Advancing regenerative biomaterials and translational strategies in orthopaedic traumatology

Abstract

The 2025 Orthopaedic Trauma Association's Basic Science Focus Forum featured a symposium on regenerative biomaterials and translational strategies for orthopaedic trauma. Four experts presented complementary perspectives on the next generation of bone and soft tissue repair: Dr. J. Tracy Watson described how orthobiologics and refined spacer design can be used to engineer a better Masquelet induced membrane; Dr. Lauren Priddy presented evidence that hydroxyapatite coatings can mitigate the rapid degradation of additively manufactured magnesium scaffolds; Dr. Vaida Glatt described the development and clinical translation of the Biomimetic Hematoma scaffold for bone and muscle regeneration; and Dr. Katherine Hixon outlined how the 3R principles (Replacement, Reduction, Refinement) can serve as a framework to accelerate musculoskeletal regenerative engineering. Collectively, the symposium underscored a shift toward biologically informed, clinically predictive, and ethically responsible engineering solutions for the patients who sustain musculoskeletal trauma.

Introduction

Over the past two decades, advances in scaffold biofabrication and orthobiologic delivery mechanisms have begun to close the gap between laboratory discovery and clinical application. However, translation remains challenging: preclinical models often fail to recapitulate the biological complexity of traumatic injury, regulatory and ethical constraints shape experimental design, and clinically meaningful end points are difficult to define early in the development pipeline. Bridging these gaps requires innovative materials science coupled with frameworks that synthesize biological rationale, translational rigor, and ethical responsibility.

At the 2025 annual meeting of the Orthopaedic Trauma Association, the Basic Science Focus Forum hosted a symposium that explored this evolving research space. Invited speakers addressed engineered Masquelet membranes, degradable metallic implants, nature-inspired scaffolds, and translationally rigorous preclinical pipelines. The first talk, entitled “Inducing Better Masquelet Membranes with Orthobiologics,” was presented by Dr. J. Tracy Watson; “Hydroxyapatite Coating Mitigates Degradation of Additively Manufactured Magnesium Scaffolds” was presented by Dr. Lauren Priddy; “Nature-Inspired Healing: Clinical Potential of the Biomimetic Hematoma Scaffold” was presented by Dr. Vaida Glatt; and “Strategies to Enhance Musculoskeletal Recovery After Traumatic Injury: The 3R Principles as a Framework for Translational Regenerative Engineering” was presented by Dr. Katherine Hixon. Together, these presentations illustrated how a biologically informed, clinically responsive approach to biomaterials development can address the persistent unmet needs of patients sustaining musculoskeletal trauma. All studies included in this symposium gained approval from their respective Institutional Review Board and/or Institutional Animal Care and Use Committee, as applicable. The research was conducted in accordance with the Declaration of the World Medical Association (www.wma.net) and informed consent from human subjects was obtained as required. The work of each presenter is summarized in the following sections.

Inducing better Masquelet membranes with orthobiologics

The Masquelet technique has improved treatment of segmental critical-size bone defects. After debridement, an antibiotic spacer is placed in the defect, triggering formation of a vascularized induced membrane. At a second stage, the membrane is opened and bone graft is inserted into this biologic chamber, where the membrane helps contain the graft and supports consolidation.^1,2 Interest has now shifted from simply timing graft placement to understanding how the membrane might be improved with orthobiologic augmentation and spacer design.

Before attempting to enhance the membrane, it is important to understand its morphology, developmental sequence, growth factor profile, vascularity, stem-cell content, and the influence of spacer material and surrounding soft tissue. These factors together determine whether the induced membrane becomes an effective environment for graft incorporation and bone regeneration. Our work has demonstrated that histologically, the induced membrane has a bilayer organization.³ The inner layer, adjacent to the polymethylmethacrylate (PMMA) spacer, is highly cellular, whereas the outer layer contains fibroblasts, myofibroblasts, collagen, and a rich vascular network contiguous with the surrounding soft tissues. By about 3 weeks, 3 zones can often be distinguished: a thin cellular layer near the spacer, a collagen-rich middle layer, and a more disorganized outer vascular layer. This organization supports the concept that the membrane is not a passive capsule but an active tissue with structural and signaling functions.

