G9 - Composite Biphasic Scaffolds for Bone Regeneration: Biomimetic Matrix for Enhanced Bioactivity and Healing

Introduction: : Critical-sized bone defects remain a major clinical challenge, as current grafting strategies such as autografts and allografts are limited by donor-site morbidity, poor integration, and risk of disease transmission. Bone graft substitutes aim to overcome these issues by enhancing structural and transport properties to support tissue ingrowth1. Although engineered scaffolds provide appropriate mechanical strength and pore-architecture, they often lack sufficient bioactivity to promote mineralized matrix formation and osteointegration. Bioactive additions such as calcium phosphate (CaP), mesenchymal stem cells (MSCs), and growth factors like BMP-2 can improve outcomes but frequently result in bone formation only along the scaffold periphery, leaving the core inadequately mineralized2. We previously reported spiral-structured scaffolds incorporating nanofibers and nanohydroxyapatite (nHAP) to improve transport and osteogenesis3. However, material-based cues alone are insufficient, and growth factor-based strategies pose challenges with dosing and release kinetics. To address these limitations, we developed a biphasic scaffold that combines a mechanically robust and highly porous layered polycaprolactone (PCL) tube (Fig. 1A) with osteogenic and bioactive features. The scaffold surface was coated with CaP to enhance osteoinduction, while the core was filled with PCL nanofibers laden with decellularized extracellular matrix (dECM). The dECM was derived by culturing fibroblasts and MSCs on electrospun PCL nanofibers under osteogenic conditions, followed by decellularization (Fig. 1A). We hypothesize that the dECM-filled core will act as a chemotactic and osteogenic cue to promote cell infiltration through the porous PCL tube, enhancing osteogenesis and host tissue integration. This approach was evaluated in a rat femoral critical-sized defect model (Fig. 1B).

Materials and

Methods: : The biphasic scaffold was fabricated using combined micro and macro fabrication techniques, as illustrated in Fig. 1. Highly porous polycaprolactone (PCL) sheets with 100 µm longitudinal channels were produced using a custom mold and salt-leaching method. Approximately 10 layers of these sheets were stacked and spirally wrapped around a 21G needle to form a cylindrical structure. The scaffold ends were sealed using dichloromethane (DCM), creating a tubular scaffold 5 mm in length, with an outer diameter of 5 mm, an inner diameter of 0.84 mm, and a wall thickness of 2.08 mm. The scaffold’s core was filled with a bioactive matrix composed of decellularized extracellular matrix (dECM). The dECM was prepared by culturing rat dermal fibroblasts and MSCs on electrospun PCL nanofibers (NF+Hyb) in osteoinductive media for 21 days, followed by decellularization to remove genetic material4. This cell-derived matrix was rolled and inserted into the scaffold core to enhance bioactivity. The assembled biphasic scaffold was coated with bovine serum albumin (BSA) and immersed in simulated body fluid to promote calcium phosphate (CaP) biomineralization. Scaffolds were characterized for physicochemical properties and in vitro bioactivity by culturing rat MSCs for osteogenesis. For in vivo evaluation, the scaffolds were implanted into 10 mm critical-sized femoral defects in 63 skeletally mature male Sprague-Dawley rats, divided into seven groups as summarized in Table 1 (n=9/group). Bone healing was monitored using x-ray and micro-computed tomography (μCT), compressive mechanical strength and histological analyses (H&E, Masson’s trichrome, Safranin O, and IHC) assessed new tissue formation and scaffold integration.

Results, Conclusions, and Discussions:: Results and

Discussion: The scaffold, coated with bovine serum albumin and mineralized in simulated body fluid, exhibited high porosity (89.6%) and a compressive modulus of 123 MPa, making it suitable for load-bearing bone defect applications. The hybrid decellularized extracellular matrix (dECM) demonstrated low DNA content (4.3 ± 0.2 ng/cm²) and high total protein levels (42.1 µg/cm²), confirming preserved bioactivity. Among the various cell laden ECMs generated by an alone or combination of osteoblasts (OST), MSCs, chondrocytes (CH), endothelial (EC) and fibroblasts (FB), the FB/MSC combination showed enhanced osteogenic activity and produced significantly higher mineralized matrix synthesis by the cultured rat MSCs in vitro during 4 weeks (Fig. 2). All animals remained healthy throughout the study. At 12 weeks post-implantation, regenerated bone was harvested, and all defects remained closed except in the defect-only control group. The test group G1 (HS-CaP+NF-Hyb), which combined CaP coating with hybrid dECM, achieved complete defect bridging, confirmed by x-ray, micro-computed tomography, and mechanical testing. G1 exhibited significantly higher compressive strength (19.1 MPa), bone volume, and qualitative healing scores compared to all controls and even the gold-standard autograft (Table 1). Micro-CT analysis showed the highest bone volume (475.8 mm³) and mineral density (589.6 mg/ccm HA) in G1, indicating effective defect bridging and mineralization. Histological staining (H&E, Masson’s Trichrome, Safranin O) revealed abundant osteoid, collagen, and cartilage matrix in G1 and G4 (HS+NF-Hyb), indicating robust endochondral ossification. Immunohistochemistry showed significantly elevated collagen type I and bone sialoprotein in G1 and G4, confirming active osteogenesis. Scaffolds lacking either CaP or dECM showed inferior outcomes, highlighting the synergistic benefit of both. G1 closely resembled native bone, outperforming autografts and demonstrating strong potential for clinical translation in repairing large, load-bearing bone defects.

Conclusions: Engineered biphasic scaffolds incorporating both CaP and dECM significantly enhanced bone regeneration, demonstrating superior bone quality and volume compared to autografts. The optimized scaffold architecture and bioactive composition effectively promoted tissue integration and mineralization. These biomaterial-based scaffolds show strong potential as a reliable alternative to biological grafts for repairing large bone defects in both military and civilian settings.

Acknowledgements and/or References (Optional):: Dr. Kumbar acknowledges funding support from the U.S. Army Medical Research Acquisition Activity (USAMRAA) through the CDMRP Peer-Reviewed Medical Research Program (Award Nos. W81XWH2010321, PR230581, and HT94252410137), as well as the Department of Defense (Award No. HT94252410944, BA230234). Additional support was provided by the National Institutes of Health (Award Nos. R01NS134604, R01EB034202, R01AR078908, and R01EB030060).

References
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4. W.Z. Wang, X.; Yin, Z.; Yu, X. , Fabrication and Evaluation of Porous dECM/PCL Scaffolds for Bone Tissue Engineering, J. Funct. Biomater. 14 (2023) 343. https://doi.org/doi.org/10.3390/jfb14070343.