David H. Kohn

Surface modification of biomaterials, particularly by adding bioactive coatings, enhances cell-material interactions at the nanoscale, improving implant performance at the macroscale. One approach involves gene delivery via surface-bound coatings, allowing for controlled local release of viral particles. However, viral gene delivery systems, such as lentiviral vectors, face challenges in precision targeting and transduction efficiency. To address these, a bio-orthogonal coating was developed and used on titanium using chemical vapor deposition (CVD) polymerization. Co-presenting a cell binding peptide and immobilized lentiviral particles on the surface of Ti increased gene delivery efficiency by directing cells to the surface, making them easier to transduce. Specifically, a poly[(4-(3,4dibromomaleimide)-p-xylylene)-co-(4-pentafluorophenol ester-p-xylylene)] coating was prepared using CVD polymerization on Ti discs as a bio-orthogonal layer to tether lentiviral particles delivering GJA1, the gene for the gap junction protein Connexin 43 (Cx43) and the mesenchymal stem cell (MSC) binding peptide, DPIYALSWSGMA. The polymer coating exhibited high binding efficiency for both lentivirus and peptide, allowing for precise microcontact printing. Immobilized lentiviral transduction efficiency matched that in supernatant, with co-delivery increasing transduction efficiency by 35%. The biorthogonal coating boosted MSC binding 2.7-fold, leading to a density-dependent rise in cell–cell communication. High-density seeding enabled gap junction formation, while Cx43 transduction increased intercellular communication by 36%. In low-density culture, transduction led to an 84% increase in cell–cell communication within 4 h of in vitro culture. This work presents a simple, repeatable surface modification method for biomolecular immobilization, combining engineered viral vectors and peptides to enhance gene delivery approaches.

Bone morphogenetic proteins (BMPs) are widely recognized for their therapeutic efficacy in bone regeneration, but one side effect of these therapies is ectopic mineralization. Previous work identified a mineral‐binding peptide (pVTK, VTKHNLQI(pS)Q(pS)Y; where pS denotes a phosphoserine) with the ability to inhibit mineralization in osteoblasts. This study investigated the application of pVTK for inhibiting ectopic mineralization secondary to BMP delivery in vitro and in vivo. It was hypothesized that a mineral binding peptide could be delivered alongside BMP to limit unwanted mineralization without limiting the pro‐osteogenic effects of the BMP signaling pathway. In vitro, pVTK reduced BMP‐stimulated mineral deposition in an osteoblast cell line, as determined by a significant reduction in extracellular matrix calcium deposition with > 300 μM pVTK (p < 0.0001) (at 50 ng/mL BMP2). Importantly, pVTK inhibited mineral deposition without competing with the BMP ligand or diminishing the osteogenic phenotype of the cells in response to BMP stimulation, as demonstrated by no changes in intracellular/extracellular osteogenic protein levels with addition of pVTK. In vivo, pVTK reduced ectopic mineralization of BMP‐loaded subcutaneous implants by 92% (p = 0.0101) compared to PBS‐treated controls. In an acellular model of spontaneous mineralization, pVTK disrupted mineral deposition and reduced crystallinity and crystal organization (as measured via Raman spectroscopy), demonstrating that pVTK is not solely reliant on cell mechanisms for inhibiting mineralization. These findings support the use of a mineral binding peptide for controlling ectopic mineralization secondary to BMP therapies without interfering with the BMP osteogenic pathway, which is necessary for a regenerative effect.

Targeting specific populations of host cells with chemotactic and adhesion factors is a promising strategy for inducing bone regeneration without the use of exogenous cells. Two peptide sequences have been derived from phage display: the mesenchymal stem cell (MSC) binding DPI (DPIYALSWSGMA) sequence and the apatite binding VTK (VTKHLNQISQSY) sequence. When combined into the dual‐functional sequence, DPI‐VTK increases the adhesion strength of MSCs to apatite surfaces and the amount of bone formation with transplanted MSCs. Because many adhesion molecules can stimulate chemotaxis, and cell adhesion to peptide DPI‐VTK is mediated by integrins also critical to migration, we hypothesized that DPI‐VTK serves as an MSC‐specific chemotactic factor and can increase bone regeneration by promoting the osteogenesis of the migrated host MSCs in vivo. In transwell assays, induced pluripotent stem cell‐derived human MSCs ( p < 0.0001) and primary mouse calvarial cells ( p < 0.0001) showed significantly increased migration in vitro when DPI‐VTK was used as a chemoattractant. Further characterization of DPI‐VTK binding cells from mouse calvaria using flow cytometry showed specificity toward cells expressing MSC markers (CD29, CD73, CD90, CD105, CD106, Sca‐1, CD44, and CD200). When conjugated to a mineralized scaffold in vivo, DPI‐VTK increased the migration of CD90 and CD200 positive cells ( p < 0.05) and increased bone formation versus no‐peptide controls ( p < 0.05). These results demonstrate the utility of phage display in creating multifunctional peptides that can increase migration, adhesion, and bone formation in vivo, a strategy that could be applied to numerous different cell types and systems. Results advance biomaterials‐based bone regeneration in two ways—demonstrating the ability of the phage‐derived peptides to increase the migration of MSCs in vivo and increase host‐mediated bone regeneration—potentially bypassing cell transplantation.

