Capacitive interdigitated system of high osteoinductive/conductive performance for personalized acting-sensing implants

Replacement orthopedic surgeries are among the most common surgeries worldwide, but clinically used passive implants cannot prevent failure rates and inherent revision arthroplasties. Optimized non-instrumented implants, resorting to preclinically tested bioactive coatings, improve initial osseointegration but lack long-term personalized actuation on the bone–implant interface. Novel bioelectronic devices comprising biophysical stimulators and sensing systems are thus emerging, aiming for long-term control of peri-implant bone growth through biointerface monitoring. These acting-sensing dual systems require high frequency (HF) operations able to stimulate osteoinduction/osteoconduction, including matrix maturation and mineralization. A sensing-compatible capacitive stimulator of thin interdigitated electrodes and delivering an electrical 60 kHz HF stimulation, 30 min/day, is here shown to promote osteoconduction in pre-osteoblasts and osteoinduction in human adipose-derived mesenchymal stem cells (hASCs). HF stimulation through this capacitive interdigitated system had significant effects on osteoblasts’ collagen-I synthesis, matrix, and mineral deposition. A proteomic analysis of microvesicles released from electrically-stimulated osteoblasts revealed regulation of osteodifferentiation and mineralization-related proteins (e.g. Tgfb3, Ttyh3, Itih1, Aldh1a1). Proteomics data are available via ProteomeXchange with the identifier PXD028551. Further, under HF stimulation, hASCs exhibited higher osteogenic commitment and enhanced hydroxyapatite deposition. These promising osteoinductive/conductive capacitive stimulators will integrate novel bioelectronic implants able to monitor the bone–implant interface and deliver personalized stimulation to peri-implant tissues.

Graphics with significantly altered GO terms were obtained upon GO enrichment analyses using the FunRich software, and comprise: the percentage of MV deregulated proteins with a specific GO term associated (green bars); the -Log10 of the p value for that percentage against the percentage of all proteins from the Mus musculus proteome associated to that specific GO term (orange markers and continuous line); the -Log10 of the p value = 0.05, indicating the significance cut-off (orange dashed line; to compare with orange continuous line); the fold enrichment of the GO term in each deregulated proteins dataset compared to the enrichment of the same GO term in the Mus musculus dataset (purple markers and line    • NPEPL1 is predicted to have aminopeptidase and metalloexopeptidase activities that can be related to osteogenesis and matrix mineralization. Aminopeptidases have homogeneous activity patterns in various highly pure osteoblastic lines 3 . • Aminopeptidases in cells and matrix vesicles from fractures callus have ability to degrade natural proteoglycan substrates 4 . • Leucine aminopeptidase is highly active in young matrix-producing cells and appears to be involved in the degradation of matrix organic components to enable matrix mineralization 5 . • Glutamyl aminopeptidase is upregulated during osteogenic differentiation of rat bone marrow stromal cells, suggesting a role in osteogenesis 6 . • There is a structural relationship between an extracellular protein of mineralized tissue and keratin, with both sharing antigenic determinants 31 . • A flavonoid that promotes osteoblast mineralization in vitro, induces upregulation of cytokeratin 32 .
These 8 upregulated proteins can be associated to different aspects of bone metabolism, such as: • Osteodifferentiation: TGFB3 is highly related to osteogenesis of stem and precursor cells 7,9,10 and RNase4 may be involved in osteoblast differentiation via miRNAs regulation 29 . • Matrix mineralization and voltage excitation: NPEPL1 is a predicted aminopeptidase and specific aminopeptidases are upregulated during osteogenic differentiation 6 and are suggested to be involved in the degradation of matrix organic components to enable mineralization 5 . The mRNA of hTTYH3, a Ca 2+ -activated chloride channel, is distributed in excitable tissues, suggesting a possible relation with voltage excitation of osteoblasts 21 ; its gene deletion was associated to a disease with a low bone mineralization phenotype 20 .

Supplementary Table 2 | Microvesicles' proteins downregulated by daily high frequency stimulation.
All proteins have high FDR confidence (<1%) and q-value = 0.0, and their levels considered significantly downregulated by the Volcano plot software; proteins concomitantly presenting adjusted p-value < 0.05 are shadowed (n=4). AN, Uniprot accession number; GS, Gene Symbol; STIM / CTRL Abundance ratio, ratio of the relative protein abundance in Stim against Ctrl conditions. Functional hierarchies' classification retrieved from KEGG BRITE (genome.jp/kegg/brite.html). Possible roles determined through abstract mining in PubMed (pubmed.ncbi. nlm.nih.gov), using the terms 'bone', 'osteo', 'osseo', 'osteoblast', 'matrix' and 'mineralization'.  Table 2) were clustered by their potential involvement in relevant bone metabolic functions:

Several of the 24 proteins DReg in secreted MVs upon HF stimulation (Supplementary
• Stemness or commitment into lineages other than osteogenic: CCN5 (or WISP2) maintains cellular undifferentiated states 42,43 or induces adipogenic commitment 44 . TAGLN2 is downregulated in chondrogenic-differentiated MSCs 54 . LUM inhibits endothelial MSC differentiation through decreases in MMP14 levels 96 . PGM1 is a marker for macrophage specificity 100 .
• Osteogenic commitment and earlier osteodifferentiation states: CNTN1 regulates TNFα in bone development 87 , and interacts with proteins on the surface of osteogenic-committed cells 88 .
• Extracellular matrix remodeling and bone mineralization: PREP may be involved in ECM remodeling in osteoblasts, as reported for other endopeptidases [60][61][62] . SPARC (alias Osteonectin), the major non-collagenous component of the bone matrix, binds Ca 2+ , has affinity for hydroxyapatite and influences pathways involved in ECM assembly 74,76,77 . MAN2A1 is an alpha-mannosidase, and the circulating levels of alpha-mannosidases inversely correlate with bone mineralization in patients 46 . COMP and LUM, are two components of bone matrix that play a role in collagen secretion and fibrillogenesis 49,97 . LUM is upregulated before the mineralization onset and returns to baseline levels at the onset of mineralization 50 . During MSC differentiation, LUM was observed to influence the expression and activity of MMP14, a metalloendopeptidase collagenolytic cell surface protein (pro-osteoclastogenic) 96 also DReg by HF stimulation (Supplementary Table 2).