Off-Stoichiometric Reactions at the Cell-Substrate Biomolecular Interface of Biomaterials: In Situ and Ex Situ Monitoring of Cell Proliferation, Differentiation, and Bone Tissue Formation.

The availability of osteoinductive biomaterials has encouraged new therapies in bone regeneration and has potentially triggered paradigmatic shifts in the development of new implants in orthopedics and dentistry. Among several available synthetic biomaterials, bioceramics have gained attention for their ability to induce mesenchymal cell differentiation and successive bone formation when implanted in the human body. However, there is currently a lack of understanding regarding the fundamental biochemical mechanisms by which these materials can induce bone formation. Phenomenological studies of retrievals have clarified the final effect of bone formation, but have left the chemical interactions at the cell-material interface uncharted. Accordingly, the knowledge of the intrinsic material properties relevant for osteoblastogenesis and osteoinduction remains incomplete. Here, we systematically monitored in vitro the chemistry of mesenchymal cell metabolism and the ionic exchanges during osteoblastogenesis on selected substrates through conventional biological assays as well as via in situ and ex situ spectroscopic techniques. Accordingly, the chemical behavior of different bioceramic substrates during their interactions with mesenchymal cells could be unfolded and compared with that of biomedical titanium alloy. Our goal was to clarify the cascade of chemical equations behind the biological processes that govern osteoblastogenic effects on different biomaterial substrates.

Silicon Nitride Bioceramics Induce Chemically Driven Lysis in Porphyromonas gingivalis.

Organisms of Gram-negative phylum bacteroidetes, Porphyromonas gingivalis, underwent lysis on polished surfaces of silicon nitride (Si3N4) bioceramics. The antibacterial activity of Si3N4 was mainly the result of chemically driven principles. The lytic activity, although not osmotic in nature, was related to the peculiar pH-dependent surface chemistry of Si3N4. A buffering effect via the formation of ammonium ions (NH4(+)) (and their modifications) was experimentally observed by pH microscopy. Lysis was confirmed by conventional fluorescence spectroscopy, and the bacteria's metabolism was traced with the aid of in situ Raman microprobe spectroscopy. This latter technique revealed the formation of peroxynitrite within the bacterium itself. Degradation of the bacteria's nucleic acid, drastic reduction in phenilalanine, and reduction of lipid concentration were observed due to short-term exposure (6 days) to Si3N4. Altering the surface chemistry of Si3N4 by either chemical etching or thermal oxidation influenced peroxynitrite formation and affected bacteria metabolism in different ways. Exploiting the peculiar surface chemistry of Si3N4 bioceramics could be helpful in counteracting Porphyromonas gingivalis in an alkaline pH environment.

Antifungal activity of polymethyl methacrylate/Si3N4 composites against Candida albicans.

Previous studies using gram-positive and -negative bacteria demonstrated that hydrolysis of silicon nitride (Si3N4) in aqueous suspensions elutes nitrogen and produces gaseous ammonia while buffering pH. According to immunochemistry assays, fluorescence imaging, and in situ Raman spectroscopy, we demonstrate here that the antipathogenic surface chemistry of Si3N4 can be extended to polymethylmethacrylate (PMMA) by compounding it with a minor fraction (~8 vol.%) of Si3N4 particles without any tangible loss in bulk properties. The hydrolytic products, which were eluted from partly exposed Si3N4 particles at the composite surface, exhibited fungicidal action against Candida albicans. Using a specific nitrative stress sensing dye and highly resolved fluorescence micrographs, we observed in situ congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while these radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. These in situ observations suggest that the surface chemistry of Si3N4 mimics the antifungal activity of macrophages, which concurrently produce NO radicals and superoxide anions (O2•-) resulting in the formation of candidacidal ONOO-. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit the uncontrolled growth of Candida albicans and ensuing candidiasis while being synergic with chemoprophylaxis. STATEMENT OF SIGNIFICANCE: In a follow-up of previous studies of gram-positive and gram-negative bacteria, we demonstrate here that the antipathogenic surface chemistry of Si3N4 could be extended to polymethylmethacrylate (PMMA) containing a minor fraction (~8 vol.%) of Si3N4 particles without tangible loss in bulk properties. Hydrolytic products eluted from Si3N4 particles at the composite surface exhibited fungicidal action against Candida albicans. Highly resolved fluorescence microscopy revealed congestion of peroxynitrite (ONOO-) radicals in the mitochondria of the Candida cells exposed to the PMMA/Si3N4 composite, while radicals were absent in the mitochondria of identical cells exposed to monolithic PMMA. The fungicidal properties of PMMA/Si3N4 composites could be used in dental appliances to inhibit uncontrolled growth of Candida albicans and ensuing candidiasis in synergy with chemoprophylaxis.

