The field of orthopedic medicine grapples with a significant clinical gap in the treatment of both chronic and acute bone disorders. Conventional therapeutic approaches, while effective for symptom management, often fail to address the underlying genetic or cellular dysfunctions that drive these pathologies (Shoback, 2007; Li et al., 2025). For example, chronic conditions and osteoporosis are managed with drugs such as bisphosphonates, which can partially ease symptoms or slow bone resorption but do not correct the fundamental genetic defect responsible for the disease (Chindamo et al., 2020; Lima et al., 2019). Similarly, the healing of large segmental bone defects and non-union fractures presents a formidable challenge, where current surgical techniques and bone grafts may be inadequate, costly, and prone to complications (Frączek et al., 2024). These limitations highlight the urgent need for innovative therapeutic approaches that can provide more permanent therapeutic solutions by targeting the underlying cause of the disorder. Gene therapy has emerged as a promising strategy to meet this need, offering the potential for long-term therapeutic effects from a single or limited number of administrations. The efficacy and safety of gene therapy hinge critically on the choice of delivery vehicle, commonly referred to as a vector. These can be broadly categorized into viral and non-viral systems (Fig. 1), each possessing unique molecular characteristics and clinical implications (Douglas, 2008). The concept of targeted drug delivery to bone, using compounds with an inherent affinity for bone, such as bisphosphonates (Brown et al., 2014), tetracyclines (Saikali and Singh, 2003), and calcium phosphates (Uskoković and Uskoković, 2011), emerged decades ago. Although this approach was fundamental, it lacked the precision and versatility offered by more advanced drug delivery platforms. The identification and subsequent development of these advanced vector systems have started a new era of targeted therapies (Chindamo et al., 2020; Dogbey et al., 2023).

Viral vectors have had a significant historical impact in this field (Franceschi, 2005), having evolved over millennia to skillfully bypass cellular defenses and efficiently deliver their genetic cargo into host cells. This inherent, natural ability to invade and deliver a modified genetic payload for therapeutic purposes makes them incredibly potent tools, explaining their widespread adoption in research and clinical trials (Ulrich-Vinther, 2007). Their sophisticated cellular entry mechanisms and often high transduction efficiencies enable robust and sustained gene expression, which is crucial for many therapeutic applications, particularly when long-term cellular changes are desired (Lundstrom and Boulikas, 2003; Lundstrom, 2018).

In contrast, non-viral vectors, which are essentially naked strands of RNA or DNA, face a much more difficult battle. They lack the intrinsic cellular trickery that viruses possess, making it considerably more challenging for them to deliver their genetic material into specific cell types effectively (Wegman et al., 2013). Simply introducing isolated nucleic acids into a biological system often results in rapid degradation by enzymes, poor cellular uptake, and inefficient transport to the nucleus (Ramamoorth and Narvekar, 2015; Zu and Gao, 2021).

To overcome these significant hurdles, researchers have had to get creative, developing sophisticated strategies to enhance the delivery of these non-viral payloads. One common approach involves complexing the RNA or DNA with specialized delivery vehicles. These vehicles, such as cationic lipids (which form positively charged structures that can bind to negatively charged DNA/RNA and facilitate entry into cells) or cationic polymers (synthetic molecules that condense nucleic acids into nanoparticles), shield the genetic material from degradation and improve its ability to traverse the cell membrane (de Ilarduya et al., 2010) (Fig. 2). These complexes essentially act as mini-delivery systems, mimicking some of the protective and entry-facilitating aspects of viral capsids (Zu and Gao, 2021).

Beyond chemical complexing, physical methods are also employed to force these nucleic acids into cells. Techniques like electroporation utilize controlled electrical pulses to temporarily create microscopic pores in the cell membrane, allowing the genetic material to slip inside (Wu et al., 2012). Similarly, hydrodynamic injection involves rapidly injecting a large volume of liquid containing the genetic material, creating a transient pressure gradient that enhances cellular uptake, particularly in organs like the liver. Other physical methods under development include sonoporation (using ultrasound) and gene guns (which shoot DNA-coated particles into cells) (Ramamoorth and Narvekar, 2015; Balmayor et al., 2015).