One approach to improving this biologic environment is the use of autologous platelet concentrates such as platelet-rich plasma (PRP). PRP delivers growth factors including transforming growth factor-beta, platelet-derived growth factor, vascular endothelial growth factor, and insulin-like growth factor and may amplify the early wound-healing milieu that drives membrane maturation.^4,5 Although PRP is not independently osteogenic, it may promote cellular proliferation, osteoblastic differentiation, mineralization, and angiogenesis when applied in an appropriate setting. It can be introduced at the time of grafting, but there is growing interest in delivering it earlier, at the time of spacer placement,⁶ to influence downstream membrane development from the outset.

Vascularity is greatest in the outer membrane and lowest near the spacer surface. Revascularization increases during the first several weeks, with many studies suggesting a favorable biologic window for grafting around 4–6 weeks, when angiogenesis and growth factor activity are robust.⁷ Bone spicules and foci of membrane-directed ossification may also appear in some cases, reinforcing the view that the membrane can participate actively in regeneration.⁸ At the same time, practical surgical handling sometimes favors waiting until the membrane has sufficient thickness and durability, which may occur somewhat later.^1,9 The source of mesenchymal stem cells demonstrates that membrane-derived stem cells are multipotent and capable of differentiating into adipogenic, osteogenic, and chondrogenic lineages. Mesenchymal stem cells are present as early as 2 weeks after PMMA spacer placement but decline gradually by 4 and 6 weeks.^3,9,10

The optimal pore size for porous-coated implants appears to be approximately 300–700 µm.¹¹ Fine-tuning pore architecture may help optimize implant mechanics and improve osseointegration through membrane-induced bone formation. In our retrieved PMMA spacers containing vancomycin and tobramycin, electron microscopy demonstrated pore sizes of roughly 450–500 µm. These findings suggest that particulate antibiotics may be preferable to liquid formulations as the antibiotics can function as a porogen.^12,13 Practically, the antibiotic powder should be mixed thoroughly and morselized completely before adding the cement powder to achieve uniform porosity within the desired range. Pore architecture may be one of the most modifiable spacer features. Our experimental work suggests an effective pore size in roughly the 300–700 µm range, and retrieved antibiotic PMMA spacers often demonstrate pores around 450–500 µm. We have also evaluated whether membrane formation can be influenced by spacer surface properties and material characteristics.^3,9,10,14 Our research demonstrated that microscale rougher surfaces elicit a more proinflammatory response after 4 weeks of implantation regardless of material. Rough spacers surfaces developed an inner membrane layer with higher factor expression.^3,9,10 This raises the important question of the optimal spacer surface properties for membrane development. Rough and porous spacers were more compliant and produced membranes capable of significantly higher tensile strain, indicating that spacer topography alters tissue mechanics.¹⁴

Many available substitute bio membranes are completely synthetic and preset with a pore size of approximately 500 µm. We have been augmenting our Masquelet membrane formation by implanting these materials at the time of wound closure over the antibiotic spacers.^6,15 Poly(lactic-co-glycolic acid) containing materials may also exert antimicrobial effects across multiple bacterial strains because their degradation products include lactic and glycolic acids, which lowers the local pH and helps combat infection along with the antibiotic spacer.^12,13 These membranes become fully ingrown and do not promote biofilm formation, making them appealing adjuncts for Masquelet membrane development.¹⁶ The scaffold supports cellular ingrowth and new tissue formation from surrounding native tissues, and as the matrix gradually resorbs, it continues to support tissue integration and neovascularization of the developing Masquelet membrane. This raises the possibility that spacer formulation, antibiotic preparation, and eventually custom-manufactured or 3D-printed devices could be used to engineer a more favorable membrane. For now, these findings mainly support careful cement preparation and thoughtful attention to how particulate additives affect spacer porosity and downstream tissue formation.¹⁴

Membrane optimization should not be viewed as a single intervention. Successful induction likely depends on coordinated control of debridement, infection management, spacer formulation, surface texture, porosity, biologic augmentation, and soft tissue support. The best membrane is probably one that is sufficiently vascular, mechanically durable, biologically active, and surgically manageable at the time of grafting. Orthobiologics such as PRP, better control of spacer architecture, and adjunctive scaffold materials all represent credible paths toward that goal.

