Tissue-Engineered Constructs for Dermal Closure Around Osseointegrated Prosthetics

Introduction: :

Introduction: The dermal barrier serves as the body’s first line of defense against the outside world. While skin is generally adept at healing post-injury, this healing is disrupted around permanent, transcutaneous implants such as osseointegrated (OI) prosthetics (Fig. 1A). OI involves the integration of a metal implant directly with the native, residual bone and through the soft tissue post-amputation for attachment to an external prosthesis. Even after soft tissue healing, this permanently ruptured barrier is vulnerable to infection, drag from soft tissue deformation (Fig. 1B), and mechanical disruption during even mild trauma (e.g., a fall). A potential solution exists in harnessing bioactive proteins and biosimilar scaffold topography to encourage cell adhesion around the aperture site. Proteins such as keratin and laminin are responsible for much of the body’s innate connective mechanisms through hemidesmosomal cellular anchoring to the basal membrane, while electrospun fibers (ESFs) mimic the structure of the extracellular matrix to encourage integration. However, protein filaments and ESFs are not macro-mechanically strong, necessitating implementation of a stiffer scaffold that can effectively distribute physical loading. Polymethyl methacrylate (PMMA) is non-biodegradable, biocompatible, and receptive to surface modification to allow for incorporation of these bioactive molecules. We therefore hypothesize that i) electrospun fibers can be fabricated and affixed within a mechanically strong framework; ii) different combinations of construct polymers and chemical/physical binding techniques will produce biostable, biocompatible, bioactive, and mechanically strong constructs; and iii) hemidesmosomal cell anchoring molecules will improve dermal adhesion in a murine subcutaneous model.

Materials and

Methods: : Materials and

Methods: Existing work from our group has focused on producing a mechanically durable component that integrates with biological tissue for OI stabilization. Experimental construct material integration has focused on varying base polymer type and chemical binding mechanisms to incorporate extracted keratin. Specifically, polymer bases (i.e., PMMA and polycarbonate) were investigated as cell-compatible, chemically modifiable, foundational components. First, complex three-dimensional (3D) support designs were computationally evaluated via SolidWorks simulation software to determine the force-distributive capabilities under clinically relevant OI forces (N=3; Fig. 2). For biological assessment, different conjugation/crosslinking mechanisms (i.e., plasma coating and chemical crosslinking) were tested for fixation of bioactive keratin and laminin structures to the construct, creating hemidesmosomal (cell-matrix) anchoring sites for long-term, sustainable cell adhesion. This material binding and biocompatibility were assessed through a 28-day degradation and a 7- and 14-day human dermal cell study, respectively. To assess these properties in an in vivo environment, tissue engineered experimental (frame and protein-modified ESFs; Fig. 3A-B) and control (frame and unmodified ESFs) constructs were implanted in 10-week-old C57BL/6 murine subcutaneous model (N=16; Fig. 3C). Mechanical and histological analyses are then performed at 2 and 4 weeks. Hematoxylin and eosin (H&E) staining was used to visualize tissue morphology. Cytokeratin Pan Type II Antibody IHC stain and a laminin 332 fluorescent antibody stain were used to visualize keratin and laminin 332 protein distribution, respectively. Mechanical assessment of dermal adhesion and infiltration strength involved a peel test to quantify the attachment of the soft tissue to the scaffold.

Results, Conclusions, and Discussions:: Results, Conclusions, and

Discussion: Computational modeling of osseointegration-specific frame designs (N=3) revealed a need for prioritization of mechanical strength around the OI fixation point. Model metrics revealed inconsistent results across the human- and rabbit-scaled designs (hexagonal – Fig. 2A, concentric – Fig. 2B, control – Fig. 2C), highlighting a need for intentional modification to balance mechanical properties and material surface area during scaling. Displacement (Fig. 2D) was successfully limited aside from the human-scaled concentric design which simulated 3.8mm of movement (two orders-of-magnitude greater than the other models). The factor of safety (FoS) results (Fig. 2E), a ratio used to ensure that a structure can withstand loads greater than its intended use, were less than optimal. The rabbit-sized hexagonal design (FoS=0.09) and the concentric human design (FoS=0.48) both fellow below passing (FoS=1.0). Overall, relative mass measurements indicate room for improvement in construct scalability as the proportions vary widely between the two sizing modalities (Fig. 2F). Previous work has identified 5 and 7 wt/v% keratin as the ideal concentrations for incorporation in ESFs to maintain consistent average pore areas of 2.3 and 1.9μm2 respectively (data not shown), where ongoing characterization is chemically conjugating bioadhesive proteins (keratin and laminin) to the scaffolds for testing in a subcutaneous murine model (Fig. 3A-D). Analysis and visualization of the scaffold structure and tissue adhesion is currently being performed with the aforementioned cellular and protein-specific staining. Mechanical testing is being implemented to test the structural properties and functional performance of the adhesion to the scaffold. This work demonstrates the strength of composite implants, where optimization of material integration forms a complex, scalable construct to support tissue adhesion while retaining patient-specific geometry. Computational modeling facilitated rapid prototyping and efficient design evaluation needed to improve the mechanical efficiency of the final design, while the murine model provided mechanism-specific data. The addition of bioadhesives pursues a path forward to identify a sustainable, permanent adhesion mechanism throughout the OI implant’s lifespan. This work establishes a foundation for the application of tissue-engineered scaffolds in OI systems. Future work continues refining construct design for use in an established, clinically relevant OI rabbit model.

Acknowledgements and/or References (Optional):: 1. Sreedharan et al., 2021, “Osseointegrated prostheses for lower limb amputees: a review of complications”
2. Harrington et al., 2024, “Thighplasty at the Time of Stage-1 Bone- Anchored Osseointegration Surgery”
3. Biorender.com