While non-viral methods often lag behind their viral counterparts in terms of raw transfection efficiency, their advantages in terms of safety (lower immunogenicity, no risk of viral replication) and manufacturing scalability continue to drive intense research. The ongoing advancements in both viral engineering and the design of intelligent non-viral delivery systems are rapidly expanding the toolkit available for gene therapy, promising a future where gene-based medicines are more accessible and effective for a wider range of diseases (Hardee et al., 2017).

Viral vectors, derived from naturally occurring viruses, have been extensively engineered to deliver genetic cargo with high efficiency and sustained expression. Their inherent ability to infect cells and introduce their genetic material makes them potent tools for gene transfer. Among viruses, Adeno-Associated Viruses (AAVs), Lentiviruses, and Adenoviruses (Ads) are the most frequently used for gene therapy due to their specific characteristics (Dogbey et al., 2023).

AAV vectors have emerged as a cornerstone in the rapidly advancing field of gene therapy, offering a powerful and often preferred means of delivering therapeutic genetic material to target cells (Naso et al., 2017; Li and Samulski, 2020).

AAV is a small, non-enveloped parvovirus, 26 nm in diameter, with a single-stranded genome of approximately 4.7 kb in length. The AAV genome encodes regulatory (Rep) and structural capsid (Cap) proteins and is flanked by two inverted terminal repeats. Replacement of the Rep and Cap genes with a transgene of interest produces a replication-defective recombinant AAV (rAAV) genome that can target tissues as a potent vector, introducing new genetic material into target tissues, acting as a powerful vector in gene therapy applications (Samulski and Muzyczka, 2014; Agbandje-McKenna and Kleinschmidt, 2011).

Their prominence is not arbitrary but rooted in a unique combination of characteristics that make them exceptionally well-suited for a wide range of clinical applications. A major benefit of these vectors is their remarkable safety profile; wild-type AAVs are naturally non-pathogenic (Qu et al., 2019). Furthermore, AAVs exhibit low immunogenicity, which translates to a reduced likelihood of triggering a strong immune response in the host (Chen et al., 2009). This allows for sustained transgene expression, a critical factor for long-term therapeutic benefit in chronic genetic conditions (Li and Samulski, 2020; Choi et al., 2006). Another significant advantage lies in their ability to provide long-term gene expression. After delivery, the AAV genome primarily remains as an episomal DNA molecule within the nucleus of the target cell (Kymalainen, 2012). This characteristic significantly reduces the risk of insertional mutagenesis, a concern with integrating viral vectors that could potentially disrupt host genes or activate oncogenes, leading to cancerous transformation. While not integrated, the episomal genome can persist and continue to express the therapeutic gene for extended periods, especially in non-dividing cells, making it an ideal choice for treating chronic disorders (Naso et al., 2017). The efficiency of AAV-mediated gene transfer is another advantage. AAVs are highly efficient at transferring a wide range of genes, effectively entering target cells and delivering their genetic payload. This high efficiency of gene transfer is crucial for achieving therapeutic levels of gene expression, especially in tissues where large numbers of cells need to be modified. Their small size and robust capsid structure also contribute to their stability and ability to navigate biological barriers, facilitating efficient delivery to target sites (Naso et al., 2017).

Despite their promise in gene therapy, AAV vectors face several significant limitations (Naso et al., 2017): One of the primary issues is their limited packaging capacity; the small size of the AAV genome restricts the delivery of larger genes, typically only accommodating those up to 4.7 kb (McCarty, 2008). Another major hurdle is pre-existing immunity. Many people have already been exposed to AAVs and have antibodies that can neutralize the therapeutic vector, thus reducing its effectiveness (Greenberg et al., 2016). Although AAVs are generally not highly immunogenic, high doses can stimulate an immune response, potentially leading to inflammation and a decrease in long-term gene expression (Louis et al., 2013). Additionally, there is a rare but potential risk of off-target effects, where the AAV's genetic material integrates into the host's genome, possibly disrupting genes or causing mutations (Naso et al., 2017). Finally, the manufacturing process for high-quality, clinical-grade AAV vectors remains a complex and costly challenge (Naso et al., 2017).

