Publications by authors named "Furqan A Shah"

36 Publications

A stochastic micro to macro mechanical model for the evolution of bone-implant interface stiffness.

Acta Biomater 2021 09 12;131:415-423. Epub 2021 Jun 12.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden.

Upon placement of an implant into living bone, an interface is formed through which various biochemical, biological, physical, and mechanical interactions take place. This interface evolves over time as the mechanical properties of peri-implant bone increase. Owing to the multifactorial nature of interfacial processes, it is challenging to devise a comprehensive model for predicting the mechanical behavior of the bone-implant interface. We propose a simple spatio-temporally evolving mechanical model - from an elementary unit cell comprising randomly oriented mineralized collagen fibrils having randomly assigned stiffness all the way up to a macroscopic bone-implant interface in a gap healing scenario. Each unit cell has an assigned Young's modulus value between 1.62 GPa and 25.73 GPa corresponding to minimum (i.e., 0) and maximum (i.e., 0.4) limits of mineral volume fraction, respectively, in the overlap region of the mineralized collagen fibril. Gap closure and subsequent stiffening are modeled to reflect the two main directions of peri-implant bone formation, i.e., contact osteogenesis and distance osteogenesis. The linear elastic stochastic finite element model reveals highly nonlinear temporal evolution of bone-implant interface stiffness, strongly dictated by the specific kinetics of contact osteogenesis and distance osteogenesis. The bone-implant interface possesses a small stiffness until gap closure, which subsequently evolves into a much higher stiffness, and this transition is reminiscent of a percolation transition whose threshold corresponds to gap closure. The model presented here, albeit preliminary, can be incorporated into future calculations of the bone-implant system where the interface is well-defined mechanically. STATEMENT OF SIGNIFICANCE: A simple, physically informed model for the mechanical characteristics of the bone-implant interface is still missing. Here, we start by extending the reported mechanical characteristics of a one cubic micrometre unit cell to a 250 µm long interface made of 1 µm thick layers. The stiffness of each cell (based on mineral content) is assigned randomly to mimic bone micro-heterogeneity. The numerical study of this interface representative structure allows for the simultaneous determination of the spatio-temporal evolution of the mechanical response at local (discrete element) and global (overall model) scales. The proposed model is the first of this kind that can easily be incorporated into realistic future models of bone-implant interaction with emphasis on implant stability and different loading conditions.
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http://dx.doi.org/10.1016/j.actbio.2021.06.019DOI Listing
September 2021

Multimodal Analysis of the Tissue Response to a Bone-Anchored Hearing Implant: Presentation of a Two-Year Case Report of a Patient With Recurrent Pain, Inflammation, and Infection, Including a Systematic Literature Review.

Front Cell Infect Microbiol 2021 30;11:640899. Epub 2021 Mar 30.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Osseointegration is a well-established concept used in applications including the percutaneous Bone-Anchored Hearing System (BAHS) and auricular rehabilitation. To date, few retrieved implants have been described. A systematic review including cases where percutaneous bone-anchored implants inserted in the temporal bone were retrieved and analyzed was performed. We also present the case of a patient who received a BAHS for mixed hearing loss. After the initial surgery, several episodes of soft tissue inflammation accompanied by pain were observed, leading to elective abutment removal 14 months post-surgery. Two years post-implantation, the implant was removed due to pain and subjected to a multiscale and multimodal analysis: microbial DNA using molecular fingerprinting, gene expression using quantitative real-time polymerase chain reaction (qPCR), X-ray microcomputed tomography (micro-CT), histology, histomorphometry, backscattered scanning electron microscopy (BSE-SEM), Raman spectroscopy, and fluorescence hybridization (FISH). Evidence of osseointegration was provided micro-CT, histology, BSE-SEM, and Raman spectroscopy. Polymicrobial colonization in the periabutment area and on the implant, including that with and , was determined using a molecular analysis a 16S-23S rDNA interspace [IS]-region-based profiling method (IS-Pro). The histology suggested bacterial colonization in the skin and in the peri-implant bone. FISH confirmed the localization of and coagulase-negative staphylococci in the skin. Ten articles (54 implants, 47 patients) met the inclusion criteria for the literature search. The analyzed samples were either BAHS (35 implants) or bone-anchored aural epitheses (19 implants) between 2 weeks and 8 years. The main reasons for elective removal were nonuse/changes in treatment, pain, or skin reactions. Most samples were evaluated using histology, demonstrating osseointegration, but with the absence of bone under the implants' proximal flange. Taken together, the literature and this case report show clear evidence of osseointegration, despite prominent complications. Nevertheless, despite implant osseointegration, chronic pain related to the BAHS may be associated with a chronic bacterial infection and raised inflammatory response in the absence of macroscopic signs of infection. It is suggested that a multimodal analysis of peri-implant health provides possibilities for device improvements and to guide diagnostic and therapeutic strategies to alleviate the impact of complications.
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http://dx.doi.org/10.3389/fcimb.2021.640899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8042154PMC
July 2021

Magnesium whitlockite - omnipresent in pathological mineralisation of soft tissues but not a significant inorganic constituent of bone.

Authors:
Furqan A Shah

Acta Biomater 2021 04 18;125:72-82. Epub 2021 Feb 18.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Electronic address:

Whitlockite is a calcium phosphate that was first identified in minerals collected from the Palermo Quarry, New Hampshire. The terms magnesium whitlockite [Mg-whitlockite; CaMg(HPO)(PO)] and beta-tricalcium phosphate [β-TCP; β-Ca(PO)] are often used interchangeably since Mg-whitlockite is not easily distinguished from β-Ca(PO) by powder X-ray diffraction although their crystalline structures differ significantly. Being both osteoconductive and bioresorbable, Mg-whitlockite is pursued as a synthetic bone graft substitute. In recent years, advances in development of synthetic Mg-whitlockite have been accompanied by claims that Mg-whitlockite is the second most abundant inorganic constituent of bone, occupying as much as 20-35 wt% of the inorganic fraction. To find evidence in support of this notion, this review presents an exhaustive summary of Mg-whitlockite identification in biological tissues. Mg-whitlockite is mainly found in association with pathological mineralisation of various soft tissues and dental calculus, and occasionally with enamel and dentine. With the exception of high-temperature treated tumoural calcified deposits around interphalangeal and metacarpal joints and rhomboidal Mg-whitlockite crystals in post-apoptotic osteocyte lacunae in human alveolar bone, this unusual mineral has never been detected in the extracellular matrix of mammalian bone. Characterisation techniques capable of unequivocally distinguishing between different calcium phosphate phases, such as high-resolution imaging, crystallography, and/or spectroscopy have exclusively identified bone mineral as poorly crystalline, ion-substituted, carbonated apatite. The idea that Mg-whitlockite is a significant constituent of bone mineral remains unsubstantiated. Contrary to claims that such biomaterials represent a bioinspired/biomimetic approach to bone repair, Mg-whitlockite remains, exclusively, a pathological biomineral. STATEMENT OF SIGNIFICANCE: Magnesium whitlockite (Mg-whitlockite) is a unique calcium phosphate that typically features in pathological calcification of soft tissues; however, an alarming trend emerging in the synthetic bioceramics community claims that Mg-whitlockite occupies 20-35 wt% of bone mineral and therefore synthetic Mg-whitlockite represents a biomimetic approach towards bone regeneration. By providing an overview of Mg-whitlockite detection in biological tissues and scrutinising a diverse cross-section of literature relevant to bone composition analysis, this review concludes that Mg-whitlockite is exclusively a pathological biomineral, and having never been reported in bone extracellular matrix, Mg-whitlockite does not constitute a biomimetic strategy for bone repair.
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http://dx.doi.org/10.1016/j.actbio.2021.02.021DOI Listing
April 2021

Mapping Bone Surface Composition Using Real-Time Surface Tracked Micro-Raman Spectroscopy.

