfully elicited the BMP2 adverse outcomes (i.e. adipogenesis and inflammation) in the mandibular defect by applying high dose BMP2. Phenamil treatment significantly improves the quality of newly formed bone by inhibiting BMP2 induced fatty cyst-like structure and inflammatory soft-tissue swelling. The observed positive phenamil effects were associated with upregulation of tribbles homolog 3 (Trib3) that suppressed adipogenic differentiation and inflammatory responses by negatively regulating PPARγ and NF-κB transcriptional activities. Thus, use of BMP2 along with phenamil stimulation or Trib3 augmentation may be a promising strategy to improve clinical efficacy and safety of current BMP therapeutics.

The use of small molecular drugs with gene manipulation offers synergistic therapeutic efficacy by targeting multiple signaling pathways for combined treatment. Stimulation of mesenchymal stem cells (MSCs) with osteoinductive small molecule phenamil combined with suppression of noggin is a promising therapeutic strategy that increases bone morphogenetic protein (BMP) signaling and bone repair. Our cationic Sterosome formulated with stearylamine (SA) and cholesterol (Chol) is an attractive co-delivery system that not only forms stable complexes with small interfering RNA (siRNA) molecules but also solubilizes hydrophobic small molecules in a single vehicle, for directing stem cell differentiation. Herein, we demonstrate the ability of SA/Chol Sterosomes to simultaneously deliver hydrophobic small molecule phenamil and noggin-directed siRNA to enhance osteogenic differentiation of MSCs both in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. These results suggest a novel liposomal platform to simultaneously deliver therapeutic genes and small molecules for combined therapy.Statement of significanceApplication of phenamil, a small molecular bone morphogenetic protein (BMP) stimulator, combined with suppression of natural BMP antagonists such as noggin is a promising therapeutic strategy to enhance bone regeneration. Here, we present a novel strategy to co-deliver hydrophobic small molecule phenamil and noggin-targeted siRNA via cationic Sterosomes formed with stearylamine (SA) and high content of cholesterol (Chol) to enhance osteogenesis and bone repair. SA/Chol Sterosomes demonstrated high phenamil encapsulation efficiency, supported sustained release of encapsulated drugs, and significantly reduced drug dose requirements to induce osteogenic differentiation of mesenchymal stem cells (MSCs). Simultaneous deliver of phenamil and noggin siRNA in a single vehicle synergistically enhanced MSC osteogenesis and calvarial bone repair. This study suggests a new non-phospholipid liposomal formulation to simultaneously deliver small molecules and therapeutic genes for combined treatment.

Growth factor-based therapeutics using bone morphogenetic protein 2 (BMP-2) presents a promising strategy to reconstruct craniofacial bone defects such as mandible. However, clinical applications require supraphysiological BMP doses that often increase inappropriate adipogenesis, resulting in well-documented, cyst-like bone formation. Here we reported a novel complementary strategy to enhance osteogenesis and mandibular bone repair by using small-molecule phenamil that has been shown to be a strong activator of BMP signaling. Phenamil synergistically induced osteogenic differentiation of human bone marrow mesenchymal stem cells with BMP-2 while suppressing their adipogenic differentiation induced by BMP-2 in vitro. The observed pro-osteogenic and antiadipogenic activity of phenamil was mediated by expression of tribbles homolog 3 (Trb3) that enhanced BMP-smad signaling and inhibited expression of peroxisome proliferator-activated receptor gamma (PPARγ), a master regulator of adipogenesis. The synergistic effect of BMP-2+phenamil on bone regeneration was further confirmed in a critical-sized rat mandibular bone defect by implanting polymer scaffolds designed to slowly release the therapeutic molecules. These findings indicate a new complementary osteoinductive strategy to improve clinical efficacy and safety of current BMP-based therapeutics.

UnlabelledAlthough adipose-derived stem cells (ASCs) are an attractive cell source for bone tissue engineering, direct use of ASCs alone has had limited success in the treatment of large bone defects. Although bone morphogenetic proteins (BMPs) are believed to be the most potent osteoinductive factors to promote osteogenic differentiation of ASCs, their clinical applications require supraphysiological dosage, leading to high medical burden and adverse side effects. In the present study, we demonstrated an alternative approach that can effectively complement the BMP activity to maximize the osteogenesis of ASCs without exogenous application of BMPs by regulating levels of antagonists and agonists to BMP signaling. Treatment of ASCs with the amiloride derivative phenamil, a positive regulator of BMP signaling, combined with gene manipulation to suppress the BMP antagonist noggin, significantly enhanced osteogenic differentiation of ASCs through increased BMP-Smad signaling in vitro. Furthermore, the combination approach of noggin suppression and phenamil stimulation enhanced the BMP signaling and bone repair in a mouse calvarial defect model by adding noggin knockdown ASCs to apatite-coated poly(lactic-coglycolic acid) scaffolds loaded with phenamil. These results suggest novel complementary osteoinductive strategies that could maximize activity of the BMP pathway in ASC bone repair while reducing potential adverse effects of current BMP-based therapeutics.SignificanceAlthough stem cell-based tissue engineering strategy offers a promising alternative to repair damaged bone, direct use of stem cells alone is not adequate for challenging healing environments such as in large bone defects. This study demonstrates a novel strategy to maximize bone formation pathways in osteogenic differentiation of mesenchymal stem cells and functional bone formation by combining gene manipulation with a small molecule activator toward osteogenesis. The findings indicate promising stem cell-based therapy for treating bone defects that can effectively complement or replace current osteoinductive therapeutics.

Noggin is a specific antagonist of bone morphogenetic proteins (BMPs) that can prevent the interaction of BMPs with their receptors. RNA interfering molecules have been used to downregulate noggin expression and thereby stimulate BMP signaling and osteogenesis. Cationic liposomes are considered one of the most efficient non-viral systems for gene delivery. In the past decade, non-phospholipid liposomes (Sterosomes) formulated with single-chain amphiphiles and high content of sterols have been developed. In particular, Sterosomes composed of stearylamine (SA) and cholesterol (Chol) display distinct properties compared with traditional phospholipid liposomes, including increased positive surface charges and enhanced particle stability. Herein, we report SA/Chol Sterosome and small interfering RNA (siRNA) complexes that significantly enhanced cellular uptake and gene knockdown efficiencies in adipose derived mesenchymal stem cells with minimal cytotoxicity compared with commercially available lipofectamine 2000. Furthermore, we confirmed osteogenic efficacy of these Sterosomes loaded with noggin siRNA in in vitro two- and three-dimensional settings as well as in a mouse calvarial defect model. The delivery of siRNA via novel SA/Chol Sterosomes presents a powerful method for efficient gene knockdown. These distinct nanoparticles may present a promising alternative approach for gene delivery.