The utility of AAV vectors in gene therapy largely stems from the remarkable diversity of their serotypes. To date, scientists have identified at least 13 natural serotypes and over 100 variants, each possessing unique characteristics that dictate their ability to target specific cells and tissues. This inherent tropism means that different AAV serotypes exhibit varying efficiencies in delivering genes to particular organs and tissues throughout the body. This extensive diversity is a key advantage, allowing gene therapy researchers to select or engineer the most appropriate AAV serotype to precisely target the desired cell or tissue type for therapeutic intervention (Hammond et al., 2017; Wu et al., 2006). AAV8 and AAV9 serotypes show strong tropism for skeletal and cardiac muscles (Burke et al., 2025; Issa et al., 2023).

AAVs are being explored for the treatment of rare skeletal diseases, such as fibrodysplasia ossificans progressiva (FOP) and osteogenesis imperfecta (OI). FOP is an ultra-rare and severely disabling genetic disorder characterized by progressive heterotopic ossification (HO), where soft connective tissues (muscles, tendons, ligaments) gradually turn into bone, forming a second skeleton. This process is primarily driven by a recurrent, activating mutation (R206H) in the ACVR1 (ALK2) gene, leading to aberrant signaling in the bone morphogenetic protein (BMP) pathway. AAV vectors are being investigated to deliver therapeutic genes that can either: 1) Inhibit the mutant ACVR1 pathway: This involves delivering gene constructs that block the overactive signaling of the mutated receptor. 2) Deliver gene editing tools: AAVs can transport CRISPR-Cas9 components to correct the ACVR1 mutation in affected cells specifically. 3) Deliver inhibitory RNA: This approach aims to silence the expression of the mutated ACVR1 gene. The challenge in FOP treatment using AAVs is to achieve localized and controlled delivery to prevent HO, especially in response to trauma, while minimizing off-target effects. Preclinical studies have demonstrated success in reducing HO in mouse models using AAVs, often incorporating tissue-specific promoters or microRNA-mediated repression to enhance specificity (Yang et al., 2024; Lin et al., 2024).

OI, often called brittle bone disease, is a rare genetic disorder primarily caused by autosomal dominant mutations in the COL1A1 or COL1A2 genes, which encode the α1 and α2 chains of type I collagen, the major structural protein in bone. This leads to defective collagen production, resulting in fragile bones, recurrent fractures, short stature, and other systemic issues (Lin et al., 2024). AAV9 vectors are being developed to deliver functional copies of the mutated collagen genes or to facilitate gene editing using CRISPR-Cas9 to correct the mutations in osteoblasts. The goal is to restore normal collagen synthesis, improve bone quality, and reduce fracture incidence. Preclinical studies have shown promising results in mouse models, demonstrating improved bone architecture and reduced fragility. Clinical trials are underway, with some aiming to assess the safety and efficacy of AAV-based therapies in human patients (Yang et al., 2024).

Beyond rare conditions, AAVs are also being investigated for prevalent bone diseases, including Osteoporosis, Bone fracture healing, Critical-sized bone defects, and Osteoarthritis.-

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to increased bone fragility and a higher risk of fractures (Falaschi et al., 2021). It often occurs due to an imbalance between bone formation and bone resorption, with osteoclast activity outstripping osteoblast activity (Falaschi et al., 2021). AAV vectors have been used to deliver artificial microRNAs (miRNAs) that silence genes like sclerostin (SOST) and schnurri-3 (SHN3), both of which are potent inhibitors of bone formation. This targeted gene silencing promoted WNT signaling, enhancing osteoblast function and reversing bone loss in mouse models of both postmenopausal and senile osteoporosis (Lin et al., 2024; Oh et al., 2023).