Hydroxyapatite coating mitigates degradation of additively manufactured magnesium scaffolds

Traumatic bone injuries such as complex fractures necessitate surgical intervention for repair, commonly involving titanium or stainless-steel implants. Although these nondegradable materials can be sufficient to heal the fracture, their mechanical properties are much higher than those of bone, which can cause the implant to bear most mechanical loads.¹⁷ This stress shielding phenomenon can lead to bone resorption and poor osseointegration, increasing the risk of implant failure and bone refracture, ultimately requiring a revision surgery to replace the implant.^18,19 Even when bone healing is successful, patients can experience debilitating, chronic pain at the implant site. Notably, for pediatric patients, the presence of an implant long-term can inhibit function and impede bone growth,²⁰ with implant removal occurring in roughly half of all cases.^19,21 Although pediatric patients reported a reduction in pain after elective implant removal,²² additional surgeries are associated with increased patient morbidity²³ and remain controversial.^24,25 Thus, there is an urgent need to engineer degradable orthopaedic implants to abrogate the risks associated with permanent implants and implant removal surgeries.

As an alternative to traditional bioinert (nondegradable) implants, degradable and osteogenic biomaterials such as magnesium (Mg) alloys are attractive as next-generation orthopaedic implants. Besides their biodegradable nature, Mg alloys are advantageous over traditional metallic biomaterials because of their mechanical properties (e.g., elastic modulus)^26–28 and densities,²⁶ which closely match those of bone, and their osteogenic²⁸ and antimicrobial²⁴ properties.

Additive manufacturing processes such as laser-powder bed fusion, which involves melting of metallic powder by a laser in a layer-by-layer fashion, have gained considerable attention as alternative methods for producing customized (e.g., porous) components for advanced medical, aerospace, and automotive applications. Compared with conventional manufacturing methods (e.g., casting), which cannot precisely control the geometry nor achieve sufficient mechanical properties,²⁹ additive manufacturing enables customization of geometries and mechanical properties at high resolution, to tailor the implant to the needs of the patient. Introducing porosity into Mg-based implants is an effective way to shift their mechanical properties even closer to those of bone and holds much potential for situations in which a solid, permanent implant may be detrimental, for example, pediatric patients who would outgrow the implant or elderly patients with lower density (osteoporotic) bone.^26,30,31

Despite the potential, the biodegradation of Mg is a significant barrier to clinical implementation of Mg implants because of rapid degradation (corrosion) of Mg and unpredictability of structural integrity over time.³² Coating of Mg with the bioceramic hydroxyapatite (HA), which is the mineral component of bone, is advantageous because of HA's resistance to degradation,^33,34 osteoconductivity,^35,36 and ability to enhance implant performance.³⁷ Indeed, we have demonstrated HA coating of solid, pure Mg discs via hydrothermal solution deposition slowed Mg degradation through 30 days in vitro.³⁸ Building upon these findings, we characterized the effects of HA coating on the degradation kinetics of additively manufactured WE43 Mg alloy scaffolds. Our hypothesis was HA coating of WE43 scaffolds would effectively enhance corrosion resistance compared with noncoated WE43 scaffolds in vitro. WE43 Mg alloy scaffolds (6 × 1.4 mm [dxh]) with diamond unit cells (400 μm strut width; 600 μm pore size) were additively manufactured via laser-powder bed fusion³⁹ and coated with hydroxyapatite using hydrothermal solution deposition at 90°C, pH 8.9,³⁸ with the deposition time extended to 16 hours on each side. Noncoated WE43 scaffolds served as the control.

