The osteoblastic differentiation of bone marrow stromal cells (bMSCs), critical to the osseointegration of titanium implants, is enhanced on titanium surfaces with biomimetic topography, and this is further enhanced when the surfaces are hydrophilic. This is a result of changing the surface free energy to change protein adsorption, improving cell attachment and differentiation, and improving bone-to-implant contact in patients. In this study, we examined different methods of plasma treatment, a well-accepted method of increasing hydrophilicity, and evaluated changes in surface properties as well as the response of bMSCs in vitro. Commercially pure Ti and titanium–aluminum–vanadium (Ti6Al4V) disks were sand-blasted and acid-etched to impart microscale and nanoscale roughness, followed by treatment with various post-processing surface modification methods, including ultraviolet light (UV), dielectric barrier discharge (DBD)-generated plasma, and plasma treatment under an argon or oxygen atmosphere. Surface wettability was based on a sessile water drop measurement of contact angle; the elemental composition was analyzed using XPS, and changes in topography were characterized using scanning electron microscopy (SEM) and confocal imaging. The cell response was evaluated using bMSCs; outcome measures included the production of osteogenic markers, paracrine signaling factors, and immunomodulatory cytokines. All plasma treatments were effective in inducing superhydrophilic surfaces. Small but significant increases in surface roughness were observed following UV, DBD and argon plasma treatment. No other modifications to surface topography were noted. However, the relative composition of Ti, O, and C varied with the treatment method. The cell response to these hydrophilic surfaces depended on the plasma treatment method used. DBD plasma treatment significantly enhanced the osteogenic response of the bMSCs. In contrast, the bMSC response to argon plasma-treated surfaces was varied, with an increase in OPG production but a decrease in OCN production. These results indicate that post-packaging methods that increased hydrophilicity as measured by contact angle did not change the surface free energy in the same way, and accordingly, cells responded differently. Wettability and surface chemistry alone are not enough to declare whether an implant has an improved osteogenic effect and do not fully explain how surface free energy affects cell response.

Spine fusion devices fabricated from polyether ether ketone (PEEK) using traditional machining or with surface pores created by salt leaching result in a fibrous connective tissue interface. To overcome this limitation, we used fused strand deposition (FSD), which elevates fused filament fabrication (FFF) or fused deposition modeling (FDM) to generate a first-of-its-kind PEEK implant with an architecture designed to mimic trabecular bone. We examined the responses of human bone marrow stromal cells (MSCs) and macrophages isolated from the femurs of C57/Bl6 male mice to the additively manufactured porous PEEK (PP) and porous PEEK coated with HA (PP-HA) to determine if these modifications would improve cell response compared to solid PEEK (SP). PP and PP-HA constructs had similar 3D architectures but differed in hydrophilicity (PP-HA > PP). MSCs and macrophages were cultured on PP, PP-HA, and SP, and osteoblast differentiation and M1/M2 polarization were assessed. MSCs attached to the SP surface and the PP and PP-HA fibers, and synthesized osteoblast proteins in a surface-dependent manner. Notably, MSCs and macrophages produced VEGF mRNA and protein on PP-HA at levels higher than those on PP or SP. Macrophages grown on PP and PP-HA exhibited reduced expression of pro-inflammatory cytokines compared to cells on SP and increased levels of anti-inflammatory cytokines, but they did not exhibit a distinct M1 or M2 phenotype. These results show that additive manufacturing of a unique, fully porous PEEK implant using FSD, results in a surface that promotes MSC differentiation and decreases the pro-inflammatory response of macrophages to the surface, suggesting that PP and PP-HA implants will improve regeneration and eventually osseointegration in vivo. Importantly, the mechanisms involved are not the same as would be expected when testing Ti6Al4V substrates under the same experimental conditions and may have been obscured if the cells had been cultured using osteogenic media (OM). We correlated our in vitro findings with the clinical use of PP-HA implants in four patients, each of whom was treated with a different bone graft material; in all cases, fusion was achieved.

