The Effect of Stereocomplexation and Crystallinity on the Degradation of Polylactide Nanoparticles.

Polymeric nanoparticles (PNPs) are frequently researched and used in drug delivery. The degradation of PNPs is highly dependent on various properties, such as polymer chemical structure, size, crystallinity, and melting temperature. Hence, a precise understanding of PNP degradation behavior is essential for optimizing the system. This study focused on enzymatic hydrolysis as a degradation mechanism by investigation of the degradation of PNP with various crystallinities. The aliphatic polyester polylactide ([C3H4O2]n, PLA) was used as two chiral forms, poly l-lactide (PlLA) and poly d-lactide (PdLA), and formed a unique crystalline stereocomplex (SC). PNPs were prepared via a nanoprecipitation method. In order to further control the crystallinity and melting temperatures of the SC, the polymer poly(3-ethylglycolide) [C6H8O4]n (PEtGly) was synthesized. Our investigation shows that the PNP degradation can be controlled by various chemical structures, crystallinity and stereocomplexation. The influence of proteinase K on PNP degradation was also discussed in this research. AFM did not reveal any changes within the first 24 h but indicated accelerated degradation after 7 days when higher EtGly content was present, implying that lower crystallinity renders the particles more susceptible to hydrolysis. QCM-D exhibited reduced enzyme adsorption and a slower degradation rate in SC-PNPs with lower EtGly contents and higher crystallinities. A more in-depth analysis of the degradation process unveiled that QCM-D detected rapid degradation from the outset, whereas AFM exhibited delayed changes of degradation. The knowledge gained in this work is useful for the design and creation of advanced PNPs with enhanced structures and properties.

The antimicrobial effect of calcium-doped titanium is activated by fibrinogen adsorption.

Directly targeting bacterial cells is the present paradigm for designing antimicrobial biomaterial surfaces and minimizing device-associated infections (DAIs); however, such pathways may create problems in tissue integration because materials that are toxic to bacteria can also be harmful to mammalian cells. Herein, we report an unexpected antimicrobial effect of calcium-doped titanium, which itself has no apparent killing effect on the growth of pathogenic bacteria (Pseudomonas aeruginosa, Pa, ATCC 27853) while presenting strong inhibition efficiency on bacterial colonization after fibrinogen adsorption onto the material. Fine X-ray photoelectron spectroscopy and Fourier-transform infrared spectroscopy analyses reported calcium-dependent shifts of the binding energy in nitrogen and oxygen involved groups and wavenumbers in the amide I and II bands of the adsorbent fibrinogen, demonstrating that locally delivered calcium can react with the carboxy-terminal regions of the Aα chains and influence their interaction with the N-termini of the Bß chains in fibrinogen. These reactions facilitate the exposure of the antimicrobial motifs of the protein, indicating the reason for the surprising antimicrobial efficacy of calcium-doped titanium. Since protein adsorption is an immediate intrinsic step during the implantation surgery, this finding may shift the present paradigm on the design of implantable antibacterial biomaterial surfaces.

Performance of Calcium Phosphate Cements in the Augmentation of Sheep Vertebrae-An Ex Vivo Study.

Oil-based calcium phosphate cement (Paste-CPC) shows not only prolonged shelf life and injection times, but also improved cohesion and reproducibility during application, while retaining the advantages of fast setting, mechanical strength, and biocompatibility. In addition, poly(L-lactide-co-glycolide) (PLGA) fiber reinforcement may decrease the risk for local extrusion. Bone defects (diameter 5 mm; depth 15 mm) generated ex vivo in lumbar (L) spines of female Merino sheep (2-4 years) were augmented using: (i) water-based CPC with 10% PLGA fiber reinforcement (L3); (ii) Paste-CPC (L4); or (iii) clinically established polymethylmethacrylate (PMMA) bone cement (L5). Untouched (L1) and empty vertebrae (L2) served as controls. Cement performance was analyzed using micro-computed tomography, histology, and biomechanical testing. Extrusion was comparable for Paste-CPC(-PLGA) and PMMA, but significantly lower for CPC + PLGA. Compressive strength and Young's modulus were similar for Paste-CPC and PMMA, but significantly higher compared to those for empty defects and/or CPC + PLGA. Expectedly, all experimental groups showed significantly or numerically lower compressive strength and Young's modulus than those of untouched controls. Ready-to-use Paste-CPC demonstrates a performance similar to that of PMMA, but improved biomechanics compared to those of water-based CPC + PLGA, expanding the therapeutic arsenal for bone defects. O, significantly lower extrusion of CPC + PLGA fibers into adjacent lumbar spongiosa may help to reduce the risk of local extrusion in spinal surgery.

Biopolymer surface modification of PLGA fibers enhances interfacial shear strength and supports immobilization of rhGDF-5 in fiber-reinforced brushite cement.