Surface functionalization of PEEK with silicon nitride.

Surface roughness, bioactivity, and antibacterial properties are desirable in skeletal implants. We hot-pressed a mix of particulate sodium chloride (NaCl) salt and silicon nitride (ß-Si3N4) onto the surface of bulk PEEK. NaCl grains were removed by leaching in water, resulting in a porous PEEK surface embedded with sim15 vol% ß-Si3N4particles. This functionalized surface showed the osteogenic and antibacterial properties previously reported in bulk silicon nitride implants. Surface enhancement of PEEK with ß-Si3N4could improve the performance of spinal fusion cages, by facilitating arthrodesis and resisting bacteria.

3D-additive deposition of an antibacterial and osteogenic silicon nitride coating on orthopaedic titanium substrate.

A 3D-additive manufacturing approach produced a dense Si3N4 ceramic coating on a biomedical grade commercially pure titanium (cp-Ti) substrate by an automatic laser-sintering procedure. Si3N4 coatings could be prepared with thicknesses from the single to the tens of microns. A coating thickness, t = 15 ± 5 µm, was selected for this study, based on projections of homogeneity and scratching resistance. The Si3N4 coating met the 20 N threshold required for biomaterial applications, according to the standard scratch testing (ASTM C1624-05). The Si3N4 coating imparted both the antibacterial and osteogenic properties of bulk Si3N4 to the cp-Ti substrate. Both properties were comparable to those previously described for bulk Si3N4 biomedical implants. The newly developed Si3N4-coating was applied to commercially available Ti-alloy acetabular shells for total hip arthroplasty. A "glowing" test based on luciferase gene transformation was applied to visualize the colonization of gram-negative Escherichia coli on Si3N4-coated and uncoated Ti-alloy acetabular shells. The results showed that the coating technology conferred resistance to Staphylococcus epidermidis and Escherichia coli adhesion onto the bulk acetabular sockets.

Enhanced bioactivity of Si3N4 through trench-patterning and back-filling with Bioglass®.

Using a simple and innovative sandblasting process, disks of monolithic biomedical silicon nitride (ß-Si3N4) were texturized with a matrix of regular, discrete square trenches with a total depth in the range of hundreds of microns. The process consisted of sandblasting Si3N4 substrates through a stainless-steel wire-mesh (150 or 200 µm) using abrasive silicon carbide powders (α-SiC, ∼40 µm) under 1,034 kPa (150 psi) of gas pressure. The depth of the porosities could be controlled varying both the treatment time and the distance from the surface. Part of the samples were then filled with 45S5 Bioglass® powders to improve the osteointegration and stimulate the production of bone tissue. Due to the increased macroscopic and microscopic roughness, biological testing using human osteosarcoma cells (SaOS-2) showed improved cell proliferation and greater production of both mineral (hydroxyapatite) and organic (collagen) phases on the patterned surfaces compared to untreated ß-Si3N4 or to the biomedical titanium control samples. Both of these effects were further enhanced when the porosities were filled with Bioglass®.

Proliferation and function of MC3T3-E1 cells on freeze-cast hydroxyapatite scaffolds with oriented pore architectures.

Previous work by the authors showed that hydroxyapatite (HA) scaffolds with different types of oriented microstructures and a unique 'elastic-plastic' mechanical response could be prepared by unidirectional freezing of suspensions. The objective of the present work was to evaluate the in vitro cellular response to these freeze-cast HA scaffolds. Unidirectional scaffolds with approximately the same porosity (65-70%) but different pore architectures, described as 'lamellar' (pore width = 25 +/- 5 microm) and 'cellular' (pore diameter = 100 +/- 10 microm), were evaluated. Whereas both groups of scaffolds showed excellent ability to support the proliferation of MC3T3-E1 pre-osteoblastic cells on their surfaces, scaffolds with the cellular-type microstructure showed far better ability to support cell proliferation into the pores and cell function. These results indicate that freeze-cast HA scaffolds with the cellular-type microstructure have better potential for bone repair applications.