Cells Tissues Organs 2020 4;209(4-6):266-275. Epub 2021 Feb 4.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

The surface of bone tells a story - one that is worth a thousand words - of how it is built and how it is repaired. Chemical (i.e., composition) and physical (i.e., morphology) characteristics of the bone surface are analogous to a historical record of osteogenesis and provide key insights into bone quality. Analysis of bone chemistry is of particular relevance to the advancement of human health, cell biology, anthropology/archaeology, and biomedical engineering. Although scanning electron microscopy remains a popular and versatile technique to image bone across multiple length scales, limited chemical information can be obtained. Micro-Raman spectroscopy is a valuable tool for nondestructive chemical/compositional analysis of bone. However, signal integrity losses occur frequently during wide-field mapping of non-planar surfaces. Samples for conventional Raman imaging are, therefore, rendered planar through polishing or sectioning to ensure uniform signal quality. Here, we demonstrate ν1 PO43- and ν1 CO32- peak intensity losses where the sample surface and the plane of focus are offset by over 1-2 μm when underfocused and 2-3 μm when overfocused at 0.5-1 s integration time (15 mW, 633 nm laser). A technique is described for mapping the composition of the inherently irregular/non-planar surface of bone. The challenge posed by the native topology characteristic of this unique biological system is circumvented via real-time focus-tracking based on laser focus optimization by continuous closed-loop feedback. At the surface of deproteinized and decellularized/defatted sheep tibial cortical bone, regions of interest up to 1 mm2 were scanned at micrometer and submicrometer resolution. Despite surface height deviations exceeding 100 μm, it is possible to seamlessly probe local gradients in organic and inorganic constituents of the extracellular matrix as markers of bone metabolism and bone turnover, blood vessels and osteocyte lacunae, and the rope-like mineralized bundles that comprise the mineral phase at the bone surface.
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http://dx.doi.org/10.1159/000511079DOI Listing
February 2021

Early plaque formation on PTFE membranes with expanded or dense surface structures applied in the oral cavity of human volunteers.

Clin Exp Dent Res 2021 04 9;7(2):137-146. Epub 2020 Nov 9.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Objectives: This clinical randomized study aimed to evaluate the early plaque formation on nonresorbable polytetrafluoroethylene (PTFE) membranes having either a dense (d-PTFE) or an expanded (e-PTFE) microstructure and exposed to the oral cavity.

Material And Methods: Twelve individuals were enrolled in this study. In a split-mouth design, five test membranes (e-PTFE) with a dual-layer configuration and five control membranes (d-PTFE) were bonded on the buccal surfaces of posterior teeth of each subject. All study subjects refrained from toothbrushing during the study period. Specimens were detached from the teeth at 4 and 24 hr and subjected to viability counting, confocal microscopy, and scanning electron microscopy. Plaque samples were harvested from neighboring teeth at baseline, 4, and 24 hr, as control. Wilcoxon signed rank test was applied.

Results: No bond failure of the membranes was reported. Between the early and late time points, viable bacterial counts increased on all membranes, with no difference between the test and control. The number of Staphylococcus spp. decreased on the tooth surfaces and increased on both membranes overtime, with a significant difference compared to teeth. The total biomass and average biofilm thickness of live and dead cells were significantly greater at the d-PTFE barriers after 4 hr.

Conclusion: This study demonstrated that the e-PTFE membrane was associated with a lesser degree of biofilm accumulation during the initial exposure compared to the d-PTFE membrane. The present experimental setup provides a valuable toolbox to study the in vivo behavior of different membranes used in guided bone regeneration (GBR).
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http://dx.doi.org/10.1002/cre2.344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019762PMC
April 2021

Regenerating the Pulp-Dentine Complex Using Autologous Platelet Concentrates: A Critical Appraisal of the Current Histological Evidence.

Tissue Eng Regen Med 2021 02 4;18(1):37-48. Epub 2020 Nov 4.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, 405 30, Gothenburg, Sweden.

Background: Autologous platelet concentrates such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) have gained overwhelming popularity in regenerative endodontics. Clinical evidence reveals the lack of a particular advantage of using PRP or PRF over an evoked blood clot in promoting canal wall thickening and/or continued root development in immature necrotic teeth. Moreover, despite stimulating tissue repair and repopulating the root canals of immature and mature permanent teeth, the new vital tissue may not possess the functional activity of the native pulp tissue.

Methods: To better understand the origin, nature, and long-term fate of the tissue types found within the pulp space, we critically examine all available histo-/morphological evidence for pulp-dentine complex regeneration using PRP and/or PRF, alone or together with an evoked blood clot, specialised or unspecialised primary cells, and other biomaterials.

Results: Histological data from clinical studies is scant. Reportedly, the inner dentinal surface supports cementum-like tissue formation, but this interface likely deviates in structure and function from the native cementodentinal junction. Presence of bone-like tissue within the pulp space is intriguing since de novo osteogenesis requires closely coordinated recruitment and differentiation of osteoprogenitor cells. Compared to untreated necrotic teeth, an evoked blood clot (with/without PRF) improves fracture resistance. Tooth regeneration using PRF and dental bud cells is unreliable and the constituent neoformed tissues are poorly organised.

Conclusion: PRP/PRF fail to demonstrate a significant advantage over an induced blood clot, alone. The true nature of neoformed tissues remains poorly characterised while their response to subsequent insult/injury is unexplored.
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http://dx.doi.org/10.1007/s13770-020-00291-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862478PMC
February 2021

In situ bone regeneration of large cranial defects using synthetic ceramic implants with a tailored composition and design.

Proc Natl Acad Sci U S A 2020 10 12;117(43):26660-26671. Epub 2020 Oct 12.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, 40530 Gothenburg, Sweden;

The repair of large cranial defects with bone is a major clinical challenge that necessitates novel materials and engineering solutions. Three-dimensionally (3D) printed bioceramic (BioCer) implants consisting of additively manufactured titanium frames enveloped with CaP BioCer or titanium control implants with similar designs were implanted in the ovine skull and at s.c. sites and retrieved after 12 and 3 mo, respectively. Samples were collected for morphological, ultrastructural, and compositional analyses using histology, electron microscopy, and Raman spectroscopy. Here, we show that BioCer implants provide osteoinductive and microarchitectural cues that promote in situ bone regeneration at locations distant from existing host bone, whereas bone regeneration with inert titanium implants was confined to ingrowth from the defect boundaries. The BioCer implant promoted bone regeneration at nonosseous sites, and bone bonding to the implant was demonstrated at the ultrastructural level. BioCer transformed to carbonated apatite in vivo, and the regenerated bone displayed a molecular composition indistinguishable from that of native bone. Proof-of-principle that this approach may represent a shift from mere reconstruction to in situ regeneration was provided by a retrieved human specimen, showing that the BioCer was transformed into well-vascularized osteonal bone, with a morphology, ultrastructure, and composition similar to those of native human skull bone.
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http://dx.doi.org/10.1073/pnas.2007635117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604495PMC
October 2020

Towards refining Raman spectroscopy-based assessment of bone composition.

