Aksu, A. | Search bioRxiv

bioRxiv2026

Reconstruction of critical-sized mandibular defects in a sheep model using a PLLA-PGA-CC scaffold

IntroductionCritical-sized bone defects cannot heal spontaneously, requiring additional, often burdensome, treatment. Thus, various synthetic substitute materials have been investigated regarding their treatment capacity. Poly-L-lactic acid (PLLA) and polyglycolic acid (PGA) have emerged as promising biodegradable scaffold materials. The addition of inorganic materials such as calcium carbonate (CC) has also been shown to be advantageous. This study investigates the effect on bone regeneration of PLLA-PGA-CC scaffolds in critical-sized bone defects over a two-year observation period using sheep as an animal model. MethodsCritical-sized mandible angle defects were created in twelve female merino sheep. Mandibular defects were reconstructed with PLLA-PGA-CC scaffolds in four sheep, while the remaining eight served as negative control (defects left empty). The scaffolds were manufactured using computer-aided design and manufacturing, incorporating an interconnected porous structure and fixated with polyether ether ketone cages. Bone regeneration was evaluated using computed tomography (CT) imaging at 3, 12, and 24 months postoperatively. Bone volume was assessed quantitatively. Additionally, a histological analysis was performed. ResultsSurgical procedures were successful and without major complications. CT assessment showed more bone regeneration in the scaffold group (mean volume: 7,472 mm3) than in the control group (4,168 mm3, p = 0.1) at 24 months postoperatively. Resorption of the scaffolds and formation of compact lamellar bone tissue were confirmed by histological analysis. However, the osteoconductive properties of the scaffolds were limited, with only minimal ingrowth of bone tissue into the porous structure. In both groups, fibrous tissue infiltration and the formation of cyst-like cavities in the defect region were observed. ConclusionPLLA-PGA-CC scaffolds were found to be biocompatible and enhanced bone regeneration compared to the control group. Due to fibrous tissue infiltration and the lack of osteoconductivity, the suitability of the material for critical-sized bone defect reconstruction is limited.

bioengineering↗

bioRxiv2026

Fibrin Selective Alteplase with Improved Thrombolysis and Inhibition Resistance Engineered by Rational Design

Thrombolytic enzymes represent an important class of proteolytic biocatalysts for medical applications, yet currently used FDA-approved variants, including alteplase and tenecteplase, remain limited by suboptimal catalytic efficiency, off-target activity, and susceptibility to inhibition. These limitations reflect the complexity of enzyme function in physiological environments, where therapeutic performance depends on the simultaneous optimization of multiple catalytic and biophysical properties. Here, we introduce a multi-objective enzyme engineering strategy for the design of next-generation thrombolytic proteases, explicitly targeting multiple properties required for therapeutic performance. Our approach combines computer-aided design, evolutionary reconstruction, and literature-guided mutation selection to improve catalytic activity, fibrin selectivity, inhibition resistance, and functional lifetime within a single workflow. This framework is coupled with systematic biochemical characterization, in vitro evaluation of clot penetration and fibrinolytic activity, and in vivo validation of efficacy and safety. By addressing multiple performance parameters simultaneously, this strategy enables efficient navigation of trade-offs that typically limit enzyme optimization. Using this approach, we identify Brnoteplase as a lead variant with enhanced fibrin selectivity, improved resistance to inhibition, and superior clot penetration, resulting in increased effective catalytic lifetime and enabling bolus administration. In vivo studies demonstrate enhanced thrombolysis and recanalization with reduced hemorrhagic complications. These findings provide a broadly applicable framework for designing proteolytic biocatalysts suitable for complex biological environments.

biochemistry↗