In vivo bone regeneration on titanium devices using serum-free grown adipose-derived stem cells, in a sheep femur model.

AIM: The aim of this study was to investigate the capacity of adipose-derived stem cells (ADSC), grown in serum-free conditions, to regenerate bone around titanium discs with different titanium surfaces. MATERIAL AND METHODS: Ovine ADSC (oADSC) were isolated from seven sheep and cultured using serum-free and osteogenic conditions. Prior to in vivo testing, the growth and osteogenic behaviour of these cells were analysed in vitro using cell proliferation and extracellular matrix mineralisation assays. The bone regenerative capacity of autologous oADSC was evaluated in vivo on titanium discs in a sheep femur epicondyle model. Machined (MTi) and alumina-blasted (ABTi) titanium discs were used. Bone regeneration within the defects was evaluated after 1 month using histology and histomorphometry. PKH26 cell-tracking dye was used to verify the persistence of oADSC in the surgical wound. RESULTS: oADSC sourced from five of seven sheep differentiated into osteoblast-like cells. Cellular proliferation was reduced only for osteogenically induced oADSC (oOS-ADSC) grown on ABTi, compared to non-induced oADSC grown on ABTi and tissue culture polystyrene (P = 0.03 and 0.02 respectively). There was no significant difference for in vitro mineralisation assays comparing oADSC with oOS-ADSC, regardless of implant surface type. oADSC labelled with PKH26 were detected 1 month after surgery within the defect. There was no difference in bone regeneration between the bone defects treated with oADSC vs. just blood clot. CONCLUSION: After 1-month healing, the use of autologous oADSC did not improve bone regeneration in defects containing titanium devices with different surfaces.

Histomorphometric and histologic evaluation of titanium-zirconium (aTiZr) implants with anodized surfaces.

The choice of implant surface has a significant influence on osseointegration. Modification of TiZr surface by anodization is reported to have the potential to modulate the osteoblast cell behaviour favouring more rapid bone formation. The aim of this study is to investigate the effect of anodizing the surface of TiZr discs with respect to osseointegration after four weeks implantation in sheep femurs. Titanium (Ti) and TiZr discs were anodized in an electrolyte containing DL-α-glycerophosphate and calcium acetate at 300 V. The surface characteristics were analyzed by scanning electron microscopy, electron dispersive spectroscopy, atomic force microscopy and goniometry. Forty implant discs with thickness of 1.5 and 10 mm diameter (10 of each-titanium, titanium-zirconium, anodized titanium and anodized titanium-zirconium) were placed in the femoral condyles of 10 sheep. Histomorphometric and histologic analysis were performed 4 weeks after implantation. The anodized implants displayed hydrophilic, porous, nano-to-micrometer scale roughened surfaces. Energy dispersive spectroscopy analysis revealed calcium and phosphorous incorporation into the surface of both titanium and titanium-zirconium after anodization. Histologically there was new bone apposition on all implanted discs, slightly more pronounced on anodised discs. The percentage bone-to-implant contact measurements of anodized implants were higher than machined/unmodified implants but there was no significant difference between the two groups with anodized surfaces (P > 0.05, n = 10). The present histomorphometric and histological findings confirm that surface modification of titanium-zirconium by anodization is similar to anodised titanium enhances early osseointegration compared to machined implant surfaces.

Spark anodization of titanium-zirconium alloy: surface characterization and bioactivity assessment.

