Backscattered electron imaging and electron backscattered diffraction in the study of bacterial attachment to titanium alloy structure.

The application of secondary electron (SE) imaging, backscattered electron imaging (BSE) and electron backscattered diffraction (EBSD) was investigated in this work to study the bacterial adhesion and proliferation on a commercially pure titanium (cp Ti) and a Ti6Al4V alloy (Ti 64) with respect to substrate microstructure and chemical composition. Adherence of Gram-positive Staphylococcus epidermidis 11047 and Streptococcus sanguinis GW2, and Gram-negative Serratia sp. NCIMB 40259 and Escherichia coli 10418 was compared on cp Ti, Ti 64, pure aluminium (Al) and vanadium (V). The substrate microstructure and the bacterial distribution on these metals were characterised using SE, BSE and EBSD imaging. It was observed that titanium alloy-phase structure, grain boundaries and grain orientation did not influence bacterial adherence or proliferation at microscale. Adherence of all four strains was similar on cp Ti and Ti 64 surfaces whilst inhibited on pure Al. This work establishes a nondestructive and straight-forward statistical method to analyse the relationship between microbial distribution and metal alloy structure.

Immediate restoration of fixed partial prostheses supported by one-piece narrow-diameter selective laser sintering implants: a 2-year prospective study in the posterior jaws of 16 patients.

PURPOSE: Selective laser sintering (SLS) is a new technique that allows implants to be produced by annealing titanium powder microparticles in a focused laser beam, according to a computer-generated model. The aim of this study was to evaluate the survival and success rate of immediately restored 1-piece narrow-diameter SLS implants, placed in the posterior jaws, after 2 years of functional loading. MATERIALS AND METHODS: At the 2-year follow-up examination, several parameters were assessed. Success criteria included absence of pain, suppuration, exudation, implant mobility; distance between the implant shoulder and the first visible bone-to-implant contact <2.0 mm; and absence of prosthetic complications. RESULTS: Thirty-seven implants were placed in the posterior jaws (14 maxilla, 23 mandible) of 16 patients (9 males, 7 females). The prosthetic restorations comprised 16 immediately restored fixed partial prostheses. No implant failure occurred, resulting in a 100% survival rate. The implant success was 94.6%, and the mean distance between the implant shoulder and the first visible bone-to-implant contact (DIB) was 0.4 ± 0.3 mm. CONCLUSIONS: This study supports the hypothesis that 1-piece narrow-diameter SLS implants can be used in fixed prosthetic rehabilitations in the posterior regions of both jaws with a predictable positive outcome.

Esthetic evaluation of single-tooth Morse taper connection implants placed in fresh extraction sockets or healed sites.

The aim of this study was to compare the esthetic outcome of single implants placed in fresh extraction sockets with those placed in fully healed sites of the anterior maxilla. This retrospective study was based on data from patients treated with single-tooth Morse taper connection implants placed in fresh extraction sockets and in fully healed sites of the anterior maxilla. Only single implant treatments were considered with both neighboring teeth present. Additional prerequisites for immediate implant treatment were intact socket walls and a thick gingival biotype. The esthetic outcome was objectively rated using the pink esthetic/white esthetic score (PES/WES). The Mann-Whitney U test was used to compare the PES and the WES between the 2 groups. Twenty-two patients received an immediate implant, and 18 patients had conventional implant surgery. The mean follow-up was 31.09 months (SD 5.57; range 24-46) and 34.44 months (SD 7.10; range 24-48) for immediately and conventionally inserted implants, respectively. No implants were lost. All implants fulfilled the success criteria. The mean PES/WES was 14.50 (SD 2.52; range 9-19) and 15.61 (SD 3.20; range 8-20) for immediately and conventionally placed implants, respectively. Immediate implants had a mean PES of 7.45 (SD 1.62; range 4-10) and a mean WES of 7.04 (SD 1.29; range 5-10). Conventional implants had a mean PES of 7.83 (SD 1.58; range 4-10) and a mean WES of 7.77 (SD 1.66; range 4-10). The difference between the 2 groups was not significant. Immediate and conventional single implant treatment yielded comparable esthetic outcomes.

Three-dimensional geometry of honeycomb collagen promotes higher beating rate of myocardial cells in culture.