In the first degradation study, samples were placed in DMEM (Dulbecco's Modified Eagle Medium, ISO 10993-12) on a shaker plate at 90 RPM, 37°C³⁸ for 20 days. Measures of scaffold mass and solution pH and visualization of surface morphology via scanning electron microscopy were performed longitudinally through 20 days. Surface profilometry scans were performed on day 20. Accelerated, dynamic degradation of scaffolds was performed in a closed-loop bioreactor over 24 hours in revised simulated body fluid at 0.3 mL/min at 37°C.⁴⁰ In addition to degradation rate, Mg²⁺ release in media extracts was measured. Media extracts were also applied to preosteoblasts (MC3T3-E1) for 24 hours to measure indirect cell viability. To visualize direct cell viability, degraded scaffolds were seeded with preosteoblasts and then cultured for 24 hours dynamically in the bioreactor system.

Mass for HA-WE43 coated scaffolds remained constant throughout the 20-day shaker plate degradation study, whereas noncoated WE43 scaffolds had a ∼35% reduction in mass. Likewise, pH was higher for WE43 scaffolds at days 10 and 20 compared with that for HA-WE43 scaffolds. The initially rod-like hydroxyapatite coating was partially dissolved by day 20, exposing the WE43 substrate, which had small cracks visible by day 10. Large cracks in the WE43 group appeared on day 4 and grew through day 20. Line and surface roughness were not different between HA-WE43 and WE43 scaffolds on day 20. In contrast, the average step height, maximum height, and minimum height were higher for the HA-WE43 scaffolds, further indicating they had undergone less degradation.

Results from the 24-hour bioreactor degradation study were consistent with the shaker plate degradation results, with HA-WE43 scaffolds degrading at a slower rate and releasing less magnesium than WE43 scaffolds. A reduction in cell viability was observed from preosteoblasts cultured with extract from WE43 scaffolds compared with that from HA-WE43 scaffolds. Similarly, cells remained viable on degraded HA-WE43 scaffolds, whereas significant cell death was observed on degraded WE43 scaffolds.

Herein, we demonstrate the utility of HA coating for mitigating degradation of additively manufactured, porous Mg scaffolds. Leveraging these experimental degradation datasets, finite element simulations were used to model the degradation of HA-coated and noncoated Mg using a continuum damage/pitting model, which enables the prediction of long-term degradation patterns and structural integrity of these materials.⁴¹ Additive manufacturing and hydroxyapatite coating of degradable, osteogenic biomaterials such as magnesium alloys with customizable geometries and mechanical properties will enhance the functionality of these materials for osseointegration and bone repair and ultimately accelerate the translation of next-generation orthopaedic implants.

Nature-inspired healing: clinical potential of biomimetic hematoma scaffold

Management of severe bone^42,43 and muscle injuries⁴⁴ remains a major challenge in orthopaedic surgery, as these injuries often fail to heal and can result in chronic pain, functional impairment, and long-term disability. Approximately 15% of fractures result in nonunion,⁴⁵ with rates reaching 20%–30% in tibial fractures.⁴⁶ Similarly, 10%–15% of severe extremity injuries result to volumetric muscle loss that exceeds the body's natural regenerative capacity. The clinical and economic burden of these conditions is substantial, with treatment cost often exceeding $200,000 per patient.^46,47 Despite advances in fixation techniques, bone grafting, biologics, and limb salvage procedures, no current therapy reliably regenerates complex musculoskeletal injuries.

Many currently available orthobiologics fail to promote true tissue regeneration and instead function primarily as passive fillers that support scar-mediated repair rather than restoration of native tissue. Conventional bone graft substitutes, including hydroxyapatite- and calcium phosphate–based materials, are biocompatible but largely biologically inert, lacking the cellular and molecular signals required to actively stimulate regeneration.⁴⁸ Their slow degradation may further impede remodeling and healing. Similarly, collagen sponges used to deliver human recombinant bone morphogenetic protein-2 (rhBMP-2), such as those used in Infuse (Medtronic, Minneapolis, MN) require high protein doses because of rapid diffusion and poor local retention, increasing the risk of inflammation and ectopic bone formation.^49,50 In muscle injuries, the limitations of existing therapies are even more pronounced, as acellular scaffolds frequently result in fibrosis rather than regeneration of functional muscle tissue.⁵¹

