**1. Introduction**

There have been many developments in engineering biocompatible and biodegradable bone implant sca ffolds for use as an alternative to autografts and allografts for bone defects over the last couple of decades, especially when bone defects are large and donor morbidity is a risk [1–4]. Bone sca ffolds have been extensively studied to promote native bone tissue growth and surrounding cell

proliferation by optimizing nutrient transportation and mimicking native mechanical properties while minimizing damage to the surrounding tissues [1,2,5–7]. Bone scaffolds have been constructed using various materials such as metals, bioglasses, ceramics, and polymers, and are typically fabricated from a composite of the latter two [1,2,5,7]. Specifically, β-tricalcium phosphate (β-TCP), a polymorph of tricalcium phosphate and a biomimetic ceramic, and polycaprolactone (PCL), a biocompatible polymer, are two commonly used, clinically available biodegradable materials in bone scaffold engineering. TCP has a comparable resorption rate to bone regeneration [1,8,9]. Additionally, when compared to five other commonly used FDA-approved poly(α-hydroxy esters), PCL was one of two that demonstrated the best structural integrity and cellular response [10].

The composite construct of β-TCP and PCL combines the respective benefits of each: osteoconductivity—or bone growth on a surface such as an implant scaffold [11–14]—and easy handling, both of which have only begun to be explored in further depth. The β-TCP/PCL composite is composed of the osteoconductive β-TCP ceramic particles suspended in the bioresorbable PCL polymer matrix [15,16]. The composite material's ability to be extruded into a filament and then 3D-printed enables the creation of controlled, patient-specific scaffolds to optimize its integration within and support for native bone tissue regeneration [17]. In addition to 3D printing, other scaffold fabrication methods include electrospinning, solvent casting, particle leaching, thermally-induced phase separation, and various molding techniques [5,6,18].

Although various factors for optimizing bone scaffolds have been studied, examining the degradation profiles of these constructs is particularly crucial for evaluating the success of bone implants for clinical applications. A bone scaffold should subsist long enough to induce the maximum therapeutic effect at the bone defect site, but also degrade when healing is underway. Poly (α-hydroxy esters)—and by association, composites with polymers in this group—undergo hydrolytic degradation via two methods: surface or bulk [19]. Ideally, degradation and resorption times for bone scaffolds should match bone regeneration rates, depending on the bone defect size. For large bone defects, the degradation and resorption duration for bone scaffolds can be greater than two years [20]. Slow-degrading scaffolds have been shown to prevent tears, allow a slow reintegration of movement, and minimize toxicity at the site of interest when compared to fast-degrading scaffolds [21].

Since sterilization is necessary for the clinical realization of a bone scaffold, it is important to then study how sterilization may change degradation, which further affects the structural integrity and mechanical profiles of bone scaffolds. Various sterilization methods exist for bone scaffolds, including heat-based ethylene oxide immersion and irradiation via ultraviolet, gamma, and electron beam (E-beam) irradiation [22]. Submersion in solvents, such as 70% ethanol, has also been used to sterilize scaffolds, but is insufficient as a sterilization method alone because ethanol has minimal sterilizing power over bacterial spores [22]. Out of all of these methods, E-beam is the most optimal for pre-packaged biomaterials with low melting points, which is relevant for β-TCP/PCL scaffolds [22]. Additionally, E-beam has higher dosage rates than both ultraviolet and gamma irradiation methods, resulting in less exposure time [22]. This is particularly important for polymers like PCL, because irradiation methods like E-beam and gamma have been shown to increase the polydispersity of PCL chains and affect mechanical properties and degradation rates [22–24]. This is a result of PCL ester–ester chain scissioning, in addition to crosslinking, or the formation of chemical bonds to connect polymer chains [23,24].

In our previous study, we found a 14% increase in the initial Young's modulus and a 25% faster in vitro degradation profile for scaffolds that received E-beam compared to those that did not [23]. The increased Young's modulus values after E-beam were likely due to crosslinking, which strengthens the β-TCP/PCL composite structure, while the increase in degradation rate after E-beam in vitro was likely due to chain scissioning, which is thought to weaken the composite structure [23]. Furthermore, since β-TCP particles are merely suspended in the polymer matrix, degradation of β-TCP/PCL scaffolds in any given solution is mainly driven by polymer degradation via the hydrolytic cleavage or scissioning of ester–ester linkages [19,20,23,25]. Previous studies, including ours, have

focused solely on 20% TCP/80% PCL [19,20,23,26,27], so this study extends the work by examining the in vivo degradation profiles of various β-TCP/PCL composite ratios by mass (0:100, 20:80, 40:60, and 60:40) in a rat model, particularly studying the effect of E-beam sterilization among these different ratios on in vivo degradation. We have chosen to use extruded filament samples over scaffold samples for this in vivo study for simplification and as a screening test for chemical compositions. While we recognize that scaffolds confer additional properties, such as porosity, that can also influence degradation, the main purpose of our in vivo study is to test how the chemical composition and E-beam affect degradation. This can be achieved using extruded filament samples, while also saving time and cost. In addition, these extruded filaments can help predict the degradation of extrusion-based printed devices and grafts.

### **2. Materials and Methods**
