Novel Osteogenic Behaviors around Hydrophilic and Radical-Free 4-META/MMA-TBB: Implications of an Osseointegrating Bone Cement.

Poly(methyl methacrylate) (PMMA)-based bone cement, which is widely used to affix orthopedic metallic implants, is considered bio-tolerant but lacks osteoconductivity and is cytotoxic. Implant loosening and toxic complications are significant and recognized problems. Here we devised two strategies to improve PMMA-based bone cement: (1) adding 4-methacryloyloxylethyl trimellitate anhydride (4-META) to MMA monomer to render it hydrophilic; and (2) using tri-n-butyl borane (TBB) as a polymerization initiator instead of benzoyl peroxide (BPO) to reduce free radical production. Rat bone marrow-derived osteoblasts were cultured on PMMA-BPO, common bone cement ingredients, and 4-META/MMA-TBB, newly formulated ingredients. After 24 h of incubation, more cells survived on 4-META/MMA-TBB than on PMMA-BPO. The mineralized area was 20-times greater on 4-META/MMA-TBB than PMMA-BPO at the later culture stage and was accompanied by upregulated osteogenic gene expression. The strength of bone-to-cement integration in rat femurs was 4- and 7-times greater for 4-META/MMA-TBB than PMMA-BPO during early- and late-stage healing, respectively. MicroCT and histomorphometric analyses revealed contact osteogenesis exclusively around 4-META/MMA-TBB, with minimal soft tissue interposition. Hydrophilicity of 4-META/MMA-TBB was sustained for 24 h, particularly under wet conditions, whereas PMMA-BPO was hydrophobic immediately after mixing and was unaffected by time or condition. Electron spin resonance (ESR) spectroscopy revealed that the free radical production for 4-META/MMA-TBB was 1/10 to 1/20 that of PMMA-BPO within 24 h, and the substantial difference persisted for at least 10 days. The compromised ability of PMMA-BPO in recruiting cells was substantially alleviated by adding free radical-scavenging amino-acid N-acetyl cysteine (NAC) into the material, whereas adding NAC did not affect the ability of 4-META/MMA-TBB. These results suggest that 4-META/MMA-TBB shows significantly reduced cytotoxicity compared to PMMA-BPO and induces osteoconductivity due to uniquely created hydrophilic and radical-free interface. Further pre-clinical and clinical validations are warranted.

Tuning of Titanium Microfiber Scaffold with UV-Photofunctionalization for Enhanced Osteoblast Affinity and Function.

Titanium (Ti) is an osteoconductive material that is routinely used as a bulk implant to fix and restore bones and teeth. This study explored the effective use of Ti as a bone engineering scaffold. Challenges to overcome were: (1) difficult liquid/cell infiltration into Ti microfiber scaffolds due to the hydrophobic nature of Ti; and (2) difficult cell attachment on thin and curved Ti microfibers. A recent discovery of UV-photofunctionalization of Ti prompted us to examine its effect on Ti microfiber scaffolds. Scaffolds in disk form were made by weaving grade 4 pure Ti microfibers (125 µm diameter) and half of them were acid-etched to roughen the surface. Some of the scaffolds with original or acid-etched surfaces were further treated by UV light before cell culture. Ti microfiber scaffolds, regardless of the surface type, were hydrophobic and did not allow glycerol/water liquid to infiltrate, whereas, after UV treatment, the scaffolds became hydrophilic and immediately absorbed the liquid. Osteogenic cells from two different origins, derived from the femoral and mandibular bone marrow of rats, were cultured on the scaffolds. The number of cells attached to scaffolds during the early stage of culture within 24 h was 3-10 times greater when the scaffolds were treated with UV. The development of cytoplasmic projections and cytoskeletal, as well as the expression of focal adhesion protein, were exclusively observed on UV-treated scaffolds. Osteoblastic functional phenotypes, such as alkaline phosphatase activity and calcium mineralization, were 2-15 times greater on UV-treated scaffolds, with more pronounced enhancement on acid-etched scaffolds compared to that on the original scaffolds. These effects of UV treatment were associated with a significant reduction in atomic carbon on the Ti microfiber surfaces. In conclusion, UV treatment of Ti microfiber scaffolds tunes their physicochemical properties and effectively enhances the attachment and function of osteoblasts, proposing a new strategy for bone engineering.