In contrast to these static biomaterials, nature provides a highly effective regenerative scaffold in a form of the hematoma.^52,53 After bone fracture or muscle injury, bleeding at the injury site leads to formation of a hematoma that servers as the initial template to tissue repair. Rather than acting as an inert filler, the hematoma is a dynamic and biologically active microenvironment that orchestrates the early stages of healing.⁵⁴ Its fibrin network traps platelets, inflammatory and progenitor cells, while concentrating growth factors and cytokines that trigger regeneration. It also provides a 3-dimensional micro-architecture that supports cell migration, vascular ingrowth, and new tissue formation. Studies have demonstrated that removal of the hematoma delays healing,⁵⁵ highlighting its critical role in tissue regeneration. The structural characteristics of the hematoma, including its fibrin mesh and porosity, are also essential in guiding the repair process.⁵³

This insight led to the development of the Biomimetic Hematoma (BH), an autologous scaffold designed to replicate the biological, structural, and physical properties of a native fracture hematoma while serving as a delivery platform for therapeutic molecules that promote true tissue regeneration.^53,56 Initially developed for growth factor delivery in bone regeneration, BH is prepared intraoperatively by combining the patient's whole blood with clotting agents. Low doses of rhBMP-2 are incorporated into the blood before coagulation, allowing the resulting fibrin matrix to naturally bind, retain, and localize the growth factor at the injury site. In addition to initiating clot formation, the coagulants enhance the osteogenic activity of rhBMP-2, creating a synergistic environment for bone regeneration. Similar to a native fracture hematoma, BH establishes a localized reservoir of regenerative cells and signaling molecules. The blood provides cells and biological signals, the fibrin scaffold provides a temporary extracellular matrix that guides tissue formation, and low-dose BMP-2 amplifies the regenerative response, creating an in situ regenerative microenvironment that supports tissue repair and healing.

The BH scaffold has demonstrated strong regenerative potential in demanding preclinical models. In a rat femoral defect model, BH loaded with 10- to 20-fold less rhBMP-2 than the standard collagen sponge achieved complete and reproducible bone regeneration by 8 weeks.⁵⁶ In a 2.5-cm goat tibial defect model, BH containing 42 or 210 µg of rhBMP-2 led to earlier and superior healing compared with 2.1 mg of rhBMP-2 delivered on a collagen sponge, while using 40-fold less protein.⁵⁷ The regenerated bone was comparable to native bone in both quality and predicted mechanical strength. Preliminary studies in volumetric muscle loss further suggest that BH may support regeneration of skeletal muscle. BH alone or in combination with mRNA encoding R-spondin 2—a key regulator of myogenesis⁵⁸ and neuromuscular junction formation,⁵⁹ restored muscle morphology and function, resulting in near-normal tissue organization and ∼70–85% recovery of tetanic force at 12 weeks, compared with ∼40% in untreated controls.

The translational potential of the BH scaffold is underscored by early clinical use in patients with complex orthopaedic injuries. BH delivering low-dose rhBMP-2 has been successfully used in over 100 cases, including aseptic and infected nonunions, segmental bone defects, and ankle fusions with substantial bone loss. In these cases, complete bone regeneration and union were achieved within 6 months, with no reported treatment-related adverse events.^60,61 Many patients had long-standing, previously nonhealing conditions that had failed multiple prior interventions. In one representative case, a 49-year-old man with a tibial nonunion complicated with chronic osteomyelitis and 20 prior surgical procedures achieved complete bone union within 6 months after treatment with BH delivering low-dose rhBMP-2, and local antibiotics, ultimately avoiding amputation.⁶¹ These early clinical findings suggest that BH may represent a promising strategy for the treatment of large bone defects and recalcitrant nonunions, including cases traditionally associated with poor healing potential.

