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Biomaterials

Biomaterials - Poster Session C & D

J2 - Response of Osteoblasts to Cellulase-Based Plant Scaffold Degradation Methods for Tissue Engineering Applications

Friday, October 10, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Despite recent advances in fabrication technology, synthetic scaffolds for tissue engineering are limited by their inability to recapitulate the structural complexities of natural extracellular matrices (ECMs) that provide structural cues to regulate cell-matrix interactions and tissue formation. Decellularized plant scaffolds with physical and structural similarities to natural ECMs have recently emerged as viable platforms for cell proliferation and tissue engineering. For instance, decellularized bamboo scaffolds retain their complex hierarchical structures (dense exterior, less dense interior), which resemble the native bone ECM, and maintain their structural and mechanical cues. Prior studies have demonstrated that decellularized bamboo scaffolds support the proliferation of osteoblasts and bone tissue engineering. Although attractive, the primary challenge in using decellularized plant scaffolds lies in their limited degradation in human tissues due to the lack of cellulases to attack the β-1,4 linkages in cellulose, the primary component of plant structures. Scaffold degradation is crucial to the success of implantable scaffolds for tissue regeneration, which requires scaffolds to degrade at controlled rates that match tissue formation and remodeling. As a result, the design of plant scaffold degradation strategies has garnered intense interest, with various studies proposing cellulase-based methods to regulate degradation. However, little is known about the effects of these methods on the viability and tissue-production capacity of cells. Hence, in this study, we present the preliminary responses (cell viability and ECM secretion) of osteoblasts to two cellulase-based degradation strategies, namely, free cellulase and sustained-release of cellulase from PLGA microparticles.

Materials and

Methods: : Initially, cellulase from Aspergillus niger (Sigma-Aldrich) was dissolved in citrate buffer (pH 5.0) to prepare a 10 mg/mL stock solution. The cellulase was added to a PLGA solution in dichloromethane and homogenized to produce PLGA-cellulase microparticles in a single-emulsion process. The microparticles were then washed with deionized water to remove residual solvents and lyophilized to obtain powdered microparticles. Afterward, the release profile of cellulase from the PLGA-cellulase microparticles was studied under simulated physiological conditions (phosphate-buffered saline, pH 7.4, 37°C). The daily amount of cellulase enzyme released was measured using the BCA protein assay. We then investigated cell response to two cellulase-based strategies: (a) free cellulase (0.5 mg/mL and 1.0 mg/mL) and (b) sustained release of cellulase from PLGA-cellulase microparticles. To achieve this, human fetal osteoblasts (hFOB) were cultured in 24-well plates with growth medium (DMEM/F-12 supplemented with 10% FBS) containing (i) no additions (control), (ii) 0.5 mg/mL cellulase, (iii) 1.0 mg/mL cellulase, and (iv) 1.0 mg/mL PLGA-cellulase microparticles. Cell viability and the amount of ECM protein produced were measured using the alamar blue assay and BCA protein assay (Thermo Fisher) after 0, 24, 48, and 72 hours of treatment. Flow cytometry analysis was conducted to determine the mechanism of cell death.

Results, Conclusions, and Discussions:: The PLGA-cellulase microparticles were spherical, as shown in the SEM image in Figure 1A. Under simulated physiological conditions, the release profile of cellulase from the PLGA-cellulase microparticles (Figure 1B) was almost linear, suggesting the sustained release of approximately equal amounts of cellulase daily. Upon exposure of the osteoblasts to free cellulase (0.5 and 1.0 mg/mL) and 1.0 mg/mL PLGA-cellulase microparticles, there were no significant differences in cell viability among the treatment groups and the control for 48 hours (Figure 2A). After 72 hours, the viability of cells treated with free cellulase PLGA-cellulase microparticles was significantly lower. However, cells treated with 0.5 mg/mL free cellulase exhibited higher viability than those exposed to 1.0 mg/mL free cellulase and 1.0 mg/mL PLGA-cellulase, with the latter two having similar viabilities. This suggests that treatment of osteoblasts with increased cellulase concentrations decreased cell viability proportionally. Flow cytometry analysis (Figure 2B&C) indicated that cell death was predominantly due to necrosis with free cellulase, while sustained cellulase release induced apoptosis. To determine the effects of the cellulases on the ECM production capacity of osteoblasts, we measured the amount of ECM proteins secreted (Figure 3). After 24 hours of exposure, there were no significant differences in de novo protein content between the treatment groups. The protein content of the control (cells only) increased slightly after 48 hours, while that of the cells treated with free cellulase and PLGA-cellulase was significantly lower. However, after 72 hours of treatment, there were no differences between the cells only (control) and cells treated with 0.5 mg/mL free cellulase and PLGA-cellulase microparticles, while treatment with 1.0 mg/mL free cellulase significantly decreased the amount of proteins secreted by the osteoblast cells. This suggests that the exposure of osteoblasts to lower cellulase concentrations did not significantly influence their secretion of ECM proteins. Collectively, these preliminary results demonstrate that the ECM protein secretion capacity of osteoblasts over the short term is not significantly affected by low cellulase concentrations (either in the free form or slowly released from PLGA microparticles), although they significantly decrease cell viability. Future work will explore long-term effects on osteoblasts.

Acknowledgements and/or References (Optional): :