Myocardial cells were isolated from newborn rats, cultured on a novel three-dimensional (3-D) honeycomb collagen scaffold (HC) and their morphology and beating rates compared with ones on conventional plastic dishes. On the first day, the cells attached to HC had already started beating. As time went on, the rate of beating increased as the pores of HC gradually filled with the cells, which integrated to form the cell-matrix complex. At day 8, beating reached the highest frequency of 162 beats per minute, which was twice that of the control cells on plastic dishes. It was concluded that 3-D geometry of the HC is conducive to functional growth of the myocardial tissues, and will potentially be useful for tissue engineering of myocardial regeneration.

Biofilm viability checker: An open-source tool for automated biofilm viability analysis from confocal microscopy images.

Quantifying biofilm formation on surfaces is challenging because traditional microbiological methods, such as total colony-forming units (CFUs), often rely on manual counting. These are laborious, resource intensive techniques, more susceptible to human error. Confocal laser scanning microscopy (CLSM) is a high-resolution technique that allows 3D visualisation of biofilm architecture. In combination with a live/dead stain, it can be used to quantify biofilm viability on both transparent and opaque surfaces. However, there is little consensus on the appropriate methodology to apply in confocal micrograph processing. In this study, we report the development of an image analysis approach to repeatably quantify biofilm viability and surface coverage. We also demonstrate its use for a range of bacterial species and translational applications. This protocol has been created with ease of use and accessibility in mind, to enable researchers who do not specialise in computational techniques to be confident in applying these methods to analyse biofilm micrographs. Furthermore, the simplicity of the method enables the user to adapt it for their bespoke needs. Validation experiments demonstrate the automated analysis is robust and accurate across a range of bacterial species and an improvement on traditional microbiological analysis. Furthermore, application to translational case studies show the automated method is a reliable measurement of biomass and cell viability. This approach will ensure image analysis is an accessible option for those in the microbiology and biomaterials field, improve current detection approaches and ultimately support the development of novel strategies for preventing biofilm formation by ensuring comparability across studies.

High speed imaging of biofilm removal from a dental implant model using ultrasonic cavitation.

OBJECTIVES: Current instruments cannot clean in between dental implant threads and effectively remove biofilm from the rough implant surface without damaging it. Cavitation bubbles have the potential to disrupt biofilms. The aim of this study was to see how biofilms can be disrupted using non-contact cavitation from an ultrasonic scaler, imaged inside a restricted implant pocket model using high speed imaging. METHODS: Streptococcus sanguinis biofilm was grown for 7 days on dental implants. The implants were placed inside a custom made restricted pocket model and immersed inside a water tank. An ultrasonic scaler tip was placed 0.5mm away from the implant surface and operated at medium power or high power for 2s. The biofilm removal process was imaged using a high speed camera operating at 500 fps. Image analysis was used to calculate the amount of biofilm removed from the high speed images. Scanning electron microscopy was done to visualize the implant surface after cleaning. RESULTS: Cavitation was able to remove biofilm from dental implants. More biofilm was removed at high power. Scanning electron microscopy showed that the implant surface was clean at the points where the cavitation was most intense. High speed imaging showed biofilm removal underneath implant threads, in areas next to the ultrasonic scaler tip. SIGNIFICANCE: A high speed imaging protocol has been developed to visualize and quantify biofilm removal from dental implants in vitro. Cavitation bubbles from dental ultrasonic scalers are able to successfully disrupt biofilm in between implant threads.

A review of co-culture models to study the oral microenvironment and disease.

Co-cultures allow for the study of cell-cell interactions between different eukaryotic species or with bacteria. Such an approach has enabled researchers to more closely mimic complex tissue structures. This review is focused on co-culture systems modelling the oral cavity, which have been used to evaluate this unique cellular environment and understand disease progression. Over time, these systems have developed significantly from simple 2D eukaryotic cultures and planktonic bacteria to more complex 3D tissue engineered structures and biofilms. Careful selection and design of the co-culture along with critical parameters, such as seeding density and choice of analysis method, have resulted in several advances. This review provides a comparison of existing co-culture systems for the oral environment, with emphasis on progression of 3D models and the opportunity to harness techniques from other fields to improve current methods. While filling a gap in navigating this literature, this review ultimately supports the development of this vital technique in the field of oral biology.

Which Parameters Affect Biofilm Removal with Acoustic Cavitation? A Review.

