(A-31) Decellularized lucky bamboo scaffolds as viable platforms for bone regeneration.

Introduction:: Many of the proposed alternative strategies to bone grafts for repairing non-union bone defects rely on developing synthetic scaffolds for bone regeneration. However, despite recent advances in fabrication technology, synthetic scaffolds developed for bone regeneration are limited by their structure and scale-up/cost implications. With its well-organized, hierarchical, and functionally graded structure and potential for large-scale cultivation for bone regeneration, lucky bamboo (Dracaena sanderiana) offers a cost-effective, scalable “green” alternative for scaffold development. It has been shown in previous studies^1,2 that human cells can grow on decellularized plant scaffolds vis-à-vis cross-kingdom interactions. However, processes to engineer the long-term culture of osteoblasts, regenerate functional bone tissues and facilitate plant scaffold biodegradation to develop lucky bamboo into a viable platform for bone regeneration are yet to be reported. Hence, in the first part of this study, we demonstrate that decellularized lucky bamboo scaffolds could support the long-term growth of human fetal osteoblasts and the regeneration of bone tissues under in vitro and in vivo conditions. In the second part, the degradation of the decellularized scaffolds is engineered using injectable cellulase-encapsulated poly(lactic-co-glycolic acid) (PLGA) microspheres, as human enzymes have limited ability to degrade cellulosic materials.


Materials and Methods:: Lucky bamboo stems were decellularized using detergents (sodium dodecyl sulfate and Triton X-100), washed with deionized water and Tris buffer, and functionalized with dopamine-conjugated RGD peptides (RGDOPA). Human fetal osteoblast cells (hFOB) were subsequently cultured for 28 days on the scaffolds. Cell proliferation (DNA content assay and cell metabolic activities), alkaline phosphatase activity, de novo total protein content (BCA assay), de novo calcium content (calcium colorimetric assay), alizarin red S staining, and scanning electron microscopy were used to assess in vitro osteogenesis. Subcutaneous implantation of the scaffolds (seeded with hFOB cells and containing 12 µg/ml rhBMP-2) into athymic nude mice for 12 weeks (following an approved IACUC protocol), followed by ex vivo histopathological studies (H & E, alizarin red S, and trichrome staining) was used to evaluate the in vivo bone regeneration potential of the decellularized scaffolds. In the second part, cellulase-encapsulated poly(lactic-co-glycolic acid) (PLGA) microspheres were fabricated using the single-emulsion technique. The sustained release of the cellulase enzymes from the PLGA microspheres in phosphate-buffered saline (pH 7.4, 37°C) was monitored, and the ensuing enzymatic degradation of the decellularized scaffolds was assessed via changes in weight and compressive mechanical properties.


Results, Conclusions, and Discussions:: The hFOB cells attached and proliferated on the decellularized bamboo scaffolds. Cell proliferation, measured by the percentage alamar blue reduction (Figure 1) and DNA content, increased on the scaffolds with time. On the other hand, ALP activity increased gradually up to day 21, corresponding to the onset of osteogenesis, before declining on day 28. ECM production, quantified by the amount of de novo proteins on the scaffolds, also gradually increased with time due to the increase in cell population and functionality. The amount of de novo calcium increased significantly from day 21, indicating the onset of significant mineralization of the ECM produced by the hFOB cells. Overall, decellularized scaffolds functionalized with RGD exhibited a significant increase in seeding efficiency versus untreated scaffolds, which translated into improved osteogenesis metrics (cell proliferation, ALP activity, protein content, and calcium content).

In the in vivo study, over a period of 12 weeks, the cells colonized the scaffolds and produced tissues with calcified deposits as shown in the ex vivo histopathological studies, including H & E staining (Figure 2 a, b), alizarin red S staining (Figure 2 c, d) and trichrome staining. The results showed the regeneration of mineralized bone-like tissues on the decellularized scaffolds. However, their slow degradation under physiological conditions could lead to a mismatch between bone formation and scaffold degradation and compromise the mechanical integrity of the defect site. As a result, we designed cellulase-encapsulated PLGA microspheres to be injected into the defect site to accelerate scaffold degradation after implantation. About 80% of the enzymes were released from the PLGA microspheres in PBS (pH 7.4, 37°C) over 6 weeks. The scaffolds lost almost 70% of their weight over 6 months (Figure 3a) and 97% of their compressive strength over 14 weeks (Figure 3b) when exposed to the cellulase-encapsulated microspheres, demonstrating that we could engineer controlled biodegradation of the decellularized scaffolds. The results of this study provide evidence for using decellularized lucky bamboo scaffolds as viable platforms for long-term osteoblast culture and bone regeneration, which could potentially circumvent the challenges associated with conventional scaffolds in an environmentally and economically sustainable way.


Acknowledgements (Optional): :

References (Optional): : 1. Fontana G et al. Adv. Healthcare Mater. 2017, 6, 1601225.

2. Gershlak JR et al. Biomaterials 2017, 125, 13-22.