Fabrication and testing of silicon nitride bearings in total hip arthroplasty: winner of the 2007 "HAP" PAUL Award.

Total hip arthroplasty (THA) bearings were fabricated from silicon nitride (Si(3)N(4)) powder. Mechanical testing showed that Si(3)N(4) had improved fracture toughness and fracture strength over modern alumina (Al(2)O(3)) ceramic. When tested with Si(3)N(4) cups in a hip simulator, both cobalt-chromium (CoCr) and Si(3)N(4) femoral heads produced low wear rates that were comparable to Al(2)O(3)-Al(2)O(3) bearings in THA. This study offers experimental support for a novel metal-ceramic THA bearing couple that combines the reliability of CoCr femoral heads with the wear advantages of ceramic surfaces.

Tibial post failures in a condylar posterior cruciate substituting total knee arthroplasty.

In posterior-stabilized total knee arthroplasties, a femoral cam and polyethylene tibial post are commonly used to restore posterior stability after sacrifice of the posterior cruciate ligament. This article reports a high incidence of early tibial post failures in one design of prosthesis and examines the variables that may have contributed to such. Five hundred sixty-four consecutive posterior-stabilized total knees were implanted in 512 patients, using a total knee prosthesis with a polyethylene tibial post and femoral cam. Clinical and radiographic outcomes were measured at a mean follow-up of 40 months after surgery (range, 24-83 months). At follow-up, 70 knees in 62 patients (12%) had undergone revision surgery because of symptoms related to catastrophic failure of the tibial post.

Development of a SiYAlON glaze for improved osteoconductivity of implantable medical devices.

The application of bioactive coatings onto orthopaedic appliances is commonly performed to compensate for the otherwise bioinert nature of medical devices and to improve their osseointegration. Calcium phosphates, hydroxyapatite (HAp), and bioglasses are commercially available for this purpose. Until recently, few other inorganic compounds have been identified with similar biofunctionality. However, silicon nitride (Si3 N4 ) has emerged as a new orthopaedic material whose unique surface chemistry also enhances osteoconductivity. Recent research has confirmed that its minority intergranular phase, consisting of silicon yttrium aluminum oxynitride (SiYAlON), is principally responsible for this improvement. As a result, it was hypothesized that SiYAlON itself might serve as an effective osteoconductive coating or glaze for medical devices. To test this hypothesis, a process inspired by traditional ceramic whiteware glazing was developed. A slurry containing ingredients similar to the intergranular SiYAlON composition was applied to a Si3 N4 surface, which was then subjected to a heat treatment to form a glaze. Various analytical tools were employed to assess its chemistry and morphology. It was found that the glaze was comprised predominately of Y5 Si3 O12 N, a compound commonly referred to as N-apatite, which is isostructural to native HAp. Subsequent exposure of the glazed surface to acellular simulated body fluid led to increased deposition of biomimetic HAp-like crystals, while exposure to Saos-2 osteosarcoma cells in vitro resulted in greater HAp deposition relative to control samples. The observation that SiYAlON exhibits enhanced osteoconductivity portends its potential as a therapeutic aid in bone and tissue repair. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1084-1096, 2018.

Incorporating Si3 N4 into PEEK to Produce Antibacterial, Osteocondutive, and Radiolucent Spinal Implants.

Polyetheretherketone (PEEK) is a popular polymeric biomaterial which is primarily used as an intervertebral spacer in spinal fusion surgery; but it is developed for trauma, prosthodontics, maxillofacial, and cranial implants. It has the purported advantages of an elastic modulus which is similar to native bone and it can be easily formed into custom 3D shapes. Nevertheless, PEEK's disadvantages include its poor antibacterial resistance, lack of bioactivity, and radiographic transparency. This study presents a simple approach to correcting these three shortcomings while preserving the base polymer's biocompatibility, chemical stability, and elastic modulus. The proposed strategy consists of preparing a PEEK composite by dispersing a minor fraction (i.e., 15 vol%) of a silicon nitride (Si3 N4 ) powder within its matrix. In vitro tests of PEEK composites with three Si3 N4 variants-ß-Si3 N4 , α-Si3 N4 , and ß-SiYAlON-demonstrate significant improvements in the polymer's osteoconductive versus SaOS-2 cells and bacteriostatic properties versus gram-positive Staphylococcus epidermidis bacteria. These properties are clearly a consequence of adding the bioceramic dispersoids, according to chemistry similar to that previously demonstrated for bulk Si3 N4 ceramics in terms of osteogenic behavior (vs both osteosarcoma and mesenchymal progenitor cells) and antibacterial properties (vs both gram-positive and gram-negative bacteria).