Authors:
Furqan A Shah

Sci Rep 2020 10 7;10(1):16662. Epub 2020 Oct 7.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Various compositional parameters are derived using intensity ratios and integral area ratios of different spectral peaks and bands in the Raman spectrum of bone. The [Formula: see text]-, [Formula: see text]-,[Formula: see text]-, [Formula: see text] PO, and [Formula: see text] CO bands represent the inorganic phase while amide I, amide III, Proline, Hydroxyproline, Phenylalanine, δ(CH), δ(CH), and [Formula: see text](C-H) represent the organic phase. Here, using high-resolution Raman spectroscopy, it is demonstrated that all PO bands of bone either partially overlap with or are positioned close to spectral contributions from the organic component. Assigned to the organic component, a shoulder at 393 cm compromises accurate estimation of [Formula: see text] PO integral area, i.e., phosphate/apatite content, with implications for apatite-to-collagen and carbonate-to-phosphate ratios. Another feature at 621 cm may be inaccurately interpreted as [Formula: see text] PO band broadening. In the 1020-1080 cm range, the ~ 1047 cm [Formula: see text] PO sub-component is obscured by the 1033 cm Phenylalanine peak, while the ~ 1076 cm [Formula: see text] PO sub-component is masked by the [Formula: see text] CO band. With [Formula: see text] PO peak broadening, [Formula: see text] PO integral area increases exponentially and individual peaks comprising the [Formula: see text] PO band merge together. Therefore, [Formula: see text] PO and [Formula: see text] PO band profiles are sensitive to changes in mineral crystallinity.
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http://dx.doi.org/10.1038/s41598-020-73559-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541616PMC
October 2020

Extracellular Vesicles Influence the Growth and Adhesion of Under Antimicrobial Selective Pressure.

Front Microbiol 2020 2;11:1132. Epub 2020 Jul 2.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

causes infections associated with orthopedic implants due to its ability to establish persistent biofilms, making infections chronic and hard to treat. Extracellular vesicles (EVs) are part of the bacterial communication system, but the role of -derived EVs in biofilm formation processes and survival is completely unknown. The aims of this study were (i) to investigate the effect of subinhibitory concentrations of antibiotics on vesiculation in and evaluate the role of EVs in bacterial survival and adhesion under antimicrobial selective pressure and (ii) to evaluate whether EVs derived from a gentamicin-resistant strain influence the susceptibility and adhesion of a gentamicin-susceptible strain. A gentamicin-susceptible (GEN ) strain isolated from implant-associated osteomyelitis was cultured with EVs previously isolated from the same strain growing with subinhibitory concentrations of GEN (0, 0.03, and 0.06 μg × mL) or with EVs from a gentamicin-resistant (GEN ) strain. EVs were characterized regarding their size, number and protein content. The growth of cultured with increasing concentrations of GEN (<=> MIC of 0.12 μg × mL) was recorded, viability was determined by quantitative culturing and fluorescence staining, and biofilm biomass on polystyrene was quantified by crystal violet staining. Cells grown in subinhibitory concentrations of GEN produced a larger number of EVs of similar size but with greater protein content than cells grown in control (Ctrl) conditions (0 GEN). Under antimicrobial pressure, EVs promoted different mechanisms of antimicrobial tolerance depending on the EV and GEN concentrations. Cell adhesion to polystyrene decreased in the presence of 0 and 0.03 μg × mL GEN upon EV stimulation. Compared with Ctrl cells, cells treated with EVs from a GEN strain showed increased cell division during the exponential growth phase, faster maximal growth rate, shorter doubling time (8-33 min), and dramatically inhibited cell adhesion. These findings suggest that vesiculation in is a survival response to subinhibitory concentrations of gentamicin. EVs may contribute to bacterial survival through their involvement (1) in the modulation of the growth rate, affecting cell division, and (2) in cell adhesion, decreasing cell attachment to polystyrene and glass.
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http://dx.doi.org/10.3389/fmicb.2020.01132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346684PMC
July 2020

Characterization of Synthetic Hydroxyapatite Fibers Using High-Resolution, Polarized Raman Spectroscopy.

Authors:
Furqan A Shah

Appl Spectrosc 2021 Apr 28;75(4):475-479. Epub 2020 Jul 28.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

In the Raman spectrum of B-type carbonated apatites, the ν CO mode (at ∼1070 cm) overlaps the ν PO band. The latter is readily observed where the CO content is low (up to ∼3 wt%). The CO content of bone is considerably higher (∼7-9 wt%). As a result, the ν PO band becomes completely obscured. The 1000-1100 cm spectral range of carbonated apatite is frequently considered a combined ν PO and ν CO region. Here, high-resolution polarized Raman spectroscopy (step size of 0.74 ± 0.04 cm) provides new insights into synthetic hydroxyapatite (HAp) obtained as micrometer-sized fibers. Compared to bone mineral (deproteinized bovine bone), spectral features of HAp fibers are highly resolved. In particular, the ν PO band resolves into nine distinct sub-components: 1028, 1032, 1040, 1043, 1047, 1053, 1055, 1062, and 1076 cm. Parameters including full width half-maximum, intensity, area fraction, intensity ratio, and area fraction ratio vary between parallel and perpendicular polarized configurations. It is likely that the ν CO band of B-type carbonated apatites may contain a small but not insignificant contribution from the 1076 cm sub-component of the ν PO band. Furthermore, the 1076 cm/1047 cm ratio changes between parallel and perpendicular scattering configurations, suggesting that the contribution of the 1076 cm sub-component may vary as a function of local orientation of bone mineral, thus skewing the ν CO band and compromising accurate estimation of carbonate-to-phosphate ratios in B-type CO substituted apatite.
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http://dx.doi.org/10.1177/0003702820942540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8027940PMC
April 2021

Transformation of bone mineral morphology: From discrete marquise-shaped motifs to a continuous interwoven mesh.