Titanium (Ti) and its alloys have been popularly used as implant biomaterial for decades. Recently, titanium-zirconium (TiZr) alloy has been developed as an alternative implant material with improved strength in load bearing areas. Surface modification is one of the key factors to alter the surface properties to hasten osseointegration. Spark anodic oxidation (anodization) is one such method that is reported to enhance the bone formation around implants. This study aims to anodize TiZr and study its surface characteristics and cytocompatibility by cell culture experiments using osteoblast-like cells. Titanium (Ti) and TiZr discs were anodized in an electrolyte containing DL-α-glycerophosphate and calcium acetate (CA) at 300 V. The surface characteristics were analyzed by scanning electron microscopy, electron dispersive spectroscopy, X-ray diffraction (XRD), atomic force microscopy and goniometry. Using osteoblast-like cells viability, proliferation, differentiation and mineralization was assessed. The anodized surfaces demonstrated increased oxygen, entrapped calcium and phosphorous from the electrolyte used. XRD analysis confirmed the presence of anatase in the oxide layer. Average roughness increased and there was a significant decrease in contact angle (P < 0.01) following anodization. The anodized TiZr (aTiZr) surfaces were more nano-porous compared to anodized Ti (aTi). No significant difference was found in the viability of cells, but after 24 h the total number of cells was significantly higher (P < 0.01). Proliferation, alkaline phosphatase activity and calcium deposits were significantly higher on anodized surfaces compared to machined surfaces (P < 0.05, ANOVA). Anodization of TiZr resulted in a more nanoporous and hydrophilic surface than aTi, and osteoblast biocompatibility appeared comparable to aTi.

In vivo healing of low temperature deproteinized bovine bone xenograft in a rabbit cranial model.

The physicochemical properties of grafting materials affect the quality of the osteointegration, resorption rate, and the new bone (NB) formation. This study assessed the physicochemical properties and integration of a low temperature deproteinized bovine bone xenograft (BBX), referred to as optimized MoaBone® (OMB). This novel BBX was physiochemically characterized both pre and post chemical bleaching and sterilization by gamma irradiation. OMB was compared to two commercial BBX; Bio-Oss® (BO) and MoaBone® (MB) using a rabbit cranial model. Residual graft and NB were quantified using histology and micro-computed tomography. Results showed that chemical treatment and gamma irradiation had limited effect on the surface texture. A significant decrease in the collagen content was detected post chemical treatment and in the carbonate content post gamma irradiation. There was no evidence of inflammatory infiltrate, necrosis, or connective tissue encapsulation, and a significant increase of NB in all grafted sites as compared to untreated defects could be observed. However, there was no statistically significant difference between the grafted sites. We conclude that chemical treatment and terminal sterilization strongly impact the final graft's properties. OMB graft showed equivalence with BO for in vivo bone formation and potentially results in lower levels of graft retention.

The Effect of Low-Temperature Thermal Processing on Bovine Hydroxyapatite Bone Substitutes, toward Bone Cell Interaction and Differentiation.

Ideal bone grafting scaffolds are osteoinductive, osteoconductive, and encourage osteogenesis through the remodeling processes of bone resorption, new bone formation, and successful integration or replacement; however, achieving this trifecta remains challenging. Production methods of bone grafts, such as thermal processing, can have significant effects on the degree of cell-surface interactions via wide-scale changes in the material properties. Here, we investigated the effects of small incremental changes at low thermal processing temperatures on the degree of osteoclast and osteoblast attachment, proliferation, and differentiation. Bovine bone scaffolds were prepared at 100, 130, 160, 190, and 220 °C and compared with a commercial control, Bio-Oss®. Osteoclast attachment and activity were significantly higher on lower temperature processed bone and were not present ≥190 °C. The highest osteoblast proliferation and differentiation were obtained from treatments at 130 and 160 °C. Similarly, qRT2-PCR assays highlighted osteoblasts attached to bone processed at 130 and 160 °C as demonstrating the highest osteogenic gene expression. This study demonstrated the significant effects of small-scale processing changes on bone graft materials in vitro, which may translate to a tailored approach of cellular response in vivo.

The Effect of Low-Processing Temperature on the Physicochemical and Mechanical Properties of Bovine Hydroxyapatite Bone Substitutes.