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Bone Fractures are common injuries. While most heal spontaneously, some result in delayed union or non-union, where the fracture fails to heal adequately, leading to chronic pain and disability. Large segmental bone defects also pose significant reconstructive challenges. Gene therapy with AAV vectors aims to accelerate and improve bone healing by delivering genes encoding potent osteogenic factors directly to the fracture site or bone defect. AAVs carrying genes for bone morphogenetic protein-2 (BMP-2) or bone morphogenetic protein-7 (BMP-7) are extensively studied. These proteins play a crucial role in initiating and promoting bone formation. The localized delivery of AAV-BMPs can stimulate mesenchymal stem cells at the injury site to differentiate into osteoblasts and produce new bone. Other growth factors and signaling molecules involved in bone regeneration are also targets for AAV delivery. This approach can provide a sustained, localized release of therapeutic proteins, overcoming limitations of recombinant protein delivery (short half-life, need for high doses). AAVs can transduce both dividing and non-dividing cells, which is advantageous for bone repair (Lee et al., 2019; Gafni et al., 2004).

AAV vectors are a versatile platform for gene therapy in bone disorders, targeting diverse pathologies from rare genetic conditions to common degenerative diseases and complex bone injuries. The ongoing research and clinical trials reflect the significant potential of this technology to revolutionize orthopedic medicine. The substantial clinical success and regulatory approvals of AAV-based gene therapies highlight their practical advantages. The extensive research and development over decades have culminated in numerous AAV-based gene therapies progressing through clinical trials, with several already receiving FDA approval. These approved therapies, targeting conditions such as spinal muscular atrophy and certain forms of inherited blindness, serve as powerful testaments to the efficacy and safety of AAV vectors in real-world clinical settings. This growing track record of success solidifies AAVs as a leading platform in gene therapy, attracting significant investment and fostering further innovation in the field (Lin et al., 2024; Mullard, 2023; Mendell et al., 2021).

AAV-based gene therapies are in various stages of clinical development for bone-related conditions. For instance, GNSC-001, an AAV vector expressing an optimized human interleukin-1 receptor antagonist (IL-1Ra), has recently received Regenerative Medicine Advanced Therapy (RMAT) designation from the FDA for the treatment of knee osteoarthritis, highlighting its potential to offer sustained IL-1 inhibition. While direct clinical trial data for AAV gene therapy specifically for bone fractures or severe skeletal dysplasia in humans are less widely publicized than for other monogenic diseases, preclinical studies consistently demonstrate their ability to deliver osteogenic factors and promote bone repair (Defois et al., 2024).

Adenovirus (Ad) vectors, especially replication-deficient type 5 Ads, are widely studied for bone-related gene therapy due to their high transduction efficiency in both dividing and non-dividing cells and their capacity for strong, short-term expression. They deliver therapeutic genes extra chromosomally, avoiding integration risks but raising concerns about transient expression and immunogenicity. Ad infects cells that express the coxsackievirus-adenovirus receptor (CAR). CAR is upregulated in immature osteoblasts during fracture healing, enabling targeted transfer to key repair cells (Ito et al., 2003).

Ads vectors offer several advantages for gene therapy, including their high efficiency in delivering genes to both dividing and non-dividing bone and stromal cells. Their ability to provide transient gene expression is particularly useful for temporary therapeutic needs, such as promoting fracture healing. Additionally, their potential for localized delivery helps minimize systemic exposure and off-target effects (Spector et al., 2000). However, Ad vectors also have significant limitations. A major concern is their immunogenicity, as they can trigger an inflammatory response, especially at high doses. Also, Immunogenicity can impair the healing process and limit the possibility of repeat dosing. Furthermore, Ad vectors often show variable results in large animal studies, possibly due to species-specific immune reactions. Finally, their short-term expression makes them unsuitable for treating chronic or long-term conditions that require sustained gene delivery (Ito et al., 2003; Evans, 2011; Egermann et al., 2006).