Preparation and bioactive characteristics of a porous 13-93 glass, and fabrication into the articulating surface of a proximal tibia.

The silicate-based 45S5 bioactive glass, typically in particulate form, has been widely investigated for bone repair. However, its application as a scaffold for bone tissue engineering is limited due to the difficulty of forming porous three-dimensional constructs with complex shapes. In this study, the use of another silicate-based bioactive glass, referred to as 13-93, was investigated for the preparation of porous constructs. Particles of 13-93 glass (255-325 microm) were consolidated and sintered to form cylindrical constructs. Characterization of these constructs was performed using mercury porosimetry, scanning electron microscopy (SEM), and mechanical testing. Constructs with porosities of 40-45% and pore sizes in the range 100-300 microm were found to have a compressive strength of 22 +/- 1 MPa. The bioactivity of the 13-93 glass was studied by immersing disks in a simulated body fluid at 37 degrees C and characterizing the reaction products. X-ray diffraction, Fourier transform infrared (FTIR) spectroscopy, and SEM showed the formation of a crystalline hydroxyapatite layer on the glass surface after approximately 7 days. The ability to fabricate the complex geometrical shape of the articulating surface of a human tibia from 13-93 glass particles was demonstrated.

Ceramic bearings in total knee arthroplasty.

Ceramic bearings have been used extensively in total hip arthroplasty, both in ceramic-on-polyethylene and in ceramic-on-ceramic articulations. Ceramic articulations in total hip arthroplasties offer the advantage of lower wear rates compared to the standard metal-polyethylene coupling. In total knee arthroplasty, few data have examined the application of ceramic bearings. The long-term failure mode of total knee arthroplasties is related to wear and failure of the polyethylene in the articulation. Wear data from knee simulators and in vitro investigations suggest ceramics may offer a superior countersurface in total knee bearings through decreased polyethylene wear rates and a lower incidence of long-term failures associated with wear. This article reviews the existing data related to the application of ceramic biomaterials in total knees and evaluates the role ceramic surfaces may have in extending the longevity of total knee arthroplasty.

Surface topography of silicon nitride affects antimicrobial and osseointegrative properties of tibial implants in a murine model.

While silicon nitride (Si3 N4 ) is an antimicrobial and osseointegrative orthopaedic biomaterial, the contribution of surface topography to these properties is unknown. Using a methicillin-resistant strain of Staphylococcus aureus (MRSA), this study evaluated Si3 N4 implants in vitro utilizing scanning electron microscopy (SEM) with colony forming unit (CFU) assays, and later in an established in vivo murine tibia model of implant-associated osteomyelitis. In vitro, the "as-fired" Si3 N4 implants displayed significant reductions in adherent bacteria versus machined Si3 N4 (2.6 × 104 vs. 8.7 × 104 CFU, respectively; p < 0.0002). Moreover, SEM imaging demonstrated that MRSA cannot directly adhere to native as-fired Si3 N4 . Subsequently, a cross-sectional study was completed in which sterile or MRSA contaminated as-fired and machined Si3 N4 implants were inserted into the tibiae of 8-week old female Balb/c mice, and harvested on day 1, 3, 5, 7, 10, or 14 post-operatively for SEM. The findings demonstrated that the antimicrobial activity of the as-fired implants resulted from macrophage clearance of the bacteria during biofilm formation on day 1, followed by osseointegration through the apparent recruitment of mesenchymal stem cells on days 3-5, which differentiated into osteoblasts on days 7-14. In contrast, the antimicrobial behavior of the machined Si3 N4 was due to repulsion of the bacteria, a phenomenon that also limited osteogenesis, as host cells were also unable to adhere to the machined surface. Taken together, these results suggest that the in vivo biological behavior of Si3 N4 orthopaedic implants is driven by critical features of their surface nanotopography. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3413-3421, 2017.

