A biomimetic engineered bone platform for advanced testing of prosthetic implants

The study addresses the need for reliable, human-relevant methods to predict osseointegration and mechanical stability of prosthetic implants. Current 2D cell culture, polyurethane foam, cadaveric bone tests, and animal models have limited biological relevance, poor predictive power, or ethical and cost constraints. Osseointegration depends on implant surface chemistry/topography and local biology; failures such as peri-implantitis motivate surface and material innovations that require better testing platforms. The authors hypothesize that a 3D biomimetic human bone platform engineered from human induced mesenchymal stem cells within decellularized trabecular bone scaffolds can recapitulate key molecular, cellular, and tissue-level events of osseointegration in vitro and predict material-dependent differences (titanium vs stainless steel) in integration strength observed in vivo.

Background literature indicates: (1) High clinical use of dental/orthopedic implants with ~95% success, yet failures (e.g., peri-implantitis) remain; surface modifications can mitigate risk. (2) Standard tests (PU foams, cadaveric bone, animal models) assess primary stability or provide partial biological relevance but are limited by species differences and poor in vitro–in vivo correlation. (3) Prior work by the authors established human iPSC-derived bone tissue systems and decellularized bone scaffolds as osteogenic substrates comparable to human bone. (4) Material-dependent outcomes are known: titanium generally achieves stronger direct bone anchorage than stainless steel. These observations justify a human 3D in vitro platform capable of molecular-to-macroscale assessment to improve prediction of clinical performance.

Study design: Model titanium (Ti; ASTM F67 GR4) and stainless steel (SS; 316L) M2 implants (2 mm diameter, 7 mm length; self-tapping edge 1.5 mm; 0.4 mm pitch; 2 mm outer thread; 0.225 mm depth) were fabricated with matched design and similar roughness. Surfaces assessed by optical profilometry (Wyko NT-1100, VSI mode) and SEM. Scaffolds: Decellularized trabecular calf bone plugs (8 mm diameter, cut to 5 mm height) were prepared via sequential washes (trypsin/EDTA, Tris/EDTA, SDS, DNase/RNase), ethanol dehydration, freeze-drying. Scaffold density calculated and matched (0.25–0.45 mg/mm³). Cells: Human induced MSCs (iMSCs; line 1013A) expanded in KO-DMEM-based medium with 10% FBS, bFGF, supplements. Seeding at 2×10^6 cells per construct via an optimized droplet method with constructs inverted to maximize attachment; seeding efficiency quantified. Platform assembly and culture: Sterilized implants were anchored into scaffolds; constructs were conditioned and then cultured 7 weeks in osteogenic medium (DMEM high-glucose + 10% FBS, ascorbic acid 50 μM, dexamethasone 1 μM, β-glycerophosphate 10 mM) with thrice-weekly medium changes. Assessments: - Cell viability/distribution: LIVE/DEAD assay at day 1 and week 7; epifluorescence/confocal imaging. - Histology: Non-demineralized (resin-embedded; Stevenel's blue/van Gieson) and demineralized (paraffin; H&E, Masson trichrome). Immunohistochemistry for osteopontin, osteocalcin, bone sialoprotein. - Molecular profiling: RNAseq of tissue post-pullout (TruSeq Stranded mRNA; HiSeq2500; STAR alignment to GRCh37; DESeq2 for differential expression). Enrichment via GOseq with GO, KEGG, Reactome; REVIGO reduction; IPA pathway analysis; STRING network analysis for ossification-related genes. - qRT-PCR: Expression of COL1A1, OPN, RUNX2, ALPL, IBSP, SOX9, PPARG normalized to GAPDH in bulk tissue and interface (implants after pullout). - Secreted factors and enzymes: Weekly glucose, lactate, pH (Vi-CELL MetaFLEX), LDH cytotoxicity assay; VEGFA and cytokines by ProcartaPlex (day 1, week 3, week 7); ALP activity (colorimetric, nM/h); osteocalcin (EIA, fg/mL). - Imaging and chemistry: MicroCT pre- and day 49 (SkyScan 1172; 6 μm voxel; BV/TV, Tb.N, Tb.Th). ToF-SIMS mapping of calcium at interface on resin sections. Post-pullout SEM and EDS for residual biological material and Ca/P mapping on implant surfaces. - Biomechanics: Pullout tests pre- and day 49 (Instron DynaMite 8841; axial tensile, 60 mm/min; max load as pullout strength). Linear regression of pullout vs scaffold density. - Statistics: Paired/unpaired Student’s t-tests with Bonferroni post-hoc where applicable; significance at p<0.05.