Effect of UV Photofunctionalization on Osseointegration in Aged Rats.

OBJECTIVES: This study evaluated the effect of photofunctionalization on osseointegration under the biologically adverse conditions of aging. MATERIALS: First of all, bone marrow-derived osteoblastic cells from young (8 weeks old) and aged (15 months old) rats were biologically characterized. Then, the osteoblasts from aged rats were seeded on titanium discs with and without photofunctionalization, and assessed for initial cell attachment and osteoblastic functions. Titanium mini-implants, with and without photofunctionalization, were placed in the femur of aged rats, and the strength of osseointegration was measured at week 2 of healing. Periimplant tissue was examined morphologically and chemically using scanning electron microscopy and energy dispersive x-ray spectroscopy, respectively. RESULTS: Cells from the aged rats showed substantially reduced biological capabilities compared with those derived from young rats. The cells from aged rats showed significantly increased cell attachment and the expression of osteoblastic function on photofunctionalized titanium than on untreated titanium. In addition, the strength of osseointegration was increased by 40% in aged rats carrying the photofunctionalized implants. Robust bone formation was observed around the photofunctionalized implants with strong elemental peaks of calcium and phosphorus, whereas the tissue around untreated implants showed weaker calcium and phosphate signals than titanium ones. CONCLUSION: These in vivo and in vitro results corroboratively demonstrate that photofunctionalization is effective for enhancing osseointegration in aged rats.

Bone Generation Profiling Around Photofunctionalized Titanium Mesh.

PURPOSE: The aim of this study was to evaluate whether photofunctionalization of titanium mesh enhances its osteoconductive capability. MATERIALS AND METHODS: The titanium mesh (0.2 mm thickness) used in this study was made of commercially pure grade-2 titanium and had hexagonal apertures (2 mm width). Photofunctionalization was performed by treating titanium mesh with UV light for 12 minutes using a photo device immediately before use. Untreated or photofunctionalized titanium mesh was placed into rat femurs, and bone generation around titanium mesh was profiled using three-dimensional (3D) microcomputed tomography (micro-CT). A set of in vitro experiments was conducted using bone marrow-derived osteoblasts. RESULTS: Photofunctionalized titanium mesh surfaces were characterized by the regenerated hydrophilicity and significantly reduced surface carbon. Bone generation profiling at week 3 of healing showed that the hexagonal apertures in photofunctionalized mesh were 95% filled, but they were only 57% filled in untreated mesh, particularly with the center zone remaining as a gap. Bone profiling in slices parallel to the titanium surface showed that photofunctionalized titanium mesh achieved 90% bone occupancy 0 to 400 µm from the surface, compared with only 35% for untreated mesh. Bone occupancy remained as high as 55% 800 to 1,200 µm from photofunctionalized titanium mesh surfaces, compared with less than 20% for untreated mesh. In vitro, photofunctionalized titanium mesh expedited and enhanced attachment and spread of osteoblasts, and increased ALP activity and the rate of mineralization. CONCLUSION: This study may provide novel and advanced metrics describing the osteoconductive property of photofunctionalized titanium mesh. Specifically, photofunctionalization not only increased the breadth, but also the 3D range, of osteoconductivity of titanium mesh, enabling space-filling and far-reaching osteoconductivity. Further translational and clinical studies are warranted to establish photofunctionalized titanium mesh as a novel clinical tool for better bone regeneration and augmentation.

Engineering bone-implant integration with photofunctionalized titanium microfibers.

