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TBM (Figures 6J and K, 7I and J), indicating effective bone biocompatibility and biodegradability. Moreover, BCP,
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formation. a mixture of hydroxyapatite and tricalcium phosphate
Masson’s trichrome and Goldner’s trichrome staining in skeleton structures, combines the advantages of both
further confirmed that more new bone formation occurred calcium salts. As an inorganic component in bone tissue,
in defect regions treated with either bergamottin or BCP provides excellent biocompatibility and mechanical
36,37
miR-138-5p antagonist-loaded TBM (Figures 8A and strength.
B, 9A and B). The CD3 and CD68 immunohistochemical A random porous bone tissue material formed by
staining and TRAP staining revealed no significant freeze-drying cannot guarantee mechanical strength
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changes in inflammation and bone resorption in the comparable to that of natural trabecular bone. To
defect areas (Figures 8C-H, 9C-H). Meanwhile, RUNX2 address this limitation, we adopted a two-stage fabrication
immunohistochemical staining showed that either strategy during the design of TBM. In the first stage, the
bergamottin or miR-138-5p antagonist-loaded TBM material was lyophilized within a limited volume, forming
significantly enhanced osteogenic differentiation in bone a core skeleton (diameter = 0.7 – 3.0 mm) with enhanced
defect regions (Figures 8I and J, 9I and J). Collectively, mechanical strength due to water crystallization. In the
these results indicate that the TBM served as an excellent second stage, rapid freezing and lyophilization generated
bone-filling material. a multilayer pore structure (50 – 250 μm pore size),
closely resembling natural trabecular bone. This design
3.6. The therapeutic effects of the drug-loaded TBM significantly improved the TBM’s mechanical support
in fracture mouse model compared to a single-stage freeze-dried porous material.
To expand the potential applications of the TBM, we Thus, the TBM demonstrated sufficient strength derived
constructed a tibial fracture mouse model and implanted from covalent bonding between proteins in the material,
the TBM at the fracture sites. After 4 weeks, the mice were eliminating the need for crosslinking agents that might
humanely euthanized to evaluate the TBM’s therapeutic compromise biosafety. In addition, the number, size,
effects on fractures (Figure 10A). Micro-CT and calcein and distribution of micropores on the surfaces of bone-
AM/PI double staining revealed that the implantation of filling material significantly affect bone tissue metabolism
TBM loaded with either bergamottin or the miR-138-5p and proliferation. In general, pore sizes of 150 – 800 μm
antagonist significantly enhanced fracture repair, as well promote nutrient transport and metabolic waste excretion,
as increased BMD, BV/TV, BMC, and bone apposition whereas smaller pore sizes of 40 – 100 μm support non-
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rates at the fracture regions (Figures 10B-G). These mineralized tissue growth. As the size of the micropores
results demonstrate that the TBM can serve as a potential on the TBM’s surfaces falls within the range of 50 – 250 μm,
therapeutic platform for fracture repair, mediated through it demonstrates great potential to promote cell growth and
the slow release of osteogenic drugs. tissue regeneration while withstanding stress (Figure 1).

4. Discussion Hydrogels are highly favored in tissue engineering due to
their excellent plasticity, biocompatibility, and drug-loading
At present, the treatment of bone defects mainly relies on capacity. 39,40 The application of hydrogels to bone-filling
the surgical implantation of bone-filling materials and bone materials may enhance their toughness, biocompatibility,
healing via intrinsic repair capability. Ideal bone substitutes and drug delivery capacity. HAMA hydrogels, in particular,
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for defect repair must not only exhibit degradability and are widely used for cell and drug encapsulation due to
biocompatibility but also possess sufficient mechanical their spatial structures, which make them suitable for
strength to provide structural support. In addition, they cell growth and provide excellent biocompatibility and
should promote osteogenic differentiation and angiogenesis degradability characteristics. 41,42 HAMA synthesis involves
to accelerate bone formation and repair processes. Although crosslinking water-soluble HAMA with the DMSO-
autologous bone transplantation is the current gold standard soluble photoinitiator I2959, enabling the simultaneous
for bone defect treatment, it is not widely used due to donor encapsulation of both water-soluble and organic-soluble
scarcity. In this study, we designed and synthesized a drugs for modular drug delivery. In our study, HAMA was
6,7
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bone-filling material, termed TBM, which closely mimics used to encapsulate the TBM’s porous skeletons and load
the structure and composition of natural trabecular bone. either the organic-soluble bergamottin or the water-soluble
The TBM is a composite material consisting of a central miR-138-5p antagonist. 32,33 This design endowed the TBM
porous framework and a peripheral hydrogel. The porous with long-term, slow-release drug properties, promoting
framework is composed of chitosan, collagen, BCP, and silk osteogenic differentiation and demonstrating the potential
fibroin. Apart from the silk fibroin, the other components for treating various bone defects (Figures 2, 4-6).
are naturally present in bone tissue, which minimizes the To further enhance the TBM’s therapeutic efficacy in
risk of foreign body reactions and enhances the TBM’s bone defect repair, we explored its potential as an organoid.

Volume 1 Issue 2 (2025) 14 doi: 10.36922/OR025040003