- Global transcriptomics: Clear separation of Ti vs SS groups (PCA). Differential expression identified 1,846 genes total: 1,003 upregulated in Ti and 843 in SS. Enrichment showed Ti favored cholesterol/ketogenic biosynthesis and lipid raft-associated genes (CAV1, CAV2, ERLIN2, PAG1), whereas SS favored glycolysis, cytoskeletal and cytokine signaling. Ossification-related DEGs: 33 overexpressed with Ti and 42 with SS; Ti enriched for canonical/non-canonical TGF-β and FGF2 signaling; SS enriched for Wnt, BMP, and IGF signaling. - Metabolism and media analytes: SS constructs consumed glucose faster (significant at weeks 2–3 and 7; p<0.001–0.003, and p=0.001), yet produced less lactate at weeks 2 (p<0.001) and 4 (p=0.001). Ti cultures exhibited lower medium pH at weeks 1 and 5 (both p<0.001) and higher LDH at all time points (p<0.005), consistent with higher turnover and aerobic glycolysis genes (HK2, PDK1) up in Ti. - Gene/protein expression: At week 7, bulk tissue qRT-PCR showed higher expression with SS for COL1A1 (p=0.01), OPN (p=0.01), BSP (p=0.03), and PPARG (p=0.01). VEGFA release was higher in SS on day 1 (p=0.02). Cumulative ALP activity and osteocalcin release were significantly higher with Ti over the entire culture (ALP p<0.005; OC p<0.01). - Mineralization: MicroCT after 7 weeks showed greater increases with Ti vs SS: BV/TV up to +79.16% vs +18.10%; trabecular number +28.66% vs +8.43%; trabecular thickness +69.72% vs +11.12%. ToF-SIMS detected calcium deposits on Ti only, indicating faster interfacial mineralization. - Biomechanics: Primary stability pre-culture was equivalent. After 7 weeks, secondary stability (pullout) increased significantly more for Ti (p=0.0224), with average maximum pullout force increases of +97.91% (Ti) vs +22.14% (SS). Pullout strength did not correlate with scaffold density, indicating biological origin of differences. Greater mineralization in Ti correlated with higher pullout strength. - Interface characterization: Post-pullout SEM showed more residual cellular material on SS surfaces; EDS confirmed Ca and P on both materials. Interface qRT-PCR suggested SS retained higher COL1A1 and OPN at the surface compared to bulk, consistent with delayed or atypical matrix maturation and limited mineralization.

The platform reproduced material-dependent differences in osseointegration observed in vivo, with titanium eliciting stronger secondary stability than stainless steel. Integrating molecular profiling, biochemical assays, imaging, and mechanics, the study connects pathway-level differences (Ti: TGF-β/FGF2, lipid rafts; SS: Wnt/BMP/IGF, glycolysis/cytoskeletal/cytokine signaling) to phenotypic outcomes in matrix composition, mineral deposition, and interface strength. Elevated ALP/osteocalcin and faster calcium deposition at Ti interfaces translated into substantially greater increases in BV/TV, trabecular metrics, and pullout force. The lack of correlation with scaffold density isolates biology as the driver of enhanced stability. The system provides mechanistic insight into implant–tissue interactions and offers predictive value for assessing osseointegration potential of new biomaterials and surface modifications in a human-relevant context, potentially reducing reliance on animal studies.

A biomimetic human bone platform was developed that anchors model implants into decellularized trabecular bone scaffolds seeded with human iMSC-derived osteogenic cells and cultured to generate bone tissue. The platform captures key molecular, cellular, and tissue-scale features of osseointegration and differentiates the performance of Ti versus SS implants consistent with preclinical and clinical observations. Ti induced pathways and phenotypes associated with enhanced mineralization and stronger secondary stability. This proof-of-concept demonstrates that 3D human stem cell-based models can elucidate mechanisms of integration/failure, evaluate antibacterial and drug-delivery surface strategies, and enable patient-specific testing to guide the design of implants with improved clinical outcomes and reduced animal use. Future work will extend the model to additional cell types and physiological factors to simulate more phases of peri-implant healing and remodeling.

- Single cell type (human iMSCs) used; inflammatory, angiogenic, and osteoclastic cells were not included. - Localized peri-implant phenomena modeled; systemic factors (e.g., hormonal status such as estrogen), mechanical loading, synovial fluid, blood supply, surrounding soft tissues, and anatomical site effects were not reproduced. - Microbiome effects, which can impair regeneration and drive peri-implant disease, were not considered. - As an in vitro model, long-term remodeling and whole-organism interactions are not captured. Planned future work includes incorporating immune/vascular cells, applying mechanical stimulation via bioreactors, considering systemic hormonal and microbiome influences, and testing pro-regenerative drugs.