Incorporation of biodegradable poly(lactic-co-glycolic acid; PLGA) fibers into calcium phosphate cements (CPCs) has proven beneficial for their mechanical properties and the targeted delivery of bone morphogenetic proteins (BMPs). However, the deficiency of functional groups on the PLGA surface results in poor fiber-matrix interfacial strength (ISS), limiting the mechanical improvement, and insufficient surface charge to immobilize therapeutic amounts of BMPs. The present study therefore focused on the: i) functionalization of PLGA fibers using polyelectrolyte multilayers (PEMs) of biopolymers; ii) analysis of their impact on the mechanical properties of the CPC in multifilament fiber pull-out tests; and iii) testing of their applicability as carriers for BMPs using chemical-free adsorption of biotinylated recombinant human growth factor (rhGDF-5) and colorimetric assays. The PEMs were created from chitosan (Chi), hyaluronic acid (HA), and gelatin (Gel) via layer-by-layer (LbL) deposition. Four PEM nanocoatings consisting of alternating Chi/Gel and Chi/HA bilayers with a terminating layer of Chi, Gel or HA were tested. Nanocoating of the PLGA fibers with PEMs significantly enhanced the ISS with the CPC matrix to max. 3.55 ± 1.05 MPa (2.2-fold). The increase in ISS, ascribed to enhanced electrostatic interactions between PLGA and calcium phosphate, was reflected in significant improvement of the composites' flexural strength compared to CPC containing untreated fibers. However, only minor effects on the composites' work of fracture were observed. The adsorption of rhGDF-5 on the PLGA surface was supported by PEMs terminating with either positive or negative charges, without significant differences among the nanocoatings. This proof-of-principle rhGDF-5 immobilization study, together with the augmented ISS of the composites, demonstrates that surface modification of PLGA fibers with biopolymers is a promising approach for targeted delivery of BMPs and improved mechanical properties of the fiber-reinforced CPC.

Quantifying the relationship between surfaces' nano-contact point density and adhesion force of Candida albicans.

It has been recently recognized that controlled surface structuring on the nanometer scale is a successful strategy to endow different materials with antimicrobial properties. Despite many studies on bacterial interactions with nanostructured surfaces, a quantitative link between surface topography and bacterial adhesion is still missing. To quantitatively link cell adhesion data with topographical surface parameters, we performed single-cell spectroscopy on chemically identical surfaces with controlled nano-contact point density achieved by immobilization of gold nanoparticles (AuNP) on gold thin films. Such materials surfaces have previously shown antimicrobial (anti-adhesive) efficacy towards Gram-negative Escherichia coli cells. In the current study, the influence of nano-structured surfaces on the surface coverage and adhesion forces of clinically relevant Candida albicans (C. albicans), the fungus primarily associated with implant infections, was investigated to validate their antimicrobial potency against different microbial cells. The adhesion forces of C. albicans cells to nanostructured surfaces showed a decreasing trend with decreasing contact-point density and correlated well with the results of the respective C. albicans cell counts. The surfaces with the lowest contact-point density, 25 AuNP/µm², resulted in an average adhesion force of 5 nN, which was up to 5 times lower compared to control and 61 AuNP/µm² surfaces. Further, detailed analyses of force-distance curves revealed that the work of adhesion, and thus the energy required to remove the C. albicans cell from the surface is up to 10 times lower on 25 AuNP/µm² surfaces compared to unstructured surfaces. These findings show that a controlled tuning of nanostructured surfaces in terms of accessible nano-contact points is crucial to generate surface structures with enhanced antimicrobial properties. The gained knowledge can be further exploited for the design of biomaterials surfaces to prevent adhesion of some most commonly encountered pathogens.

The old sheep: a convenient and suitable model for senile osteopenia.

INTRODUCTION: Existing osteoporosis models in sheep exhibit some disadvantages, e.g., challenging surgical procedures, serious ethical concerns, failure of reliable induction of substantial bone loss, or lack of comparability to the human condition. This study aimed to compare bone morphological and mechanical properties of old and young sheep, and to evaluate the suitability of the old sheep as a model for senile osteopenia. MATERIALS AND METHODS: The lumbar vertebral body L3 of female merino sheep with two age ranges, i.e., old animals (6-10 years; n = 41) and young animals (2-4 years; n = 40), was analyzed concerning its morphological and mechanical properties by bone densitometry, quantitative histomorphometry, and biomechanical testing of the corticalis and/or central spongious region. RESULTS: In comparison with young sheep, old animals showed only marginally diminished bone mineral density of the vertebral bodies, but significantly decreased structural (bone volume, - 15.1%; ventral cortical thickness, - 11.8%; lateral cortical thickness, - 12.2%) and bone formation parameters (osteoid volume, osteoid surface, osteoid thickness, osteoblast surface, all - 100.0%), as well as significantly increased bone erosion (eroded surface, osteoclast surface). This resulted in numerically decreased biomechanical properties (compressive strength; - 6.4%). CONCLUSION: Old sheep may represent a suitable model of senile osteopenia with markedly diminished bone structure and formation, and substantially augmented bone erosion. The underlying physiological aging concept reduces challenging surgical procedures and ethical concerns and, due to complex alteration of different facets of bone turnover, may be well representative of the human condition.