BMP-2 is most studied for osteoinduction. An early in vitro/in vivo study used an Adenovirus delivering human BMP-2 cDNA into the C3H/10T 1/2 mesenchymal progenitor cell line. Transduced cells exhibited dose-dependent proliferation and osteoblastic differentiation, and formed ossicles containing trabecular bone and marrow when implanted into nude mice (Lou et al., 1999). Also, in another study, mice with rib fractures, CAR expression peaked at 10–14 days post-fracture. Adenoviral lacZ reporter injected at day 14 selectively transduced immature osteoblasts, indicating the feasibility of targeted delivery to repair cells (Ito et al., 2003). In another study, Egermann et al. showed that local injection of Ad.BMP-2 at tibial osteotomy of adult osteoporotic sheep enhanced early callus formation, increased bone density and stiffness, and accelerated healing versus controls, assessed via radiography, CT, biomechanical testing, and histology (Egermann et al., 2006). In ovariectomized (OVX) rats modeling osteoporosis, adenoviral delivery of α-calcitonin gene-related peptide (α-CGRP) locally at dental implant sites enhanced new bone formation. After 28 days, markers showed: +80.2 % bone volume/total volume, +55.1 % trabecular number, +68.7 % thickness, and −38.4 % trabecular separation compared to controls. Osteogenic factors Runx2 and ALP were elevated, and osteoclastogenesis was suppressed via lower Receptor Activator of Nuclear Factor-κB Ligand (RANKL) expression (Guozhu et al., 2025). In another study, rabbits with spinal fusion underwent in-operation transduction of autologous buffy-coat cells (or muscle/fat autografts) with Ad-LMP-1 or Ad-BMP-2 and immediate reimplantation. All achieved successful fusion/healing in a single-stage surgery (Viggeswarapu et al., 2001).

Adenovirus vectors represent a promising platform for gene therapy in bone disorders, especially fracture healing and implant integration under compromised bone conditions. The evidence from animal case studies suggests strong regenerative potential, though translational hurdles remain. With improved delivery systems, vector design, and ex vivo techniques, clinical applications may eventually emerge.

Lentivirus vectors are a significant tool in gene therapy for various conditions, including bone diseases like osteoarthritis. These vectors are derived from retroviruses, specifically HIV-1, but are engineered to be replication-incompetent, meaning they cannot cause disease. They can stably integrate their genetic material into the host cell genome, leading to durable transgene expression. This makes them attractive for modifying long-lived cells such as bone marrow mesenchymal stem cells. For instance, studies have shown that lentiviral vectors can achieve high transfection efficiencies (up to 95 %) in chondrocytes, making them more efficient than some non-viral methods (Li et al., 2004).

Lentivirus vectors offer several key advantages for gene therapy, making them a popular choice for delivering genetic material. In addition to dividing cells, Lentiviruses are also very effective at infecting non-dividing cells, which is important for treating diseases that affect tissues such as the nervous system. Their ability to integrate their DNA into the host genome ensures stable and long-term gene expression, providing a durable therapeutic effect. Additionally, Lentiviral vectors can carry relatively large genetic payloads, typically up to 8–10 kilobases (kb), which is larger than many other viral vectors such as AAV, offering flexibility for complex gene constructs. While not entirely free of immune responses, engineered lentiviruses are generally considered to have lower immunogenicity compared to other vectors like adenoviruses (Dissen et al., 2011; Joglekar and Sandoval, 2017).

However, these vectors also have significant drawbacks. The most serious concern is the risk of insertional mutagenesis, since they integrate into the host genome, there is a theoretical risk that the integration could disrupt an endogenous gene or activate an oncogene, potentially leading to adverse effects such as tumor formation. While their integration profile is considered more favorable than gamma-retroviral vectors, this remains a concern. Although engineered to minimize immune responses, viral vectors, including lentiviruses, can still elicit an inflammatory response in vivo, which could lead to side effects or reduce the efficacy of the gene therapy. Moreover, manufacturing clinical-grade lentiviral vectors can be complex and expensive, requiring specialized facilities and expertise (Dissen et al., 2011; Joglekar and Sandoval, 2017).

Sugiyama et al. showed that lentivirus-mediated gene transfer could lead to sustained expression of BMP-2 and consequently induce new bone formation both in vitro and in vivo. This approach addresses limitations of directly applying recombinant BMP-2 protein, which often requires large doses and scaffolds for localized and sustained delivery (Sugiyama et al., 2005).