Bone Rep 2020 Dec 19;13:100283. Epub 2020 May 19.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Continual bone apposition at the cranial sutures provides the unique opportunity to understand how bone is built. Bone harvested from 16-week-old Sprague Dawley rat calvaria was either () deproteinised to isolate the inorganic phase (i.e., bone mineral) for secondary electron scanning electron microscopy or () resin embedded for X-ray micro-computed tomography, backscattered electron scanning electron microscopy, and micro-Raman spectroscopy. Interdigitated finger-like projections form the interface between frontal and parietal bones. Viewed from the surface, bone mineral at the mineralisation front is comprised of nanoscale mineral platelets arranged into discrete, ~0.6-3.5 μm high and ~0.2-1.5 μm wide, marquise-shaped motifs that gradually evolve into a continuous interwoven mesh of mineralised bundles. Marquise-shaped motifs also contribute to the burial of osteoblastic-osteocytes by contributing to the roof over the lacunae. In cross-section, apices of the finger-like projections resemble islands of mineralised tissue, where new bone apposition at the surface is evident as low mineral density areas, while the marquise-shaped motifs appear as near-equiaxed assemblies of mineral platelets. Carbonated apatite content is higher towards the internal surface of the cranial vault. Up to 4 μm from the bone surface, strong Amide III, Pro, Hyp, and Phe signals, distinct PO bands, but negligible CO signal indicate recent bone formation and/or delayed maturation of the mineral. We show, for the first time, that the extracellular matrix of bone is assembled into micrometre-sized units, revealing a superstructure above the mineralised collagen fibril level, which has significant implications for function and mechanical competence of bone.
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http://dx.doi.org/10.1016/j.bonr.2020.100283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305389PMC
December 2020

Exosomes influence the behavior of human mesenchymal stem cells on titanium surfaces.

Biomaterials 2020 02 24;230:119571. Epub 2019 Oct 24.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. Electronic address:

Mesenchymal stem cells (MSCs) have important roles during osseointegration. This study determined (i) if MSC-derived extracellular vesicles (EVs)/exosomes can be immobilized on titanium (Ti) surfaces and influence the behavior of MSCs, (ii) if the response is differentially affected by EVs from expanded vs differentiated MSCs and (iii) if the EV protein cargos predict the functional features of the exosomes. EVs secreted by human adipose-derived MSCs were isolated by ultracentrifugation and analyzed using nanoparticle tracking analysis, Western blotting and relative quantitative mass spectrometry. Fluorescence microscopy, scanning electron microscopy, cell counting assay and quantitative polymerase chain reaction were used to analyze MSC adhesion, proliferation and differentiation. Exosome immobilization on Ti promoted MSC adhesion and spreading after 24 h and proliferation after 3 and 6 days, irrespective of whether the exosomes were obtained from expansion or differentiation conditions. Immobilized exosomes upregulated stromal cell-derived factor (SDF-1α) gene expression. Cell adhesion molecules and signaling molecules were abundant in the exosomal proteome. The predicted functions of the equally-abundant proteins in both exosome types were in line with the observed biological effects mediated by the exosomes. Thus, exosomes derived from MSCs and immobilized on Ti surfaces interact with MSCs and rapidly promote MSC adhesion and proliferation. These findings provide a novel route for modification of titanium implant surfaces.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119571DOI Listing
February 2020

Does Smoking Impair Bone Regeneration in the Dental Alveolar Socket?

Calcif Tissue Int 2019 12 18;105(6):619-629. Epub 2019 Sep 18.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Smoking is a major risk factor for dental implant failure. In addition to higher marginal bone loss around implants, the cellular and molecular responses to injury and implant physicochemical properties are also differentially affected in smokers. The purpose of this work is to determine if smoking impairs bone microstructure and extracellular matrix composition within the dental alveolar socket after tooth extraction. Alveolar bone biopsies obtained from Smokers (> 10 cigarettes per day for at least 10 years) and Ctrl (never-smokers), 7-146 months after tooth extraction, were investigated using X-ray micro-computed tomography, backscattered electron scanning electron microscopy, and Raman spectroscopy. Both Smokers and Ctrl exhibited high inter- and intra-individual heterogeneity in bone microstructure, which varied between dense cortical and porous trabecular architecture. Regions of disorganised/woven bone were more prevalent during early healing. Remodelled lamellar bone was predominant at longer healing periods. Bone mineral density, bone surface-to-volume ratio, mineral crystallinity, the carbonate-to-phosphate ratio, the mineral-to-matrix ratio, the collagen crosslink ratio, and the amounts of amino acids phenylalanine and proline/hydroxyproline were also comparable between Smokers and Ctrl. Bone microstructure and composition within the healing dental alveolar socket are not significantly affected by moderate-to-heavy smoking.
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http://dx.doi.org/10.1007/s00223-019-00610-4DOI Listing
December 2019

Extracellular matrix composition during bone regeneration in the human dental alveolar socket.

Bone 2019 10 6;127:244-249. Epub 2019 Jun 6.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Within the dental alveolar socket, the sequence of events following tooth extraction involves deposition of a provisional connective tissue matrix that is later replaced by woven bone and eventually by lamellar bone. Bone regeneration within the dental alveolar socket is unique since the space occupied by the root(s) of a tooth does not originally contain any bone. However, extracellular matrix composition of the healing alveolar socket has not previously been investigated. Here, alveolar bone biopsies representing early (7-46 months, < 4y) and late (48-60 months; 4-5y) healing periods were investigated using Raman spectroscopy, X-ray micro-computed tomography and backscattered electron scanning electron microscopy. Partially or completely edentulous individuals and those with a smoking habit were not excluded. Between < 4y and 4-5y, mineral crystallinity and bone mineral density increase, phenylalanine, proline/hydroxyproline, and bone surface-to-volume ratio decrease, while the carbonate-to-phosphate ratio, the mineral-to-matrix ratio, and the collagen crosslink ratio remain relatively unchanged. Observed exclusively at 4-5y, hypermineralised osteocyte lacunae contain spherical and rhomboidal mineral nodules. Spearman correlation analysis reveals several significant, high (ρ = 0.7-0.9; p ≤ 0.01) and moderate (ρ = 0.5-0.7; p ≤ 0.01) correlations. Mineral crystallinity and proline/hydroxyproline, the carbonate-to-phosphate ratio and phenylalanine, mineral crystallinity and bone surface-to-volume ratio, the carbonate-to-phosphate ratio and bone surface-to-volume ratio, proline/hydroxyproline and bone mineral density, and bone mineral density and bone surface-to-volume ratio are negatively correlated. Mineral crystallinity and bone mineral density, and proline/hydroxyproline and bone surface-to-volume ratio are positively correlated. Although bone regeneration in the dental alveolar socket follows typical bone healing patterns, the compositional and microstructural patterns reveal mature bone at <4y with indications of better mechanical competence at 4-5y.
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http://dx.doi.org/10.1016/j.bone.2019.06.003DOI Listing
October 2019

50 years of scanning electron microscopy of bone-a comprehensive overview of the important discoveries made and insights gained into bone material properties in health, disease, and taphonomy.