There are significant challenges in regenerating large volumes of bone tissue, and titanium implant therapy is extremely difficult or contraindicated when there is no supporting bone. Surface conditioning of titanium implants with UV light immediately prior to use, or photofunctionalization, improves the speed and degree of bone-implant integration. Here, we hypothesized that photofunctionalized titanium microfibers are capable of promoting bone ingrowth into the microfiber scaffold to improve bone-implant integration in bone defects. Titanium implants (1 mm in diameter, 2 mm in length) enfolded with 0.7 mm-thick titanium microfibers were placed into 2.4-mm diameter osteotomy in rat femurs. Titanium microfibers and implants were photofunctionalized by treatment with UV light for 12 min using a photo device immediately prior to surgery. Photofunctionalized microfibers and implants were hydrophilic, while as-made microfiber-enfolded implants were hydrophobic. Implant anchorage strength was 2.5 times and 2.2 times greater for photofunctionalized microfiber-enfolded implants than as-made ones at weeks 2 and 4 of healing, respectively. Robust bone formation was only seen at the implant surface of photofunctionalized microfiber-enfolded implants. Bone formation as measured by the Ca/Ti ratio was 5 to over 20 times greater for photofunctionalized than as-made microfiber scaffolds. The Ca/P ratio was 1.55-1.65 in the tissue produced in photofunctionalized microfibers and 1.1-1.3 in tissue in as-made microfibers. In vitro, the number of attached osteoblasts and their alkaline phosphatase activity, both near zero on as-received microfibers, were significantly increased on photofunctionalized microfibers. In conclusion, bone ingrowth occurred in photofunctionalized titanium microfiber scaffolds, enabling successful bone-implant integration when the microfiber-enfolded implants were placed in a site without primary bone support. The combined use of titanium microfibers and photofunctionalization may provide a novel and effective strategy to regenerate and integrate bone in a wider range of applications.

Effect of Photofunctionalization on Ti6Al4V Screw Stability Placed in Segmental Bone Defects in Rat Femurs.

PURPOSE: Ultraviolet-mediated photofunctionalization is a new technology to improve bone and titanium integration. We hypothesized that photofunctionalization would enhance the stability of titanium screws used for segmental bone defects. MATERIALS AND METHODS: Disks were prepared of a titanium alloy (Ti6Al4V) for an in vitro study to evaluate the attachment, proliferation, and differentiation of osteoblasts. Commercially available Ti6Al4V screws were used in vivo. Segmental bone defects were created in rat femurs as an immediate loading reconstruction model. The defects were reconstructed with commercially available titanium plates and Ti6Al4V screws, with or without photofunctionalization. The screw survival rates and mechanical stability were evaluated at 2 and 4 weeks, and the bone formation around the screws was analyzed. RESULTS: Osteoblasts showed greater attachment, proliferation, and differentiation on the photofunctionalized Ti6Al4V disks. Photofunctionalized screws had significantly greater survival rates and mechanical stability at 2 and 4 weeks. The bone formation around the photofunctionalized screws was significantly greater than that around the untreated screws at 4 weeks. CONCLUSIONS: The results of the present study have demonstrated the efficacy of photofunctionalization on enhancing the survival and stability of Ti6Al4V screws under a loaded condition in the reconstruction of segmental defects. This was associated with increased bioactivity and bone formation around the photofunctionalized Ti6Al4V material.

Enhancing osteoblast-affinity of titanium scaffolds for bone engineering by use of ultraviolet light treatment.

Ultraviolet (UV) treatment immediately prior to use is attracting attention as an effective surface conditioning method for titanium to improve osteoblast-affinity. The affinity of titanium to osteoblasts in two-dimensional plate culture has been well studied, but that in three-dimensional cultures remains unclear. Here, we examined the effect of UV treatment on titanium scaffolds, comprising micro-thin titanium fibers, used in bone engineering. Titanium scaffolds, with and without UV treatment, were seeded with rat bone marrow derived osteoblasts, and the number of cells attached to scaffolds and osteoblastic phenotype in the cultures were examined. UV treatment improved the wettability of scaffolds and significantly reduced the percentage of surface carbon. Along with these physicochemical changes in the scaffolds, cell attachment increased by a factor of 1.3 as compared to that of the untreated control. In addition, alkaline phosphatase activity and calcium deposition significantly increased by a factor of 2.3 and 2.0, respectively. Robust formation of mineralized structures consisting of clear peaks of calcium and phosphorus was observed in the UV-treated scaffolds. The observed increase in osteoblast affinity and capability of mineralized matrix formation indicates the potential use of UV-treated titanium scaffolds for bone engineering.