Bacterial biofilms are a cause of contamination in a wide range of medical and biological areas. Ultrasound is a mechanical energy that can remove these biofilms using cavitation and acoustic streaming, which generate shear forces to disrupt biofilm from a surface. The aim of this narrative review is to investigate the literature on the mechanical removal of biofilm using acoustic cavitation to identify the different operating parameters affecting its removal using this method. The properties of the liquid and the properties of the ultrasound have a large impact on the type of cavitation generated. These include gas content, temperature, surface tension, frequency of ultrasound and acoustic pressure. For many of these parameters, more research is required to understand their mechanisms in the area of ultrasonic biofilm removal, and further research will help to optimise this method for effective removal of biofilms from different surfaces.

Human adipose tissue-derived mesenchymal stem/stromal cells adhere to and inhibit the growth of Staphylococcus aureus and Pseudomonas aeruginosa.

We have cultured and phenotyped human adipose tissue-derived mesenchymal stem/stromal cells (AT MSCs) and inoculated these cultures with bacteria common to infected skin wounds, i.e. Staphylococcus aureus and Pseudomonas aeruginosa. Cell interactions were examined by scanning electron microscopy (SEM), whilst bacterial growth was measured by colony forming unit (c.f.u.) and biofilm assays. AT MSCs appeared to attach to the bacteria and to engulf S. aureus. Significantly fewer bacterial c.f.u. were present in AT MSC : bacterial co-cultures compared with bacteria cultured alone. Antibacterial activity, including an inhibition of P. aeruginosa biofilm formation, was observed when bacteria were treated with conditioned medium harvested from the AT MSC :  bacterial co-cultures, irrespective of the bacterial species to which the AT MSCs had been exposed to previously. Hence, we have demonstrated that AT MSCs inhibit the growth of two common bacterial species. This was associated with bacterial adhesion, potential engulfment or phagocytosis, and the secretion of antibacterial factors.

High Speed Imaging of Cavitation around Dental Ultrasonic Scaler Tips.

Cavitation occurs around dental ultrasonic scalers, which are used clinically for removing dental biofilm and calculus. However it is not known if this contributes to the cleaning process. Characterisation of the cavitation around ultrasonic scalers will assist in assessing its contribution and in developing new clinical devices for removing biofilm with cavitation. The aim is to use high speed camera imaging to quantify cavitation patterns around an ultrasonic scaler. A Satelec ultrasonic scaler operating at 29 kHz with three different shaped tips has been studied at medium and high operating power using high speed imaging at 15,000, 90,000 and 250,000 frames per second. The tip displacement has been recorded using scanning laser vibrometry. Cavitation occurs at the free end of the tip and increases with power while the area and width of the cavitation cloud varies for different shaped tips. The cavitation starts at the antinodes, with little or no cavitation at the node. High speed image sequences combined with scanning laser vibrometry show individual microbubbles imploding and bubble clouds lifting and moving away from the ultrasonic scaler tip, with larger tip displacement causing more cavitation.

A novel non line-of-sight method for coating hydroxyapatite onto the surfaces of support materials by biomineralization.

A novel method is described for the non line-of-sight coating of hydroxyapatite onto polyurethane reticulated foam and titanium discs. This utilises a biofilm of Serratia sp. NCIMB 40259 which, when challenged with a solution containing calcium chloride and phosphatase substrate, manufactures biofilm-bound material identified as hydroxyapatite by X-ray powder diffraction analysis. Non-invasive magnetic resonance imaging was used to visualize the biofilm coating throughout the foam labyrinth and to measure the thickness of the film within reticulated foam cubes in situ. The film developed within the cube matrices was similar to that measured on the surface of a glass slide. Using LaPO(4) deposition as a model system the metallised biofilm was visualised in two-dimensional slices throughout three-dimensional images acquired by magnetic resonance imaging. A similar encrustation of hydroxyapatite on the surface of biofilm grown on titanium discs was confirmed by scanning electron microscopy. Potential applications for bio-hydroxyapatite as possible bone implant precursors are discussed.

An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair.