Studies have evaluated vitamin D₃’s therapeutic potential in estrogen-responsive cancers, with conflicting findings. We have shown that the proliferation of breast cancer cells is regulated by 24R,25-dihydroxyvitamin D₃ (24R,25(OH)₂D₃) depending on estrogen receptor alpha 66 (ERα66) expression, suggesting that this could also be the case for estrogen-sensitive laryngeal cancer cells. Accordingly, we examined levels of ERα isoforms in ERα66-positive UM-SCC-12 and ERα66-negative UM-SCC-11A cells and their response to 24R,25(OH)₂D₃. 24R,25(OH)₂D₃ stimulated proliferation, increased the expression of metastatic markers, and inhibited apoptosis in UM-SCC-12 cells while having the opposite effect in UM-SCC-11A cells. To evaluate if vitamin metabolites could act via autocrine/paracrine mechanisms, we assessed the expression, protein levels, and activity of vitamin D₃ hydroxylases CYP24A1 and CYP27B1. Both cell types expressed both mRNAs; but the levels of the enzymes and their activities were differentially regulated by estrogen. ERα66-negative UM-SCC-11A cells produced more 24,25(OH)₂D₃ than UM-SCC-12 cells, but comparable levels of 1,25(OH)₂D₃ when treated with 25(OH)D₃ These results suggest that the regulation of vitamin D₃ metabolism in laryngeal cancer cells is modulated by ERα66 expression, and support a role for 24R,25(OH)₂D₃ as an autocrine/paracrine regulator of laryngeal cancer. The local metabolism of 25(OH)D₃ should be considered when determining the potential of vitamin D₃ in laryngeal cancer.

This study examined the effects of 24R,25‐dihydroxyvitamin D ₃ (24R,25(OH) ₂ D ₃ ) in estrogen‐responsive laryngeal cancer tumorigenesis in vivo, the mechanisms involved, and whether the ability of the tumor cells to produce 24R,25(OH) ₂ D ₃ locally is estrogen‐dependent. Estrogen receptor alpha‐66 positive (ER+) UM‐SCC‐12 cells and ER− UM‐SCC‐11A cells responded differently to 24R,25(OH) ₂ D ₃ in vivo; 24R,25(OH) ₂ D ₃ enhanced tumorigenesis in ER+ tumors but inhibited tumorigenesis in ER− tumors. Treatment with 17β‐estradiol (E ₂ ) for 24 h reduced levels of CYP24A1 protein but increased 24R,25(OH) ₂ D ₃ production in ER+ cells; treatment with E ₂ for 9 min reduced CYP24A1 at 24 h and reduced 24R,25(OH) ₂ D ₃ production in ER− cells. These findings suggest the involvement of E ₂ receptor(s) in addition to ERα66. To investigate if 24R,25(OH) ₂ D ₃ can act locally, ER+ and ER− cells were treated with 24R,25(OH) ₂ D ₃ after inhibiting putative 24R,25(OH) ₂ D ₃ receptors, and the cells were assessed for effects on DNA synthesis (proliferation) and p53 production (apoptosis). Specific inhibitors were used to assess downstream secondary messenger signaling pathways and requirements for palmitoylation and caveolae in both cell lines. The results show that 24R,25(OH) ₂ D ₃ binds to a complex of receptors, including TLCD3B2, VDR, and protein disulfide‐isomerase A3 (PDIA3) in ER+ UM‐SCC‐12 cells. The mechanism requires palmitoylation, and PLD, PI3K, and LPAR are involved. The anti‐tumorigenic effects of 24R,25(OH) ₂ D ₃ in ER− UM‐SCC‐11A cells involve a membrane‐receptor complex consisting of VDR, PDIA3, and ROR2 within caveolae to activate a yet‐to‐be‐elucidated downstream signaling cascade. This work demonstrates a driving mechanism for the therapeutic agent 24R,25(OH) ₂ D ₃ that may be used for laryngeal cancer patients.

