Key Takeaways & Executive Findings
- •• • 3.0% CB/BSA hydrogel achieved the highest bone volume fraction, bone mineral density, and trabecular thickness at 8 weeks (P<0.05), while reducing bone surface-to-volume ratio (P<0.05), indicating superior microarchitectural restoration compared to lower CB fractions and Bio-Oss® Collagen. • • The 3.0% CB/BSA group upregulated WNT3A, β-catenin, VEGFA, COL1A1, PECAM-1, and OCN at both gene and protein levels (P<0.05), confirming Wnt/β-catenin pathway activation as the mechanistic driver of osteogenesis and angiogenesis. • • No abnormalities in blood routine or serum liver/kidney function markers and no organ inflammation, necrosis, or fibrosis were observed, establishing a favorable in vivo safety profile for the photocrosslinked hydrogel. • • The 3.0% CB/BSA formulation exhibited the best overall healing outcome, suggesting that its degradation kinetics—via hydroxyapatite dissolution and BSA network hydrolysis—are temporally matched to bone regeneration, though dynamic degradation monitoring remains necessary.
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Abstract
Critical-sized bone defects (CSBD) remain a clinical bottleneck due to insufficient osteogenic drive and uncontrolled degradation of current grafts. This study evaluates a photocrosslinked hydrogel composed of cuttlebone (CB) and bovine serum albumin (BSA) for repairing 5 mm rat calvarial CSBD. SD rats were randomized into control, positive control (Bio-Oss® Collagen), BSA, 0.5% CB/BSA, 1.5% CB/BSA, and 3.0% CB/BSA groups (n=6). After 8 weeks, micro-CT revealed no new bone in controls, whereas all CB/BSA groups exhibited significant increases in bone volume fraction, bone mineral density, and trabecular thickness (P<0.05), with reduced bone surface-to-volume ratio (P<0.05). Histology confirmed new bone formation in hydrogel groups versus loose fibrous tissue in controls. Immunohistochemistry and immunofluorescence showed elevated COL1A1, PECAM-1, and OCN expression (P<0.05). qRT-PCR and Western blotting demonstrated upregulation of WNT3A, β-catenin, VEGFA, COL1A1, PECAM-1, and OCN (P<0.05). The 3.0% CB/BSA group exhibited the most pronounced osteogenic effect. Blood routine and serum liver/kidney function tests showed no abnormalities, and major organs displayed no inflammation, necrosis, or fibrosis. These findings indicate that CB/BSA photocrosslinked hydrogel promotes bone repair with favorable in vivo safety, likely through activation of the Wnt/β-catenin signaling pathway.
1. Introduction
Critical-sized bone defects (CSBD) present a persistent clinical challenge: autografts are limited by donor site morbidity and insufficient volume, while allografts and synthetic substitutes often fail to integrate due to poor osteoinductivity, uncontrolled degradation, or inadequate vascularization. Commercial products such as Bio-Oss® Collagen provide osteoconductive scaffolds but lack the bioactive cues needed to drive robust osteogenesis and angiogenesis, resulting in fibrous encapsulation rather than functional bone regeneration.
This study addresses the bottleneck by engineering a photocrosslinked hydrogel combining cuttlebone (CB) and bovine serum albumin (BSA). CB serves as a source of Ca2+, Sr2+, and Mg2+ ions that stimulate osteogenic signaling, while the methacrylated BSA network provides a tunable, biodegradable matrix. The hypothesis is that CB/BSA hydrogel activates the Wnt/β-catenin pathway, upregulating WNT3A, β-catenin, VEGFA, COL1A1, PECAM-1, and OCN to accelerate bone healing. The 5 mm rat calvarial CSBD model was used to evaluate efficacy, mechanism, and systemic safety over 8 weeks.
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WU Sixian, ZHUO Tao, WANG Guowei, LI Mengying, HE Yi, LIU Jianhang (2026). Effect and Mechanism of Cuttlebone/Bovine Serum Albumin Photocrosslinked Hydrogel on Promoting Bone Healing. Chinese Traditional and Herbal Drugs. https://doi.org/10.7501/j.issn.0253-2670.2026.16.20261612
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Frequently Asked Questions
What is the degradation mechanism of the CB/BSA hydrogel, and how does it match the rate of bone regeneration?
The hydrogel degrades through two concurrent processes: dissolution and ion exchange of hydroxyapatite in CB, releasing Ca2+, Sr2+, and Mg2+; and enzymatic/hydrolytic cleavage of the methacrylated BSA network. The 3.0% CB/BSA group showed optimal healing at 8 weeks, suggesting temporal coordination between degradation and osteogenesis. However, dynamic monitoring of mass loss, morphology, and spatiotemporal bone formation is required to quantify the degradation-regeneration match.
How does the osteogenic efficacy of 3.0% CB/BSA compare to the commercial positive control Bio-Oss® Collagen?
Micro-CT and histology demonstrated that CB/BSA groups, particularly 3.0% CB/BSA, significantly increased bone volume fraction, bone mineral density, and trabecular thickness (P<0.05) and reduced bone surface-to-volume ratio (P<0.05) compared to controls. While the positive control also supported healing, the 3.0% CB/BSA group exhibited the most pronounced upregulation of osteogenic and angiogenic markers (WNT3A, β-catenin, VEGFA, COL1A1, PECAM-1, OCN), indicating superior bioactive performance over the commercial graft.
What is the evidence for Wnt/β-catenin pathway activation, and are there off-target risks?
qRT-PCR and Western blotting showed significant upregulation of WNT3A and β-catenin in CB/BSA groups (P<0.05), with downstream targets COL1A1, PECAM-1, VEGFA, and OCN also elevated. No systemic toxicity was observed: blood routine, liver/kidney function, and organ histology remained normal. However, localized sustained Wnt activation requires long-term surveillance for potential ectopic ossification or oncogenic risk, which was not assessed beyond 8 weeks.
What are the scalability and manufacturing challenges for CB/BSA photocrosslinked hydrogel?
The hydrogel requires methacrylation of BSA, uniform dispersion of CB particles, and photocrosslinking under controlled light exposure. Scale-up must ensure consistent CB particle size, homogeneous mixing, and reproducible crosslinking density to maintain mechanical integrity and degradation profiles. Sterilization and shelf-life stability of the protein-based network are additional bottlenecks. No industrial-scale validation data are presented; current evidence is limited to small animal models.
Does the 3.0% CB/BSA hydrogel support angiogenesis, and how is it quantified?
Yes. Immunohistochemistry and immunofluorescence showed increased PECAM-1 (CD31) positive expression and fluorescence intensity in CB/BSA groups (P<0.05). qRT-PCR and Western blotting further confirmed upregulation of PECAM-1 and VEGFA (P<0.05), indicating enhanced angiogenic signaling. This is critical for bone regeneration, as vascularization supplies oxygen and nutrients to the defect site.
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