AIM: To compare the incorporation, growth, and chondrogenic potential of bone marrow (BM) and adipose tissue (AT) mesenchymal stem cells (MSCs) in scaffolds used for cartilage repair. METHODS: Human BM and AT MSCs were isolated, culture expanded, and characterised using standard protocols, then seeded into 2 different scaffolds, Chondro-Gide or Alpha Chondro Shield. Cell adhesion, incorporation, and viable cell growth were assessed microscopically and following calcein AM/ethidium homodimer (Live/Dead) staining. Cell-seeded scaffolds were treated with chondrogenic inducers for 28 days. Extracellular matrix deposition and soluble glycosaminoglycan (GAG) release into the culture medium was measured at day 28 by histology/immunohistochemistry and dimethylmethylene blue assay, respectively. RESULTS: A greater number of viable MSCs from either source adhered and incorporated into Chondro-Gide than into Alpha Chondro Shield. In both cell scaffolds, this incorporation represented less than 2% of the cells that were seeded. There was a marked proliferation of BM MSCs, but not AT MSCs, in Chondro-Gide. MSCs from both sources underwent chondrogenic differentiation following induction. However, cartilaginous extracellular matrix deposition was most marked in Chondro-Gide seeded with BM MSCs. Soluble GAG secretion increased in chondrogenic versus control conditions. There was no marked difference in GAG secretion by MSCs from either cell source. CONCLUSION: Chondro-Gide and Alpha Chondro Shield were permissive to the incorporation and chondrogenic differentiation of human BM and AT MSCs. Chondro-Gide seeded with BM MSCs demonstrated the greatest increase in MSC number and deposition of a cartilaginous tissue.

Histochemical, immunohistological and scanning electron microscope analysis of tissue retained on spontaneously extruded ventilation tubes.

The aim of this study was to analyse the tissue surrounding prematurely extruded ventilation tubes (grommets). Thirty-one ventilation tubes, including 21 Shah and six Shepherd tubes, that had been extruded naturally into the ear canal were examined. After formalin fixation, material adhering to the tubes was retrieved and processed to paraffin wax. Five tubes were processed for scanning electron microscope (SEM) analysis. Sections were stained using histochemical methods for collagen, keratin and keratohyaline. In addition, the presence of collagen (type I and III), keratin, vimentin, fibronectin, tenascin, factor VIII-related antigen, CD31 and CD45 was tested for by immunohistochemistry. Results showed that all specimens consisted of an acellular fibrous material, oriented in concentric rings parallel to the tube surface that was often associated with small collections of CD45+ inflammatory cells. Two specimens contained collagen that was detectable by histochemical and immunohistological methods. Twelve specimens contained identifiable desquamated epithelial cells containing keratin and keratohyaline. Only one specimen stained positively for connective tissue markers (vimentin, fibrous fibronectin, tenascin) or showed the presence of vascular epithelium. SEM revealed adherent clusters or sheets of plate-like structures, consistent with the presence of epithelial squames, on three of the five ventilation tubes examined. It is concluded that tissue retained on extruded grommets consists of orthokeratinitizing epithelium infiltrated by inflammatory cells, a finding that is consistent with the theory that tube extrusion occurs as a result of squamous epithelial proliferation, medial migration, differentiation and desquamation.

Clinical outcome of narrow-diameter (3.3-mm) locking-taper implants: a prospective study with 1 to 10 years of follow-up.

PURPOSE: To evaluate the outcome of narrow-diameter (3.3-mm) locking-taper implants used in the rehabilitation of partially and fully edentulous patients over a 10-year period. MATERIALS AND METHODS: Between January 2002 and December 2011, all patients referred to a private dental clinic for treatment with narrow-diameter implants were enrolled in this study. At each annual follow-up session, clinical and radiographic parameters were assessed; the outcome measurements were implant failure, peri-implant marginal bone loss (distance between the implant shoulder and the first visible bone-to-implant contact [DIB]), and biologic and technical complications. The cumulative survival rate (CSR) was assessed using the Kaplan-Meier survival estimator; Tarone-Ware and chi-square analyses were used to evaluate correlations between the study variables. The statistical analysis was performed at the patient- and implant-level. RESULTS: A total of 324 narrow-diameter implants were placed in 279 patients (159 men, 120 women; age: 25 to 73 years). Four implants failed, for an overall CSR of 98.5% (patient-based) and 98.7% (implant-based) at the 10-year follow-up. The survival rate did not differ significantly with respect to patients' sex, age, smoking or parafunctional habits, bone type, prosthetic restoration, or implant location, position, or length. Among the surviving implants, a mean DIB of 0.31 ± 0.23 mm, 0.45 ± 0.27 mm, and 0.69 ± 0.28 mm was observed at the 1-, 5-, and 10-year follow-up examinations, respectively. A few biologic (1.2%) and technical complications (7.5%) were reported. CONCLUSIONS: Within the limitations of this study, it can be concluded that narrow-diameter locking-taper implants represent a good treatment option for the prosthetic rehabilitation of partially and totally edentulous patients.