The Biomimetic Hematoma scaffold represents a significant advancement in orthobiologics by integrating biomaterial science with the body's natural healing mechanisms. By replicating the structure and biological activity of the native fracture hematoma, BH addresses key limitations of traditional scaffolds while actively promoting tissue regeneration. Preclinical and early clinical studies demonstrate that BH can support robust bone healing using substantially lower doses of rhBMP-2 than are typically required, potentially reducing cost and dose-related complications. Emerging evidence also suggests that BH may restore skeletal muscle structure and functional recovering after volumetric muscle loss, further highlighting its potential as a versatile regenerative platform. Grounded in the principles of biomimicry, BH offers a practical, translational approach for tissue repair that may improve clinical outcomes, reduce the need for revision procedures, and expand the future applications of regenerative medicine. Rather than replacing the body's natural healing response, BH seeks to recreate and enhance it, offering a new paradigm for the treatment of challenging musculoskeletal injuries.

Strategies to enhance musculoskeletal recovery after traumatic injury: the 3R principles as a framework for translational regenerative engineering

Musculoskeletal injuries represent a large and increasing clinical burden,^62,63 and current strategies often fail to restore full form and function in patients with critical-size bone defects, volumetric muscle loss, or tendon injuries. Conventional surgical reconstruction, autografting, and fixation technologies frequently restore structural continuity but remain insufficient to reestablish biological integrity and long-term functional performance. Tissue engineering seeks to address these challenges through biomaterials, biologics, and mechanobiological cues that guide regeneration.^64,65 Yet, despite innovation, progress remains limited in part by a persistent dependence on animal models that do not always reproduce human clinical healing environments. Applying the 3Rs (Replacement, Reduction, and Refinement) in musculoskeletal regenerative research enables ethically responsible scientific advancement while increasing translational fidelity and accelerating the path toward patient impact.^66–68

Replacement: toward human-relevant experimental systems

Traditional two-dimensional (2D) monocultures lack the mineral, matrix, and mechanical features necessary to recapitulate bone-tendon healing. Advanced in vitro platforms now enable more predictive evaluation of engineered scaffolds before in vivo testing. Cryogel and tendon scaffold systems support mechanobiology studies that mimic the physical environment of musculoskeletal tissues and reveal how cell behavior is influenced by stiffness gradients, matrix architecture, oxygen tension, biochemical cues, and dynamic loading. These models allow researchers to interrogate osteogenic differentiation, collagen alignment, and matrix deposition in a controlled setting.^69–71

Co-culture models, especially those combining endothelial and stromal populations, allow early assessment of vascularization potential, a critical determinant of bone, muscle, and tendon repair success. By capturing cell–cell signaling, angiogenic sprouting, and vessel infiltration potential up front, these platforms reduce the burden on animal studies to answer high-risk feasibility questions.^72–74 Ex vivo explants, such as tendon, maintain native hierarchical organization while enabling controlled perturbation of cell–matrix interactions over time. They serve as a bridge between simplified in vitro constructs and complex in vivo models, offering mechanistic insights with minimal animal use.^75,76

Computational methods further contribute to Replacement objectives. Finite element modeling and automated lattice optimization workflows allow engineers to rapidly screen scaffold architectures for load transfer, stress distribution, and implant fit before fabrication. Eliminating low-performing designs computationally means that only the strongest candidates move forward, reducing reliance on exploratory animal trials. Together, these advanced platforms produce more human-relevant data from the outset of discovery.^69,77,78

In the context of traumatic musculoskeletal injury, mineralized cryogel scaffolds developed for craniofacial and long-bone regeneration provide a human-relevant platform to study bone-muscle-vascular crosstalk under mechanically and biologically compromised conditions.^79–82 These systems enable mechanistic interrogation of osteogenesis, angiogenesis, and soft tissue integration before deployment in radiation-damaged or critically injured defect models.