Bone Res 2019 22;7:15. Epub 2019 May 22.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Bone is an architecturally complex system that constantly undergoes structural and functional optimisation through renewal and repair. The scanning electron microscope (SEM) is among the most frequently used instruments for examining bone. It offers the key advantage of very high spatial resolution coupled with a large depth of field and wide field of view. Interactions between incident electrons and atoms on the sample surface generate backscattered electrons, secondary electrons, and various other signals including X-rays that relay compositional and topographical information. Through selective removal or preservation of specific tissue components (organic, inorganic, cellular, vascular), their individual contribution(s) to the overall functional competence can be elucidated. With few restrictions on sample geometry and a variety of applicable sample-processing routes, a given sample may be conveniently adapted for multiple analytical methods. While a conventional SEM operates at high vacuum conditions that demand clean, dry, and electrically conductive samples, non-conductive materials (e.g., bone) can be imaged without significant modification from the natural state using an environmental scanning electron microscope. This review highlights important insights gained into bone microstructure and pathophysiology, bone response to implanted biomaterials, elemental analysis, SEM in paleoarchaeology, 3D imaging using focused ion beam techniques, correlative microscopy and in situ experiments. The capacity to image seamlessly across multiple length scales within the meso-micro-nano-continuum, the SEM lends itself to many unique and diverse applications, which attest to the versatility and user-friendly nature of this instrument for studying bone. Significant technological developments are anticipated for analysing bone using the SEM.
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http://dx.doi.org/10.1038/s41413-019-0053-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531483PMC
May 2019

Micro-Raman Spectroscopy Reveals the Presence of Octacalcium Phosphate and Whitlockite in Association with Bacteria-Free Zones Within the Mineralized Dental Biofilm.

Authors:
Furqan A Shah

Microsc Microanal 2019 02 4;25(1):129-134. Epub 2019 Feb 4.

Department of Biomaterials, Sahlgrenska Academy,University of Gothenburg,Göteborg,Sweden.

Through a correlative analytical approach encompassing backscattered electron scanning electron microscopy (BSE-SEM), energy dispersive X-ray spectroscopy (EDX), and micro-Raman spectroscopy, the composition of the mineralized biofilm around a dental implant, retrieved due to peri-implantitis, was investigated. The mineralized biofilm contains two morphologically distinct regions: (i) bacteria-containing zones (Bact+), characterized by aggregations of unmineralized and mineralized bacteria, and intermicrobial mineralization, and (ii) bacteria-free zones (Bact-), comprised mainly of randomly oriented mineral platelets. Intramicrobial mineralization, within Bact+, appears as smooth, solid mineral deposits resembling the morphologies of dental plaque bacteria. Bact- is associated with micrometer-sized Mg-rich mineral nodules. The Ca/P ratio of Bact+ is higher than Bact-. The inorganic phase of Bact+ is carbonated apatite (CHAp), while that of Bact- is predominantly octacalcium phosphate (OCP) and whitlockite (WL) inclusions. Compared with native bone, the inorganic phase of Bact+ (i.e., CHAp) exhibits higher mineral crystallinity, lower carbonate content, and lower Ca/P, C/Ca, Mg/Ca, and Mg/P ratios. The various CaPs found within the mineralized dental biofilm (CHAp, OCP, and WL) are related to the local presence/absence of bacteria. In combination with BSE-SEM and EDX, micro-Raman spectroscopy is a valuable analytical tool for nondestructive investigation of mineralized dental biofilm composition and development.
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http://dx.doi.org/10.1017/S1431927618015659DOI Listing
February 2019

Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.

FASEB J 2019 04 22;33(4):5237-5247. Epub 2019 Jan 22.

Department of Internal Medicine and Clinical Nutrition, Centre for Bone and Arthritis Research, Institute for Medicine, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

Increased vitamin A consumption is associated with decreased cortical bone mass and increased fracture risk in humans. Rodent studies have demonstrated that hypervitaminosis A increases cortical bone resorption, whereas the importance of the effects on bone formation is less well defined. We used an experimental model of increased bone formation by loading of the tibiae to investigate the effect of vitamin A on bone formation. Control [retinol activity equivalents (RAE) 4.5 µg/g chow] or vitamin A (RAE 60 µg/g chow) diets were given to female C57BL/6N mice for 4 wk, after which the tibiae were subjected to axial loading on alternate days for 2 wk, while the diets were continued. Vitamin A inhibited the loading-induced increase in trabecular and cortical bone volume. This was attributed to inhibition of loading-induced increase in osteoblast number and activity, and expression of osteoblastic genes Sp7, Alpl, and Col1a1 in cortical bone. Vitamin A, loading, and combination thereof also resulted in site-specific effects on bone composition measured by Raman spectroscopy. In summary, a clinically relevant dose of vitamin A suppresses the loading-induced gain of bone mass by decreasing bone formation. These observations may have implications for regulation of bone mass caused by physical activity and the risk of osteoporosis in humans.-Lionikaite, V., Henning, P., Drevinge, C., Shah, F. A., Palmquist, A., Wikström, P., Windahl, S. H., Lerner, U. H. Vitamin A decreases the anabolic bone response to mechanical loading by suppressing bone formation.
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http://dx.doi.org/10.1096/fj.201802040RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436664PMC
April 2019

Osseointegration and current interpretations of the bone-implant interface.

Acta Biomater 2019 01 13;84:1-15. Epub 2018 Nov 13.

Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden.

Complex physical and chemical interactions take place in the interface between the implant surface and bone. Various descriptions of the ultrastructural arrangement to various implant design features, ranging from solid and macroporous geometries to surface modifications on the micron-, submicron-, and nano- levels, have been put forward. Here, the current knowledge regarding structural organisation of the bone-implant interface is reviewed with a focus on solid devices, mainly metal (or alloy) intended for permanent anchorage in bone. Certain biomaterials that undergo surface and bulk degradation are also considered. The bone-implant interface is a heterogeneous zone consisting of mineralised, partially mineralised, and unmineralised areas. Within the meso-micro-nano-continuum, mineralised collagen fibrils form the structural basis of the bone-implant interface, in addition to accumulation of non-collagenous macromolecules such as osteopontin, bone sialoprotein, and osteocalcin. In the published literature, as many as eight distinct arrangements of the bone-implant interface ultrastructure have been described. The interpretation is influenced by the in vivo model and species-specific characteristics, healing time point(s), physico-chemical properties of the implant surface, implant geometry, sample preparation route(s) and associated artefacts, analytical technique(s) and their limitations, and non-compromised vs compromised local tissue conditions. The understanding of the ultrastructure of the interface under experimental conditions is rapidly evolving due to the introduction of novel techniques for sample preparation and analysis. Nevertheless, the current understanding of the interface zone in humans in relation to clinical implant performance is still hampered by the shortcomings of clinical methods for resolving the finer details of the bone-implant interface. STATEMENT OF SIGNIFICANCE: Being a hierarchical material by design, the overall strength of bone is governed by composition and structure. Understanding the structure of the bone-implant interface is essential in the development of novel bone repair materials and strategies, and their long-term success. Here, the current knowledge regarding the eventual structural organisation of the bone-implant interface is reviewed, with a focus on solid devices intended for permanent anchorage in bone, and certain biomaterials that undergo surface and bulk degradation. The bone-implant interface is a heterogeneous zone consisting of mineralised, partially mineralised, and unmineralised areas. Within the meso-micro-nano-continuum, mineralised collagen fibrils form the structural basis of the bone-implant interface, in addition to accumulation of non-collagenous macromolecules such as osteopontin, bone sialoprotein, and osteocalcin.
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http://dx.doi.org/10.1016/j.actbio.2018.11.018DOI Listing
January 2019

In vitro evaluation of barrier function against oral bacteria of dense and expanded polytetrafluoroethylene (PTFE) membranes for guided bone regeneration.