Maxillary sinus augmentation with adult mesenchymal stem cells: a review of the current literature.

PURPOSE: Mesenchymal stem cells (MSCs) have been applied in maxillary sinus augmentation (MSA) with clinically successful results. The purpose of this article was to evaluate the systematically acquired evidence for the effectiveness of cell-based approaches in MSA with various scaffolds, and to narratively assess evidence from additional articles that report effectiveness of cell-based approaches in MSA. MATERIALS AND METHODS: Electronic database searches were performed. Inclusion criteria were studies of cell-based approaches in MSA with various scaffolds, in humans, with at least 3 to 4 months of follow-up. Meta-analysis was performed for randomized controlled trials (RCTs) with histologic/histomorphometric evaluation. RESULTS: Fifteen studies (4 RCTs) were considered to be eligible for inclusion in the review. The meta-analysis suggested a marginal, nonstatistically significant positive effect of MSCs on the bone regrowth. CONCLUSIONS: A number of studies have demonstrated the potential for cell-based approaches in MSA; further RCTs that clearly demonstrate benefits of cell-based approach are needed.

Active screen plasma surface modification of polycaprolactone to improve cell attachment.

To tailor polycaprolactone (PCL) surface properties for biomedical applications, film samples of PCL were surface modified by the active screen plasma nitriding (ASPN) technique. The chemical composition and structure were characterized by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The wettability of the surface modified polymers was investigated by contact angle and surface energy methods. Biocompatibility of the prepared PCL samples was evaluated in vitro using MC3T3-E1 osteoblast-like cells. The degradability was assessed by determining the self-degradation rate (catalyzed by lipase). The results show that ASPN surface modification can effectively improve osteoblast cell adhesion and spreading on the surface of PCL. The main change in chemical composition is the exchange of some carboxyl groups on the surface for hydroxyl groups. The active-screen plasma nitriding technique has been found to be an effective and practical method to effectively improve osteoblast cell adhesion and spreading on the PCL surface. Such changes have been attributed to the increase in wettablity and generation of new hydroxyl groups by plasma treatment. After active-screen plasma treatment, the PCL film is still degradable, but the enzymatic degradation rate is slower compared with untreated PCL film.

Prospective evaluation of 2,549 Morse taper connection implants: 1- to 6-year data.

BACKGROUND: The aim of this study is to evaluate the implant survival, the implant-crown success, and the prosthetic complications of 2,549 Morse taper interference-fit connection implants. METHODS: A total of 2,549 Morse taper connection implants were inserted in 893 patients from January 2003 until December 2008. At each annual recall, clinical, radiographic, and prosthetic parameters were assessed. The implant-crown success criteria included the absence of pain, suppuration, and clinical mobility; an average distance between the implant shoulder and the first visible bone contact <2 mm from initial surgery; and the absence of prosthetic complications at the implant-abutment interface. Prosthetic restorations were fixed partial prostheses (462 units); fixed full-arch prostheses (60 units); single crowns (531 units); and overdentures (93 units). RESULTS: The cumulative implant survival rate was 98.23% (97.25% maxilla, 99.05% mandible). The implant-crown success was 92.49%. A few prosthetic complications at implant-abutment interface were reported (0.37%). After 6 years, distance between the implant shoulder and the first visible bone contact was 1.10 mm (± 0.30 mm). CONCLUSION: The use of Morse taper connection implants represents a successful procedure for the rehabilitation of partially and completely edentulous arches.

Fluid exudates from inflamed bone-anchored hearing aids demonstrate elevated levels of cytokines and biomarkers of tissue and bone metabolism.