Reduction: maximizing impact per animal

When in vivo studies are necessary, strategic experimental planning can drastically reduce animal usage. High-throughput in vitro screens eliminate materials without osteogenic, angiogenic, or biomechanical promise before transitioning into preclinical models.^83,84 Clear, quantitative decision gates ensure that each animal is used for testing only the most viable therapeutic candidates. Improved statistical approaches, including formal power analyses and repeated-measures frameworks enabled by longitudinal microCT imaging, increase the robustness of data obtained from each study. Functional metrics can be collected nonterminally at multiple time points, allowing fewer animals to provide more insight into healing progression.⁸⁵

At the regulatory level, federal agencies are increasingly prioritizing the reduction of animal use in preclinical research. In April 2025, the US Food and Drug Administration released a Roadmap to Reducing Animal Testing in Preclinical Safety Studies, outlining a strategic framework to reduce, refine, and ultimately replace animal testing in drug development by encouraging the adoption of advanced human-relevant methodologies such as computational modeling, organoids, tissue-chip platforms, and other new approach methodologies. This roadmap reflects a major regulatory shift toward minimizing animal use while improving translational relevance.⁸⁶

Similarly, the National Institutes of Health (NIH) announced a new agency-wide initiative to prioritize human-based research technologies and reduce reliance on animal models in federally funded biomedical research. As part of this effort, NIH plans to establish a dedicated Office of Research Innovation, Validation, and Application to coordinate development, validation, funding, and training around nonanimal experimental platforms, further reinforcing a national commitment to high-impact, human-relevant, and animal-sparing research paradigms.⁸⁷

Finally, resource sharing plays a major role in Reduction. Harmonizing defect sizes, fixation approaches, animal ages, and readout methodologies across laboratories decreases duplication and increases the efficiency of knowledge gain across the field. This shift promotes a culture of collaboration and transparency while reducing animal use globally.^88,89

Refinement: enhancing welfare and translational relevance

Refinement efforts focus on improving welfare without compromising, and often enhancing, scientific output. A key example is choosing models that accurately reflect human pathological conditions. For example, irradiated mandibular defect models mimic the compromised healing environment observed in patients undergoing head and neck cancer treatment,⁹⁰ in contrast to repaired defects in young, healthy bone that do not reflect clinical complexity. Likewise, true critical-size defects offer more predictive performance benchmarking than simple acute injury models in which healing will occur regardless of intervention.^91,92

Clinically relevant radiation-injury models further exemplify refinement in musculoskeletal research by replicating dose-dependent impairments in osteogenesis, vascularization, and cortical bone remodeling that are observed in postoncologic patients.^93,94 These refined models enable identification of dose-dependent therapeutic windows that cannot be captured in healthy bone systems.

Surgical and perioperative refinements further support animal welfare and high-quality outcomes. External fixation systems allow physiologic loading and reduce morbidity from internal fixation hardware. Advances in analgesia and humane end points minimize suffering while reducing variability in recovery and behavior.⁹⁵ Moreover, refined readouts, including vessel-specific histology, gait analysis, noninvasive monitoring, and biomechanical testing, better align preclinical results with functional outcomes meaningful to patients.⁹⁶ Collectively, these refinements ensure that each animal contributes high-value, clinically relevant information to the progress of regenerative medicine.^97–99

The 3Rs as a catalyst for innovation

The 3R principles are sometimes perceived as constraints that slow scientific progress. In practice, they serve as a powerful framework that challenges researchers to think creatively, design smarter experiments, and elevate both ethics and rigor.¹⁰⁰ Ongoing innovation in advanced biomaterials, microphysiological models, functional imaging, and computational design is making the preclinical pipeline more predictive and efficient. In musculoskeletal tissue engineering, the 3Rs directly support our core mission: improving patient outcomes and advancing surgical care. By shifting to a mindset of clinical-predictive first, animal-validated second, we prioritize relevance and reduce steps that do not contribute meaningfully to translational success.

Looking ahead, the most impactful progress will come from embedding at least one Replace, Reduce, or Refine strategy into every new research project. This commitment not only respects the ethical obligation we have toward animal use but also ensures that scientific advances translate more rapidly and successfully to those who need them most.

Data availability statement

This article summarizes research presented by the individual authors at a scientific symposium; no new data were generated in the preparation of this manuscript. Data underlying the studies discussed are available in the original publications cited or from the respective corresponding authors upon reasonable request.