A biomimetic engineered bone platform for advanced testing of prosthetic implants

The development of new prosthetic implants for dentistry and orthopedics, a multi-billion dollar market, is hindered by limitations in existing testing methods. Current methods, such as 2D cell cultures, fail to replicate the complex 3D architecture and cellular interactions of native bone tissue. These methods do not adequately assess the mechanical interaction between implant and tissue, making it difficult to predict osseointegration potential. While pullout testing in artificial materials or cadaveric bone provides some insight, preclinical animal models are resource-intensive and suffer from interspecies variability. The current study addresses this need by developing a biomimetic human bone platform that closely mirrors the *in vivo* osseointegration process, allowing for more accurate prediction of implant success. This platform builds upon previous work engineering functional bone tissue from human induced pluripotent stem cells (iPSCs). The researchers hypothesized that this new platform would accurately reflect the differences in osseointegration observed between titanium (Ti) and stainless steel (SS) implants *in vivo*, materials known to elicit distinct biological responses. The researchers selected these materials to validate the platform's predictive capabilities in replicating aspects of the *in vivo* response.

The literature extensively documents the importance of osseointegration—the formation of a strong bond between a prosthetic implant and surrounding bone tissue—for the long-term success of dental and orthopedic implants. This process involves a complex interplay of mesenchymal stem cells (MSCs), which migrate to the implant surface, differentiate into osteoblasts, and produce a mineralized bone matrix at the tissue-implant interface. Implant surface chemistry, topography, and design significantly influence osseointegration. While many implants achieve high success rates, existing testing methods are insufficient. Two-dimensional (2D) cell culture models lack the structural and functional complexity of the bone microenvironment, failing to capture crucial cell-cell and cell-matrix interactions. Animal models, although more representative of the *in vivo* environment, are costly, time-consuming, and limited by interspecies variations. Previous research has demonstrated the potential of biomimetic approaches using iPSCs to generate functional bone tissue *in vitro*. However, the application of this approach to evaluate prosthetic implant osseointegration remained unexplored, prompting the development of this novel platform.

The researchers engineered a biomimetic bone-implant testing platform by anchoring model Ti and SS implants (identical design and surface roughness) into decellularized cow bone scaffolds. Human iMSCs were seeded onto the scaffolds and cultured for seven weeks in an osteogenic environment containing osteogenic inducing factors. The use of decellularized cow bone scaffolds was justified by their similar trabecular architecture to human bone and their ability to support iMSC osteogenic differentiation. Human iMSCs were selected for their potential to model the *in vivo* response and avoid donor variability. To minimize variability, scaffolds of similar density were selected and cells were seeded using an optimized droplet technique ensuring high cell attachment. Multiple methods were then employed to evaluate the platform's effectiveness. **Cell distribution, viability and growth:** The distribution and viability of cells after one day and seven weeks were assessed using the LIVE/DEAD assay and visualized via epifluorescence and confocal microscopy. **Histology:** Non-demineralized and demineralized samples were examined using histological staining (Stevenel’s blue, van Giesson picrofuchsin, hematoxylin and eosin, Masson’s trichrome) and immunohistochemistry (osteopontin, osteocalcin, bone sialoprotein) to assess tissue formation, collagen fiber arrangement and bone matrix protein deposition. **RNA sequencing (RNAseq):** RNA was extracted from samples to identify differentially expressed genes (DEGs) between Ti and SS groups and to understand the underlying molecular mechanisms. Bioinformatic analyses (GO enrichment, KEGG pathway analysis, Reactome pathway analysis, Ingenuity Pathway Analysis (IPA)) were performed to interpret the functional significance of DEGs. **Bioanalyte analysis:** Glucose, lactate, and pH levels in the culture medium were measured weekly to assess cellular metabolic activity. **Lactate dehydrogenase (LDH) assay:** LDH levels in the culture medium were measured to assess cell death. **Multiplex immunoassay:** The release of various growth factors (VEGF-A, IL-2, IL-6, IL-8, MCP-1, and MIP-1β) was measured to understand their role in the osseointegration process. **Alkaline phosphatase (ALP) and osteocalcin assays:** ALP and osteocalcin release were assessed as markers of osteoblast activity and bone matrix mineralization. **Microcomputed tomography (µCT):** µCT was used to quantify tissue mineralization by assessing bone volume fraction (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb.Th). **Time-of-flight secondary ion mass spectrometry (ToF-SIMS):** ToF-SIMS was used to visualize calcium deposition at the tissue-implant interface. **Pullout test:** This biomechanical test was performed to measure the strength of the tissue-implant interaction. SEM and EDS were used to characterize implant surfaces after the pullout test. Real-time PCR (qRT-PCR) was used to assess the expression of osteogenic genes (RUNX2, COL1A1, ALPL, OPN) at the tissue-implant interface.

The biomimetic bone platform successfully recapitulated key aspects of the *in vivo* osseointegration process. RNAseq analysis revealed distinct molecular signatures for Ti and SS implants, with Ti implants upregulating TGF-beta and FGF2 pathways, while SS implants upregulated Wnt, BMP, and IGF pathways. µCT imaging showed significantly greater increases in BV/TV, Tb.N, and Tb.Th with Ti implants compared to SS implants, indicating enhanced tissue mineralization. ToF-SIMS confirmed that calcium deposition was more pronounced at the tissue-implant interface of Ti implants. Pullout testing demonstrated similar primary stability for both implant types, but Ti implants exhibited significantly higher secondary stability (97.91% increase in maximum pullout force versus 22.14% for SS implants), correlating with increased mineralization. Post-pullout analysis revealed more residual cellular material on SS implants, suggesting a different mode of tissue-implant interaction. qRT-PCR analysis showed differences in expression of key osteogenic genes at the tissue-implant interface of the two implant types.

The results demonstrate that the biomimetic bone platform accurately reflects the superior osseointegration potential of Ti implants compared to SS implants, as observed *in vivo*. The distinct molecular pathways activated by each implant material highlight the importance of considering material-specific biological responses in implant design. The increased mineralization and secondary stability observed with Ti implants likely contribute to its stronger bone integration. The platform's ability to reproduce these *in vivo* observations validates its potential as a powerful tool for preclinical evaluation of new implant materials and designs. The platform offers the potential to deeply investigate the biological aspects of osseointegration, potentially leading to the creation of novel implants with enhanced osseointegration properties.

This study successfully demonstrated a novel biomimetic human bone platform for *in vitro* evaluation of implant osseointegration. The platform accurately reproduced the differential osseointegration potential of Ti and SS implants observed *in vivo*, providing valuable insights into the underlying molecular and cellular mechanisms. This technology offers a powerful and ethically sound alternative to animal models in preclinical testing, accelerating the development of improved prosthetic implants with enhanced clinical outcomes. Future research should incorporate additional cell types (e.g., inflammatory cells, angiogenic cells), investigate the impact of systemic factors (hormones, microbiome), and study the effects of mechanical loading on the osseointegration process within the platform.

The current study's limitations include the use of only a single cell type (iMSCs), neglecting the contributions of other cell types involved in *in vivo* osseointegration (e.g., inflammatory cells, osteoclasts, endothelial cells). The platform does not encompass the complex systemic hormonal influences or the role of the peri-implant microbiome, both of which can affect osseointegration. The lack of mechanical loading in the current setup may also affect the results, as mechanical stimulation is crucial for bone remodeling *in vivo*. Future studies should address these limitations for more comprehensive evaluation of implant biocompatibility and performance.