OBJECTIVE: The purpose of the current study was to determine the presence of biomarkers of tissue and bone metabolism and 10 cytokines within the fluid exudate from around bone anchored hearing aids (BAHAs), with a view to understanding the mechanisms of peri-BAHA inflammation. STUDY DESIGN: Fluid exudate from around BAHAs was collected from volunteers (n = 10) with inflammation and controls (n = 10) without inflammation around the BAHA. SETTING: Studies of periodontitis and dental peri-implantitis have demonstrated that fluid exudate, arising from the hard tissue-soft tissue junction, is increased during disease; this fluid contains biomarkers of tissue metabolism, bone metabolism and inflammation. PATIENTS: Volunteers were recruited from the Queen Elizabeth Hospital, Birmingham, UK and New Cross Hospital, Wolverhampton, UK. INTERVENTIONS: This was an observational study and no interventions were carried out as part of the study. MAIN OUTCOME MEASURE: Biomarkers of tissue metabolism (MMP9, TIMP1 and 2), bone metabolism (RANKL and OPG) and cytokines (GM-CSF, interferon (IFN)-*, IL-1A, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF>) were measured in the fluid samples by Luminex multianalyte assay. RESULTS: Volumes of fluid exudate were significantly higher (p < 0.05) at sites of inflammation than from un-inflamed tissues surrounding BAHAs. IL-1*, IL-6, IL-8, TNF*, MMP9, TIMP1 and 2 were found in both inflamed and un-inflamed samples and levels were significantly higher in samples from inflamed sites. RANKL, IFN*, IL-2, IL-4, and GM-CSF were detected only at inflamed sites whereas OPG, IL-5 and IL-10 were not detected in any samples. CONCLUSION: These data demonstrate that peri-BAHA tissues showing clinical signs of inflammation are associated with increased fluid exudate, which contains elevated levels of key biomarkers of inflammation and a biomarker profile that is consistent with increased tissue and bone remodeling around BAHAs.

Microstructure and composition of biosynthetically synthesised hydroxyapatite.

Biosynthetic hydroxyapatite (HA) manufactured utilising the bacterium Serratia sp. NCIMB40259 was characterised using X-ray diffraction (XRD), Fourier transform infra-red spectroscopy (FTIR), energy dispersive X-ray analysis (EDX) scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electron diffraction (ED). SEM/EDX showed that the non-sintered material consisted mainly of calcium-deficient HA (CDHA) with a Ca/P ratio of 1.61 +/- 0.06 and crystal size (from TEM) of 50 +/- 10 nm. ED analysis of non-sintered powder showed resolvable ring patterns ascribed to (0002), (1122) and (0006) planes of crystalline HA. The crystallinity of the samples improved with heat treatment from approximately 9.4% (non-sintered) to 53% (1,200 degrees C). Samples heated at 600 degrees C and sintered at 1,200 degrees C were identified by XRD and FTIR as mainly CDHA with some sodium calcium phosphate in the sintered samples. Ca/P ratios (SEM/EDX) were 1.62 and 1.52, respectively. Single crystal spot patterns characteristic of HA were seen with commercial HA and Serratia HA heated at 600 degrees C. After sintering at 1,200 degrees C the material consisted of needle-like crystals with a length between 86 and 323 nm (from TEM) or 54-111 nm (from XRD) and lattice parameters of a = 9.441 A and c = 6.875 A. This study indicated that the material produced by Serratia bacteria was initially mainly nanophase calcium deficient hydroxyapatite, which sintered to a more highly crystalline form. With further refinements the method could be used as an inexpensive route for hydroxyapatite production for biomaterials applications.

Polyhydroxybutyrate accumulation by a Serratia sp.

A strain of Serratia sp. showed intracellular electron-transparent inclusion bodies when incubated in the presence of citrate and glycerol 2-phosphate without nitrogen source following pre-growth under carbon-limitation in continuous culture. About 1.3 mmol citrate were consumed per 450 mg biomass, giving a calculated yield of maximally 55% of stored material per g of biomass dry wt. The inclusion bodies were stained with Sudan Black and Nile Red (NR), suggesting a lipid material, which was confirmed as polyhydroxybutyrate (PHB) by analysis of molecular fragments by GC and by FTIR spectroscopy of isolated bio-PHB in comparison with reference material. Multi-parameter flow cytometry in conjunction with NR fluorescence, and electron microscopy, showed that not all cells contained heavy PHB bodies, suggesting the potential for increasing the overall yield. The economic attractiveness is enhanced by the co-production of nanoscale hydroxyapatite (HA), a possible high-value precursor for bone replacement materials.