Reconciling in vivo and in vitro kinetics of the polymorphic transformation in zirconia-toughened alumina for hip joints: I. Phenomenology.

Exploitation of the toughening effect induced by polymorphic phase transformation of zirconia in zirconia-toughened alumina (ZTA) requires the composite being properly designed and carefully manufactured. A sound algorithm for predicting phase stability along with strict control over manufacturing steps are required in order to prevent possible in vivo surface degradation or implant fracture. This paper is the first in a series of three monographs, which aim at: (i) statistically comparing the in vitro/in vivo phenomenology of surface-metastability for currently marketed ZTA femoral heads; (ii) refining pre-existing theoretical models for predicting in vivo zirconia phase metastability via the use of accelerated in vitro ageing experiments; and, (iii) providing a rationale for the mechanism(s) involved with the observed in vivo surface metastability. This initial paper of a series of three, which specifically deals with item (i), shows discrepancies between the levels of polymorphic phase transformation detected in ZTA retrievals and in vitro predictions, and attempts a phenomenological analysis of the reasons behind such discrepancies. Moreover, marked inhomogeneities are also found among as-manufactured components through different years of production. The phenomenology of retrievals' data suggests key roles for both the presence of metallic stain and the initial value of monoclinic volume fraction.

Primary TKA with a zirconia ceramic femoral component.

This article presents the 2-year clinical results of primary total knee arthroplasty (TKA) performed with a zirconia ceramic femoral component. A posterior-stabilized TKA was performed for degenerative arthritis in 36 patients (39 knees). The components included a zirconia femoral component, a cobalt-chrome alloy tibial baseplate, and a polyethylene patella; all were implanted with bone cement. The ultra-high molecular-weight polyethylene-bearing insert had a deep-dish, ultra-congruent design. At the 2-year interval, mean Western Ontario and McMaster Universities (WOMAC) osteoarthritis indices improved from 41 to 86, and mean Knee Society Scores improved from 40 to 92. Revision to constrained implants was necessary in one patient for persistent knee instability after trauma. These early results are encouraging, but more data are needed to determine whether ceramics are a suitable alternative to metal countersurfaces in TKAs.

A method for removing the polyethylene liner during revision total hip arthroplasty.

Extraction of the polyethylene liner is necessary if acetabular fixation screws and the metal acetabular shell are to be revised during total hip arthroplasty. In some situations, revision of the polyethylene liner alone may be indicated for excessive wear and osteolysis. A simple method for dissociating the liner from the shell is described in this report. No special instruments are needed with this technique.

Effect of copper-doped silicate 13-93 bioactive glass scaffolds on the response of MC3T3-E1 cells in vitro and on bone regeneration and angiogenesis in rat calvarial defects in vivo.

The release of inorganic ions from biomaterials could provide an alternative approach to the use of growth factors for improving tissue healing. In the present study, the release of copper (Cu) ions from bioactive silicate (13-93) glass scaffolds on the response of cells in vitro and on bone regeneration and angiogenesis in vivo was studied. Scaffolds doped with varying concentrations of Cu (0-2.0wt.% CuO) were created with a grid-like microstructure by robotic deposition. When immersed in simulated body fluid in vitro, the Cu-doped scaffolds released Cu ions into the medium in a dose-dependent manner and converted partially to hydroxyapatite. The proliferation and alkaline phosphatase activity of pre-osteoblastic MC3T3-E1 cells cultured on the scaffolds were not affected by 0.4 and 0.8wt.% CuO in the glass but they were significantly reduced by 2.0wt.% CuO. The percent new bone that infiltrated the scaffolds implanted for 6weeks in rat calvarial defects (46±8%) was not significantly affected by 0.4 or 0.8wt.% CuO in the glass whereas it was significantly inhibited (0.8±0.7%) in the scaffolds doped with 2.0wt.% CuO. The area of new blood vessels in the fibrous tissue that infiltrated the scaffolds increased with CuO content of the glass and was significantly higher for the scaffolds doped with 2.0wt.% CuO. Loading the scaffolds with bone morphogenetic protein-2 (1µg/defect) significantly enhanced bone infiltration and reduced fibrous tissue in the scaffolds. These results showed that doping the 13-93 glass scaffolds with up to 0.8wt.% CuO did not affect their biocompatibility whereas 2.0wt.% CuO was toxic to cells and detrimental to bone regeneration.