Osteocytes, the most abundant cell type in bone, play a crucial role in mechanosensation and signaling for bone formation and resorption. These cells reside within a complex lacuno-canalicular network (OLCN). Osteocyte signaling is reduced under diabetic conditions, and both type 1 and type 2 diabetes lead to reduced bone turnover, perturbed bone composition, and increased fracture risk. We hypothesized that this reduced bone turnover, and altered bone composition with diabetes is associated with reduced OLCN architecture and connectivity. This study aimed to elucidate: (1) the sequence of OLCN changes with diabetes related to bone turnover and (2) whether changes to the OLCN are associated with tissue composition and mechanical properties. Twelve- to fourteen-week-old male C57BL/6 mice were administered streptozotocin at 50 mg/kg for 5 consecutive days to induce hyperglycemia, sacrificed at baseline (BL), or after being diabetic for 3 (D3) and 7 (D7) wk with age-matched (C3, C7) controls (n = 10–12 per group). Mineralized femoral sections were infiltrated with rhodamine, imaged with confocal microscopy, then the OLCN morphology and topology were characterized and correlated against bone histomorphometry, as well as local and whole-bone mechanics and composition. D7 mice exhibited a lower number of peripheral branches relative to C7. The total number of canalicular intersections (nodes) was lower in D3 and D7 relative to BL (P < 0.05 for all), and a reduced bone formation rate (BFR) was observed at D7 vs C7. The number of nodes explained only 15% of BFR, but 45% of Ct.BV/TV, and 31% of ultimate load. The number of branches explained 30% and 22% of the elastic work at the perilacunar and intracortical region, respectively. Collectively, the reduction in OLCN architecture and association of OLCN measures with bone turnover, mechanics, and composition highlights the relevance of the osteocyte and the OLCN and a potential therapeutic target for treating diabetic skeletal fragility. Diabetics are at increased risk of bone fracture. Osteocytes, the most abundant cells in bone, reside in a complex lacuno-canalicular network (OLCN), which is critical to maintaining bone health. This study showed that with diabetes, there is a reduction in OLCN architecture, which is associated with reduced bone turnover, altered bone composition and reduced bone mechanical integrity.

Osteocytes are the most abundant cell type in bone and remodel their local perilacunar matrix in response to a variety of stimuli and diseases. How the perilacunar composition and mechanical properties are affected by type 1 diabetes (T1D), and the contribution of these local changes to the decline in whole-bone functional properties that occurs with diabetes remains unclear. 12–14 week old C57/BL6 male mice were administered a series of low-dose streptozotocin injections and sacrificed at baseline (BL), 3 (D3) and 7 weeks (D7) following confirmation of diabetes, along with age-matched controls (C3, C7). Femora were then subjected to a thorough morphological (μCT), mechanical (four-point bending, nanoindentation), and compositional (HPLC for collagen cross-links, Raman spectroscopy) analysis at the whole-bone and local (perilacunar and intracortical) levels. At the whole-bone level, D7 mice exhibited 10.7% lower ultimate load and 26.4% lower post-yield work relative to C7. These mechanical changes coincided with 52.2% higher levels of pentosidine at D7 compared to C7. At the local level, the creep distance increased, while modulus and hardness decreased in the perilacunar region relative to the intracortical for D7 mice, suggesting a spatial uncoupling in skeletal adaptation. D7 mice also exhibited increased matrix maturity in the 1660/1690 cm⁻¹ ratio at both regions relative to C7. The perilacunar matrix maturity was predictive of post-yield work (46%), but perilacunar measures were not predictive of ultimate load, which was better explained by cortical area (26%). These results show that diabetes causes local perilacunar composition perturbations that affect whole-bone level mechanical properties, implicating osteocyte maintenance of its local matrix in the progression of diabetic skeletal fragility.