Titanium–aluminum–vanadium (Ti6Al4V) is a material chosen for spine, orthopedic, and dental implants due to its combination of desirable mechanical and biological properties. Lasers have been used to modify metal surfaces, enabling the generation of a surface on Ti6Al4V with distinct micro- and nano-scale structures. Studies indicate that topography with micro/nano features of osteoclast resorption pits causes bone marrow stromal cells (MSCs) and osteoprogenitor cells to favor differentiation into an osteoblastic phenotype. This study examined whether the biological response of human MSCs to Ti6Al4V surfaces is sensitive to laser treatment-controlled micro/nano-topography. First, 15 mm diameter Ti6Al4V discs (Spine Wave Inc., Shelton, CT, USA) were either machined (M) or additively manufactured (AM). Surface treatments included no laser treatment (NT), nanosecond laser (Ns), femtosecond laser (Fs), or nanosecond followed by femtosecond laser (Ns+Fs). Surface wettability, roughness, and surface chemistry were determined using sessile drop contact angle, laser confocal microscopy, X-ray photoelectron spectroscopy (XPS), and scanning electron microscopy (SEM). Human MSCs were cultured in growth media on tissue culture polystyrene (TCPS) or test surfaces. On day 7, the levels of osteocalcin (OCN), osteopontin (OPN), osteoprotegerin (OPG), and vascular endothelial growth factor 165 (VEGF) in the conditioned media were measured. M NT, Fs, and Ns+Fs surfaces were hydrophilic; Ns was hydrophobic. AM NT and Fs surfaces were hydrophilic; AM Ns and Ns+Fs were hydrophobic. Roughness (Sa and Sz) increased after Ns and Ns+Fs treatment for both M and AM disks. All surfaces primarily consisted of oxygen, titanium, and carbon; Fs had increased levels of aluminum for both M and AM. SEM images showed that M NT discs had a smooth surface, whereas AM surfaces appeared rough at a higher magnification. Fs surfaces had a similar morphology to their respective NT disc at low magnification, but higher magnification revealed nano-scale bumps not seen on NT surfaces. AM Fs surfaces also had regular interval ridges that were not seen on non-femto laser-ablated surfaces. Surface roughness was increased on M and AM Ns and Ns+Fs disks compared to NT and Fs disks. OCN was enhanced, and DNA was reduced on Ns and Ns+Fs, with no difference between them. OPN, OPG, and VEGF levels for laser-treated M surfaces were unchanged compared to NT, apart from an increase in OPG on Fs. MSCs grown on AM Ns and Ns+Fs surfaces had increased levels of OCN per DNA. These results indicate that MSCs cultured on AM Ns and AM Ns+Fs surfaces, which exhibited unique roughness at the microscale and nanoscale, had enhanced differentiation to an osteoblastic phenotype. The laser treatments of the surface mediated this enhancement of MSC differentiation and warrant further clinical investigation.

Urinary incontinence (UI), the involuntary leakage of urine, is a common condition that imposes significant physical, emotional, and financial burdens. Injury to the external urethral sphincter (EUS) contributes to myogenic UI by damaging the skeletal muscle responsible for urinary control. This study evaluated the engraftment and therapeutic potential of human muscle-derived cells (hMDCs) using acute and chronic skeletal muscle injury models. Immunocompromised female rats received cardiotoxin (CTX) or volumetric muscle loss (VML) injuries to the tibialis anterior muscle. Animals were treated with 100,000 or 500,000 passage 3 or passage 6 hMDCs injected one day after CTX injury or six weeks after VML injury. VivoTrack imaging and human leukocyte antigen staining confirmed successful engraftment of hMDCs at the injection site. A total of 500,000 passage 3 hMDCs reduced muscle force, whereas later-passage 6 cells had no effect on force production. All treatment groups exhibited small, newly regenerated muscle fibers. In chronic VML injuries, hMDC engraftment using 500,000 passage 6 cells enhanced regenerative characteristics despite limited fusion with host muscle fibers. These findings suggest that hMDCs improved regenerative characteristics in a fibrotic VML model but did not restore contractile force. This work supports the therapeutic potential of hMDCs and provides a foundation for future clinical strategies to treat urinary incontinence.