Liu et al. explored whether the overexpression of miR-26a, achieved through lentivirus-mediated gene transfer into bone mesenchymal stem cells (BMSCs), could enhance bone regeneration in a critical-sized calvarial bone defect model in mice. This builds upon previous research indicating miR-26a is involved in osteogenic differentiation. They first successfully transduced mouse BMSCs with a lentiviral vector designed to overexpress miR-26a (Lv-miR-26a), demonstrating that this overexpression significantly promoted the osteogenic differentiation of these BMSCs in vitro. This was evidenced by increased alkaline phosphatase (ALP) activity, enhanced mineralization (calcium deposition), and elevated expression levels of key osteogenesis-related genes, such as Runx2, osteocalcin (OCN), and bone sialoprotein (BSP). These findings suggested that miR-26a positively regulates the differentiation of BMSCs into bone-forming cells. The study also delved into the potential molecular mechanisms, identifying that miR-26a exerts its pro-osteogenic effects by targeting specific genes, such as tumor necrosis factor alpha (TNF-α) and cyclin D1, which are known to negatively regulate osteogenesis. By suppressing these targets, miR-26a promotes the osteogenic differentiation pathway. The findings suggested that lentivirus-mediated overexpression of miR-26a in BMSCs represents a promising gene therapy strategy for facilitating bone regeneration. This approach could potentially be applied in clinical settings for the repair of large bone defects, offering a novel therapeutic avenue by enhancing the intrinsic osteogenic potential of mesenchymal stem cells (Liu et al., 2018).

Non-viral vectors have garnered considerable attention as safe and versatile platforms for the localized and sustained delivery of diverse therapeutic payloads, including small molecules, proteins, and nucleic acids. Unlike their viral counterparts, non-viral systems circumvent concerns related to immunogenicity, insertional mutagenesis, and large-scale production, rendering them attractive for clinical translation (Table 1) (Jun Loh and Lee, 2012).

Effective delivery to bone necessitates overcoming several physiological barriers, including rapid systemic clearance and the dense, avascular nature of bone tissue. Non-viral strategies primarily leverage two mechanisms for achieving bone tropism (Qadir et al., 2019; Partridge and Oreffo, 2004): 1) Passive Targeting (Enhanced permeability): In bone disorders, such as fractures or inflamed areas, local vessels often become leaky due to impaired endothelial integrity, which produces the permeability and retention (EPR) effect. Nanocarriers of appropriate size and surface characteristics can passively accumulate in these areas due to increased vascular permeability. Furthermore, materials with an inherent affinity for bone mineral, such as those containing calcium phosphate or bisphosphonate moieties, can directly bind to hydroxyapatite, leading to localized retention and sustained release of the encapsulated therapeutic agent (Tong et al., 2023). 2) Active Targeting (Ligand-Receptor Interactions): This sophisticated approach involves conjugating specific ligands to the surface of non-viral carriers. These ligands are designed to recognize and bind to receptors or components uniquely expressed or abundant in bone tissue or bone-resident cells (Tong et al., 2023).-

Classes of Non-Viral Delivery Systems:

A broad spectrum of materials has been explored for non-viral bone delivery, each with distinct advantages and limitations:

1) Polymeric Nanoparticles: Biodegradable and biocompatible polymers (such as PLGA, chitosan, hyaluronic acid) can be engineered to encapsulate various therapeutics. Their versatility allows for tunable degradation rates, controlled release profiles, and surface functionalization for active targeting. 2)Lipid-Based Nanocarriers: Liposomes and lipid nanoparticles (LNPs) offer excellent biocompatibility and low immunogenicity. They are particularly well-suited for delivering nucleic acids (such as mRNA, siRNA) and hydrophobic drugs due to their amphiphilic nature. 3) Inorganic Nanomaterials: Calcium phosphate nanoparticles, mesoporous silica nanoparticles, and magnetic nanoparticles possess intrinsic bone-targeting capabilities or can be modified for such. Calcium phosphate, being a major component of bone, offers excellent osteoconductivity and biodegradability. 4) Peptide-Based Systems: Self-assembling peptides can form nanostructures capable of encapsulating drugs or genes. Their inherent biocompatibility and ease of modification make them attractive for site-specific delivery. 5) Hydrogels and Scaffolds: These macroscopic systems provide a localized reservoir for sustained drug release directly at the bone defect site. They can be engineered to be injectable, biodegradable, and osteoconductive, often serving as matrices for cell delivery in regenerative medicine (Balmayor et al., 2015; Ranjbarnejad et al., 2022).-

Pre-Clinical studies of Non-viral Vectors:

One notable polymeric nanoparticles study conducted by Zhou et al. investigated the use of chitosan-based polymeric nanoparticles for non-viral gene delivery targeting critical-sized bone defects. The researchers designed a nanoparticle system encapsulating plasmid DNA encoding BMP-2 and evaluated its osteoinductive potential in a rat calvarial defect model. The chitosan-DNA complexes were administered locally to the defect site via an injectable hydrogel scaffold, allowing for sustained release and enhanced retention at the injury location. Histological analysis at 4- and 8-week post-treatment showed significant bone regeneration in the nanoparticle-treated group compared to both the untreated control and the group receiving naked plasmid DNA. Quantitatively, micro-CT analysis revealed over 60 % defect closure in the nanoparticle group versus less than 25 % in the control. Immunohistochemistry confirmed elevated expression of osteogenic markers such as Runx2 and osteocalcin, indicating active bone formation. Importantly, no signs of systemic toxicity or inflammation were observed, highlighting the biocompatibility and biosafety of the non-viral delivery platform. This study demonstrates the promise of polymer-based non-viral vectors for gene therapy in bone repair and their clinical translation as a safer alternative to viral approaches (Zhao et al., 2016).

A cutting-edge study by De La Vega et al. explored the therapeutic potential of chemically modified messenger RNA (cmRNA) encoding BMP-2 delivered via lipid nanoparticles (LNPs) for repairing large segmental bone defects. In their preclinical model, cmRNA was loaded onto collagen scaffolds and implanted into critical-sized femoral defects in rats. This approach enabled localized, transient expression of BMP-2 at the injury site, triggering strong osteoinductive signaling without the complications associated with recombinant BMP-2 protein, such as ectopic bone growth or excessive callus formation. Compared to the standard rhBMP-2 protein treatment, the LNP-delivered cmRNA achieved comparable or superior bone regeneration as assessed by bone volume, density, and biomechanical strength, while significantly reducing the required dosage. The localized gene expression was efficient, short-lived, and well tolerated, with no detectable systemic toxicity. This study underscores the clinical viability of LNP-mediated mRNA therapy for bone regeneration, combining the safety and precision of non-viral delivery with the robust regenerative signaling of BMP-2 (De La Vega et al., 2022).

In a revolutionary preclinical study, Basha et al. investigated the targeted silencing of the SOST gene (a negative regulator of bone formation) using LNPs to deliver siRNA directly to osteocytes, the most abundant cells embedded within bone matrix. The LNP formulation was optimized for systemic administration and enabled effective delivery of siRNA to skeletal tissue following intravenous injection in mice. The siRNA successfully suppressed SOST mRNA levels, which in turn led to a measurable reduction in the protein sclerostin, a known inhibitor of the Wnt signaling pathway that regulates osteogenesis. Remarkably, the study reported significant increases in bone formation markers and trabecular bone mass in the treated animals, confirming that SOST silencing can trigger an anabolic bone response. The approach demonstrated a favorable safety profile with no adverse systemic effects. This study highlights the potential of LNP-based siRNA therapies for treating bone diseases such as osteoporosis by modulating gene expression in hard-to-reach bone-resident cells such as osteocytes, without relying on viral vectors (Basha et al., 2016).

Kim et al. developed a minimally invasive system that combines an injectable alginate/collagen hydrogel with two distinct microparticle populations (one containing BMP-2 and the other containing IGF-1). This scaffold was applied to critical-sized cranial bone defects in a rat model. The engineered hydrogel allowed sequential release, with BMP-2 released first to initiate osteogenic differentiation, followed by IGF-1 to support cellular proliferation and vascularization. After 4 and 8 weeks, treated animals showed accelerated bone formation and increased bone volume, on par with high-dose BMP-2 delivery alone, but with notably lower BMP-2 dosage required. Micro-CT images and histological analyses confirmed robust bone bridging and tissue integration. Importantly, the combined low-dose regimen minimized side effects typically associated with high BMP-2 concentrations, such as ectopic ossification or excessive inflammation. This study illustrates the power of combining controlled dual-release systems in hydrogels for bone repair, offering improved therapeutic efficacy while reducing growth factor load and potential adverse effects (Kim et al., 2023).