Bovine bone grafts (BBX) require protein removal as part of the manufacturing process to reduce antigenicity and, in consequence, to be safely used in humans. Deproteinisation may have direct effects on the characteristics of the bone material and on in vivo material performance. This research aimed to comprehensively study the physicochemical and mechanical properties of BBX processed at low deproteinisation processing temperatures. Cubes of bovine bone (8 mm3) were treated with temperatures between 100 °C and 220 °C at 30 °C intervals and with pressures ranging from 1.01 to 24.58 Bar. The samples were characterised topographically and mechanically using scanning electron microscopy (SEM), atomic force microscopy (AFM), and uniaxial bending tests. The organic content and the chemical composition were determined using thermogravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FTIR). X-ray diffraction (XRD) and FTIR were also used to quantitatively determine the specimen crystallinity. Increasing temperature/pressure was associated with decreasing protein levels and compressive strength and increasing surface irregularities and crystallinity. The findings suggest that low-temperature processed bone is likely to exhibit a rapid in vivo degradation rate. The deproteinisation temperature can be adjusted to tailor the graft properties for specific applications.

Effect of Titanium Surfaces on the Osteogenic Differentiation of Human Adipose-Derived Stem Cells.

PURPOSE: To investigate whether human adipose-derived stem cells will form a functional bone-like matrix on titanium substrates. The behavior of human adipose-derived stem cells was examined when grown in either serum-free, xeno-free stem cell growth medium or osteogenic differentiation medium and cultured on either machined titanium (MTi) or on roughened alumina-blasted titanium (ABTi) discs. MATERIALS AND METHODS: Cellular proliferation, extracellular mineralized matrix production, osteogenic-related protein production (RUNX2 and osteocalcin), and gene expression for pluripotency and self-renewal (TERT and OCT4) and osteogenic-related (MSX2, RUNX2, and BGLAP) genes were performed. RESULTS: Human adipose-derived stem cells in serum-free medium (hADSC) proliferated at a higher rate compared with osteogenically differentiated cells (hOS-ADSC); however, the osteogenically committed cells produced more mineralized matrix on the titanium surfaces compared with either tissue culture plastic or the undifferentiated cells. The immunofluorescence analysis showed that human adipose-derived stem cells cultured in serum-free medium and osteogenic differentiation medium produced RUNX2 on both the machined titanium surface and on the alumina-blasted titanium surface after 7 days in culture. Only osteogenically differentiated cells produced osteocalcin after 21 days. Relative gene expression showed stable expression of MSX2, RUNX2, and BGLAP over time on all surfaces. Only osteogenically differentiated cells displayed osteogenic characteristics over time. CONCLUSION: This study confirmed that human adipose-derived stem cells could be successfully grown in serum-free, xeno-free culture medium suitable for clinical use. Adipose-derived stem cells thus show potential utility for bone regeneration in association with titanium surfaces.

Soft and Hard Tissue Response to Zirconia versus Titanium One-Piece Implants Placed in Alveolar and Palatal Sites: A Randomized Control Trial.

BACKGROUND: Titanium (Ti) implants have been used in the last four decades to replace missing teeth. Alternatives to Ti such as zirconia (Zr) may offer aesthetic advantages and be more acceptable to patients and clinicians concerned about Ti allergy but must show equivalent biological acceptability to Ti. PURPOSE: The research aimed to investigate soft and hard tissue response to Ti and Zr implants in edentulous patients. MATERIALS AND METHODS: The research included 24 participants (Ti = 12, Zr = 12) restored with one-piece ball-abutment implants to support overdentures. Participants received four maxillary implants (two in the premolar alveolus, one off center in the alveolar midline, and one wide-diameter implant in the anterior median palate) and three mandibular implants (one in the midline and bilateral posterior implants). RESULTS: Success rates for both Ti and Zr implants were low, 67.9% for all alveolar implants and a survival rate of 50.0% for the palatal implants. Only 11 (52.4%) of 21 palatal implants survived the follow-up period. Peri-implant health was equivalent for Ti and Zr implants and showed no statistically significant changes from loading to the 1-year follow-up. Statistically significant differences were noted in radiographic bone level between Ti and Zr implants (p = .02), with Zr showing greater bone loss. CONCLUSIONS: Although the failure rates with the one-piece Zr implants were higher than with the Ti ones, suggesting that the former's clinical usage as in this study cannot be recommended, it should be borne in mind that the fault may also lie with the novel prosthodontic design which was used.