Clin Implant Dent Relat Res 2018 Oct 24;20(5):738-748. Epub 2018 Jul 24.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden.

Aim: This study evaluates biofilm formation and barrier function against Streptococcus oralis of nonresorbable polytetrafluoroethylene (PTFE) guided bone regeneration membranes having expanded (e-PTFE) and dense (d-PTFE) microstructure.

Materials And Methods: Three e-PTFE membranes of varying openness, one d-PTFE membrane, and commercially pure titanium discs were evaluated. All e-PTFE membranes consisted of PTFE nodes interconnected by fibrils. The d-PTFE membrane was fibril-free, with large evenly spaced indentations. The surfaces were challenged with S. oralis and incubated statically for 2-48h. Bacterial colonization, viability, and penetration were evaluated.

Results: S. oralis numbers increased over time on all surfaces, as observed using scanning electron microscopy, while cell viability decreased, as measured by colony forming unit (CFU) counting. At 24h and 48h, biofilms on d-PTFE were more mature and thicker (tower formations) than on e-PTFE, where fewer layers of cells were distributed mainly horizontally. Biofilms accumulated preferentially within d-PTFE membrane indentations. At 48h, greater biofilm biomass and number of viable S. oralis were found on d-PTFE compared to e-PTFE membranes. All membranes were impermeable to S. oralis cells.

Conclusions: All PTFE membranes were effective barriers against bacterial passage in vitro. However, d-PTFE favored S. oralis biofilm formation.
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http://dx.doi.org/10.1111/cid.12629DOI Listing
October 2018

Interactions between monocytes, mesenchymal stem cells, and implants evaluated using flow cytometry and gene expression.

J Tissue Eng Regen Med 2018 07 5;12(7):1728-1741. Epub 2018 Jun 5.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.

Monocytes and mesenchymal stem cells (MSC) are evident at the implants during early healing. However, when coexisting, their interactions at different implants have not been determined. This study uses an in vitro system, consisting of monoculture and direct co-cultures of monocytes and MSC on screw-shaped machined and oxidized titanium implants in combination with scanning electron microscopy, enzyme-linked immunosorbent assay, flow cytometry, cell sorting, and quantitative polymerase chain reaction. The cell-specific adhesion and gene expression of monocytes and MSC was determined. After 24 hr, the coexistence of monocytes and MSC in co-culture led to equal proportions of adherent monocytes and MSC, irrespective of the implant type. In contrast, higher number of adherent monocytes than MSC was found on the oxidized implant in monoculture. Quantitative polymerase chain reaction analysis of fluorescent activated cell sorting-sorted cells revealed up-regulation of interleukin-1beta, in monocytes, and interleukin-1beta and C-X-C chemokine receptor type 4, in MSC, when the cell types coexisted compared with monocultures. Further, in co-culture, the expression of bone morphogenetic protein-2, stromal cell-derived factor 1, and integrin-β1 was enhanced in the implant-adherent MSC, but not monocytes. It is concluded that during the first 24 hr in an in vitro static condition, the effect of co-culture of monocytes and MSC was more prominent than the effect of the implant surface properties. The results indicate that the coexistence of monocytes and MSC on an implant alters the adhesion and expression of some genes compared with when each cell type existed alone. Further, the results show that the gene expression of major growth and recruitment factors is mainly enhanced in the implant-adherent MSC in contrast to implant-adherent monocytes in co-culture.
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http://dx.doi.org/10.1002/term.2700DOI Listing
July 2018

Osseointegration of 3D printed microalloyed CoCr implants-Addition of 0.04% Zr to CoCr does not alter bone material properties.

J Biomed Mater Res A 2018 06 5;106(6):1655-1663. Epub 2018 Mar 5.

Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden.

Electron beam melting (EBM) is a three-dimensional (3D) printing technique for the production of metal structures where complex geometries with interconnected porosities can be built. Incorporation of as little as 0.04% Zr into the CoCr alloy can significantly improve the biomechanical anchorage of constructs fabricated by EBM. Here we investigate bone material properties, including microstructure and composition, adjacent to 3D printed CoCr implants with and without addition of 0.04% Zr, after 8 weeks of healing in the rabbit femur. In low amounts, zirconium addition does not alter the microstructure and extracellular matrix composition of bone formed adjacent to the surface of EBM manufactured implants. Bone ingrowth into surface irregularities of 3D printed CoCr and CoCr + Zr implants is seen. Extensive remodeling is also evident. Osteocytes attach directly on to the implant surface. The interfacial tissue at CoCr and CoCr + Zr has similar mineral crystallinity, apatite-to-collagen ratio, carbonate-to-phosphate ratio, Ca/P ratio, bone-implant contact, percentage porosity, and osteocyte density (N.Ot/B.Ar). Compared to the native bone, the mineral crystallinity of the interfacial tissue was lower while N.Ot/B.Ar was higher for both CoCr and CoCr + Zr. Overall, the results indicate that bone tissue adjacent to CoCr and CoCr + Zr implants is highly mature and exhibits comparable healing kinetics. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1655-1663, 2018.
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http://dx.doi.org/10.1002/jbm.a.36366DOI Listing
June 2018

Multiscale characterization of cortical bone composition, microstructure, and nanomechanical properties in experimentally induced osteoporosis.

J Biomed Mater Res A 2018 04 8;106(4):997-1007. Epub 2017 Dec 8.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden.

Cortical bone plays a vital role in determining overall bone strength. We investigate the structural, compositional, and nanomechanical properties of cortical bone following ovariectomy (OVX) of 12-week-old Sprague Dawley rats, since this animal model is frequently employed to evaluate the performance of implantable biomaterials in compromised bone healing conditions. Morphological parameters and material properties of bone in the geometrical center of the femoral cortex were investigated four and eight weeks post-OVX and in unoperated controls (Ctrl), using X-ray micro-computed tomography, backscattered electron scanning electron microscopy, Raman spectroscopy, and nanoindentation. The OVX animals showed increase in body weight, diminished bone mineral density, increased intracortical porosity, but increased bone mass through periosteal apposition (e.g., increases in periosteal perimeter, cortical cross-sectional thickness, and cross-sectional area). However, osteocyte densities, osteocyte lacunar dimensions, and the nanomechanical behavior on the single mineralized collagen fibril level remained unaffected. Our correlative multiscale investigation provides structural, chemical, and nanomechanical evidence substantiating earlier reports suggesting that rats ovariectomized at 12 weeks undergo simultaneous bone loss and growth, resulting in the effects of OVX being less obvious. Periosteal apposition contradicts the conventional view of bone loss in osteoporosis but appears advantageous for the greater functional demand imposed on the skeleton by increased body weight and fragility induced by increased intracortical porosity. Through a variety of morphological changes, it is likely that 12-week-old rats are able to adapt to OVX-related microstructural and compositional alterations. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 997-1007, 2018.
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http://dx.doi.org/10.1002/jbm.a.36294DOI Listing
April 2018

Micrometer-Sized Magnesium Whitlockite Crystals in Micropetrosis of Bisphosphonate-Exposed Human Alveolar Bone.

Nano Lett 2017 10 18;17(10):6210-6216. Epub 2017 Sep 18.

Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg , SE-405 30 Göteborg, Sweden.

Osteocytes are contained within spaces called lacunae and play a central role in bone remodelling. Administered frequently to prevent osteoporotic fractures, antiresorptive agents such as bisphosphonates suppress osteocyte apoptosis and may be localized within osteocyte lacunae. Bisphosphonates also reduce osteoclast viability and thereby hinder the repair of damaged tissue. Osteocyte lacunae contribute to toughening mechanisms. Following osteocyte apoptosis, the lacunar space undergoes mineralization, termed "micropetrosis". Hypermineralized lacunae are believed to increase bone fragility. Using nanoanalytical electron microscopy with complementary spectroscopic and crystallographic experiments, postapoptotic mineralization of osteocyte lacunae in bisphosphonate-exposed human bone was investigated. We report an unprecedented presence of ∼80 nm to ∼3 μm wide, distinctly faceted, magnesium whitlockite [CaMg(HPO)(PO)] crystals and consequently altered local nanomechanical properties. These findings have broad implications on the role of therapeutic agents in driving biomineralization and shed new insights into a possible relationship between bisphosphonate exposure, availability of intracellular magnesium, and pathological calcification inside lacunae.
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http://dx.doi.org/10.1021/acs.nanolett.7b02888DOI Listing
October 2017

Evidence that Osteocytes in Autogenous Bone Fragments can Repair Disrupted Canalicular Networks and Connect with Osteocytes in de novo Formed Bone on the Fragment Surface.

Calcif Tissue Int 2017 09 10;101(3):321-327. Epub 2017 May 10.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden.

Autogenous bone fragments generated during surgery (e.g. implant site preparation) accelerate bone formation by the release of a large variety of growth factors from the extracellular matrix and the cells contained within. Osteocytes, whether viable or apoptotic, within such fragments are able to recruit osteoclasts to a site of bone remodelling. Here, using correlative scanning electron microscopy, we provide compelling evidence that at one week healing in the Sprague Dawley rat tibia, following surgery (and/or the placement of a bone-anchored implant), autogenous bone fragments support bone formation on their surface. Furthermore, osteocytes within the autogenous fragments are frequently able to repair the disrupted canalicular networks and appear to connect with osteocytes (or osteoblastic-osteocytes) in the de novo formed bone on the surface of the fragment.
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http://dx.doi.org/10.1007/s00223-017-0283-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544810PMC
September 2017

3D Characterization of Human Nano-osseointegration by On-Axis Electron Tomography without the Missing Wedge.

ACS Biomater Sci Eng 2017 Jan 1;3(1):49-55. Epub 2016 Dec 1.

Department of Materials Science and Engineering, McMaster University, Hamilton, Ontario L8S 4L8, Canada.

Three-dimensional (3D) visualization of bone-implant interfaces via electron tomography (ET) has contributed to the novel perspective of nano-osseointegration and offers evidential support for nanoscaled biomaterial surface modification. Conventional single-axis ET provides a relatively large field of view of the human bone to titanium implant interface showing bone structure arrangement near the interface. However, the "missing wedge" associated with conventional single-axis ET leads to artifacts and elongation in the reconstruction, limiting the resolution and fidelity of reconstructions, as well as the ability to extract quantitative information from nanostructured interfaces. On-axis ET, performed by 180° rotation of a needle-shaped sample, is a promising method to solve this problem. In this work, we present the first application of on-axis ET for investigation of human bone and laser-modified titanium implant interfaces without the missing wedge. This work demonstrates a near artifact-free 3D visualization of the nanotopographies of the implant surface oxide layer and bone growth into these features. Complementary electron energy-loss spectroscopy (EELS) mapping was used to illustrate the gradual intermixing of carbon and calcium (characteristic elements of bone) with the nanoscaled oxide layer of the implant surface. Ultimately, this approach serves as direct evidence of nano-osseointegration and as a potential platform to evaluate differently structured implant surfaces.
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http://dx.doi.org/10.1021/acsbiomaterials.6b00519DOI Listing
January 2017

A technique for evaluating bone ingrowth into 3D printed, porous Ti6Al4V implants accurately using X-ray micro-computed tomography and histomorphometry.

Micron 2017 Mar 21;94:1-8. Epub 2016 Nov 21.

Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden; BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Göteborg, Sweden.

This paper investigates the application of X-ray micro-computed tomography (micro-CT) to accurately evaluate bone formation within 3D printed, porous Ti6Al4V implants manufactured using Electron Beam Melting (EBM), retrieved after six months of healing in sheep femur and tibia. All samples were scanned twice (i.e., before and after resin embedding), using fast, low-resolution scans (Skyscan 1172; Bruker micro-CT, Kontich, Belgium), and were analysed by 2D and 3D morphometry. The main questions posed were: (i) Can low resolution, fast scans provide morphometric data of bone formed inside (and around) metal implants with a complex, open-pore architecture?, (ii) Can micro-CT be used to accurately quantify both the bone area (BA) and bone-implant contact (BIC)?, (iii) What degree of error is introduced in the quantitative data by varying the threshold values?, and (iv) Does resin embedding influence the accuracy of the analysis? To validate the accuracy of micro-CT measurements, each data set was correlated with a corresponding centrally cut histological section. The results show that quantitative histomorphometry corresponds strongly with 3D measurements made by micro-CT, where a high correlation exists between the two techniques for bone area/volume measurements around and inside the porous network. On the contrary, the direct bone-implant contact is challenging to estimate accurately or reproducibly. Large errors may be introduced in micro-CT measurements when segmentation is performed without calibrating the data set against a corresponding histological section. Generally, the bone area measurement is strongly influenced by the lower threshold limit, while the upper threshold limit has little or no effect. Resin embedding does not compromise the accuracy of micro-CT measurements, although there is a change in the contrast distributions and optimisation of the threshold ranges is required.
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http://dx.doi.org/10.1016/j.micron.2016.11.009DOI Listing
March 2017

Direct communication between osteocytes and acid-etched titanium implants with a sub-micron topography.

J Mater Sci Mater Med 2016 Nov 3;27(11):167. Epub 2016 Oct 3.

Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden.

The osteocyte network, through the numerous dendritic processes of osteocytes, is responsible for sensing mechanical loading and orchestrates adaptive bone remodelling by communicating with both the osteoclasts and the osteoblasts. The osteocyte network in the vicinity of implant surfaces provides insight into the bone healing process around metallic implants. Here, we investigate whether osteocytes are able to make an intimate contact with topologically modified, but micrometre smooth (S  < 0.5 µm) implant surfaces, and if sub-micron topography alters the composition of the interfacial tissue. Screw shaped, commercially pure (cp-Ti) titanium implants with (i) machined (S  = ~0.2 µm), and (ii) two-step acid-etched (HF/HNO and HSO/HCl; S  = ~0.5 µm) surfaces were inserted in Sprague Dawley rat tibia and followed for 28 days. Both surfaces showed similar bone area, while the bone-implant contact was 73 % higher for the acid-etched surface. By resin cast etching, osteocytes were observed to maintain a direct intimate contact with the acid-etched surface. Although well mineralised, the interfacial tissue showed lower Ca/P and apatite-to-collagen ratios at the acid-etched surface, while mineral crystallinity and the carbonate-to-phosphate ratios were comparable for both implant surfaces. The interfacial tissue composition may therefore vary with changes in implant surface topography, independently of the amount of bone formed. Implant surfaces that influence bone to have higher amounts of organic matrix without affecting the crystallinity or the carbonate content of the mineral phase presumably result in a more resilient interfacial tissue, better able to resist crack development during functional loading than densely mineralised bone.
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http://dx.doi.org/10.1007/s10856-016-5779-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047930PMC
November 2016

Laser-Modified Surface Enhances Osseointegration and Biomechanical Anchorage of Commercially Pure Titanium Implants for Bone-Anchored Hearing Systems.

PLoS One 2016 14;11(6):e0157504. Epub 2016 Jun 14.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden.

Osseointegrated implants inserted in the temporal bone are a vital component of bone-anchored hearing systems (BAHS). Despite low implant failure levels, early loading protocols and simplified procedures necessitate the application of implants which promote bone formation, bone bonding and biomechanical stability. Here, screw-shaped, commercially pure titanium implants were selectively laser ablated within the thread valley using an Nd:YAG laser to produce a microtopography with a superimposed nanotexture and a thickened surface oxide layer. State-of-the-art machined implants served as controls. After eight weeks' implantation in rabbit tibiae, resonance frequency analysis (RFA) values increased from insertion to retrieval for both implant types, while removal torque (RTQ) measurements showed 153% higher biomechanical anchorage of the laser-modified implants. Comparably high bone area (BA) and bone-implant contact (BIC) were recorded for both implant types but with distinctly different failure patterns following biomechanical testing. Fracture lines appeared within the bone ~30-50 μm from the laser-modified surface, while separation occurred at the bone-implant interface for the machined surface. Strong correlations were found between RTQ and BIC and between RFA at retrieval and BA. In the endosteal threads, where all the bone had formed de novo, the extracellular matrix composition, the mineralised bone area and osteocyte densities were comparable for the two types of implant. Using resin cast etching, osteocyte canaliculi were observed directly approaching the laser-modified implant surface. Transmission electron microscopy showed canaliculi in close proximity to the laser-modified surface, in addition to a highly ordered arrangement of collagen fibrils aligned parallel to the implant surface contour. It is concluded that the physico-chemical surface properties of laser-modified surfaces (thicker oxide, micro- and nanoscale texture) promote bone bonding which may be of benefit in situations where large demands are imposed on biomechanically stable interfaces, such as in early loading and in compromised conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0157504PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907497PMC
July 2017

Long-term osseointegration of 3D printed CoCr constructs with an interconnected open-pore architecture prepared by electron beam melting.

Acta Biomater 2016 05 18;36:296-309. Epub 2016 Mar 18.

Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden; BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Göteborg, Sweden.

Unlabelled: In orthopaedic surgery, cobalt chromium (CoCr) based alloys are used extensively for their high strength and wear properties, but with concerns over stress shielding and bone resorption due to the high stiffness of CoCr. The structural stiffness, principally related to the bulk and the elastic modulus of the material, may be lowered by appropriate design modifications, to reduce the stiffness mismatch between metal/alloy implants and the adjacent bone. Here, 3D printed CoCr and Ti6Al4V implants of similar macro-geometry and interconnected open-pore architecture prepared by electron beam melting (EBM) were evaluated following 26week implantation in adult sheep femora. Despite higher total bone-implant contact for Ti6Al4V (39±4%) than CoCr (27±4%), bone formation patterns were similar, e.g., densification around the implant, and gradual ingrowth into the porous network, with more bone in the outer half (periphery) than the inner half (centre). Raman spectroscopy revealed no major differences in mineral crystallinity, the apatite-to-collagen ratio, or the carbonate-to-phosphate ratio. Energy dispersive X-ray spectroscopy showed similar Ca/P ratio of the interfacial tissue adjacent to both materials. Osteocytes made direct contact with CoCr and Ti6Al4V. While osteocyte density and distribution in the new-formed bone were largely similar for the two alloys, higher osteocyte density was observed at the periphery of the porous network for CoCr, attributable to slower remodelling and a different biomechanical environment. The results demonstrate the possibility to achieve bone ingrowth into open-pore CoCr constructs, and attest to the potential for fabricating customised osseointegrated CoCr implants for load-bearing applications.

Statement Of Significance: Although cobalt chromium (CoCr) based alloys are used extensively in orthopaedic surgery, stress shielding due to the high stiffness of CoCr is of concern. To reduce the stiffness mismatch between CoCr and bone, CoCr and Ti6Al4V implants having an interconnected open-pore architecture were prepared by electron beam melting (EBM). After six months of submerged healing in sheep, both alloys showed similar patterns of bone formation, with densification around the implant and gradual ingrowth into the porous network. The molecular and elemental composition of the interfacial tissue was similar for both alloys. Osteocytes made direct contact with both alloys, with similar overall osteocyte density and distribution. The work attests to the potential for achieving osseointegration of EBM manufactured porous CoCr implants.
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http://dx.doi.org/10.1016/j.actbio.2016.03.033DOI Listing
May 2016

Commercially pure titanium (cp-Ti) versus titanium alloy (Ti6Al4V) materials as bone anchored implants - Is one truly better than the other?

Mater Sci Eng C Mater Biol Appl 2016 May 16;62:960-6. Epub 2016 Jan 16.

Department of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden; BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Göteborg, Sweden.

Commercially pure titanium (cp-Ti) and titanium alloys (typically Ti6Al4V) display excellent corrosion resistance and biocompatibility. Although the chemical composition and topography are considered important, the mechanical properties of the material and the loading conditions in the host have, conventionally, influenced material selection for different clinical applications: predominantly Ti6Al4V in orthopaedics while cp-Ti in dentistry. This paper attempts to address three important questions: (i) To what extent do the surface properties differ when cp-Ti and Ti6Al4V materials are manufactured with the same processing technique?, (ii) Does bone tissue respond differently to the two materials, and (iii) Do bacteria responsible for causing biomaterial-associated infections respond differently to the two materials? It is concluded that: (i) Machined cp-Ti and Ti6Al4V exhibit similar surface morphology, topography, phase composition and chemistry, (ii) Under experimental conditions, cp-Ti and Ti6Al4V demonstrate similar osseointegration and biomechanical anchorage, and (iii) Experiments in vitro fail to disclose differences between cp-Ti and Ti6Al4V to harbour Staphylococcus epidermidis growth. No clinical comparative studies exist which could determine if long-term, clinical differences exist between the two types of bulk materials. It is debatable whether cp-Ti or Ti6Al4V exhibit superiority over the other, and further comparative studies, particularly in a clinical setting, are required.
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http://dx.doi.org/10.1016/j.msec.2016.01.032DOI Listing
May 2016
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