In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement.

Bone regeneration of sheep lumbar osteopenia is promoted by targeted delivery of bone morphogenetic proteins (BMPs) via a biodegradable, brushite-forming calcium-phosphate-cement (CPC) with stabilizing poly(l-lactide-co-glycolide) acid (PLGA) fibers. The present study sought to quantify the release and bioactivity of BMPs from a specific own CPC formulation successfully used in previous in vivo studies. CPC solid bodies with PLGA fibers (0%, 5%, 10%) containing increasing dosages of GDF5, BB-1, and BMP-2 (2 to 1000 µg/mL) were ground and extracted in phosphate-buffered saline (PBS) or pure sheep serum/cell culture medium containing 10% fetal calf serum (FCS; up to 30/31 days). Released BMPs were quantified by ELISA, bioactivity was determined via alkaline phosphatase (ALP) activity after 3-day exposure of different osteogenic cell lines (C2C12; C2C12BRlb with overexpressed BMP-receptor-1b; MCHT-1/26; ATDC-5) and via the influence of the extracts on the expression of osteogenic/chondrogenic genes and proteins in human adipose tissue-derived mesenchymal stem cells (hASCs). There was hardly any BMP release in PBS, whereas in medium + FCS or sheep serum the cumulative release over 30/31 days was 11-34% for GDF5 and 6-17% for BB-1; the release of BMP-2 over 14 days was 25.7%. Addition of 10% PLGA fibers significantly augmented the 14-day release of GDF5 and BMP-2 (to 22.6% and 43.7%, respectively), but not of BB-1 (13.2%). All BMPs proved to be bioactive, as demonstrated by increased ALP activity in several cell lines, with partial enhancement by 10% PLGA fibers, and by a specific, early regulation of osteogenic/chondrogenic genes and proteins in hASCs. Between 10% and 45% of bioactive BMPs were released in vitro from CPC + PLGA fibers over a time period of 14 days, providing a basis for estimating and tailoring therapeutically effective doses for experimental and human in vivo studies.

The poly (l-lactid-co-glycolide; PLGA) fiber component of brushite-forming calcium phosphate cement induces the osteogenic differentiation of human adipose tissue-derived stem cells.

A brushite-forming calcium phosphate cement (CPC) was mechanically stabilized by addition of poly (l-lactid-co-glycolide; PLGA) fibers (≤10% w/w). It proved highly biocompatible and its fiber component enhanced bone formation in a sheep lumbar vertebroplasty model. However, possible effects on the osteogenic differentiation of resident mesenchymal stem cells (MSCs) remained unexplored. The present study used a novel approach, simultaneously analyzing the influence of a solid CPC scaffold and its relatively low PLGA proportion (a mimicry of natural bone) on osteogenic, chondrogenic, and adipogenic differentiation, as well as the pluripotency of human adipose tissue-derived mesenchymal stem cells (hASCs). hASCs were cultured on CPC discs with/without PLGA fibers (5% and 10%) in the absence of osteogenic medium for 3, 7, and 14 d. Gene expression of osteogenic markers (Runx2, osterix, alkaline phosphatase, collagen I, osteonectin, osteopontin, osteocalcin), chondrogenic markers (collagen II, Sox9, aggrecan), adipogenic markers (PPARG, Leptin, and FABP4), and pluripotency markers (Nanog, Tert, Rex) was analyzed by RT-PCR. The ability of hASCs to synthesize alkaline phosphatase was also evaluated. Cell number and viability were determined by fluorescein diacetate/propidium iodide staining. Compared to pure CPC, cultivation of hASCs on fiber-reinforced CPC transiently induced the gene expression of Runx2 and osterix (day 3), and long-lastingly augmented the expression of alkaline phosphatase (and its enzyme activity), collagen I, and osteonectin (until day 14). In contrast, augmented expression of all chondrogenic, adipogenic, and pluripotency markers was limited to day 3, followed by significant downregulation. Cultivation of hASCs on fiber-reinforced CPC reduced the cell number, but not the proportion of viable cells (viability > 95%). The PLGA component of fiber-reinforced, brushite-forming CPC supports long-lasting osteogenic differentiation of hASCs, whereas chondrogenesis, adipogenesis, and pluripotency are initially augmented, but subsequently suppressed. In view of parallel animal results, PLGA fibers may represent an interesting clinical target for future improvement of CPC- based bone regeneration.

Microorganisms @ materials surfaces in aircraft: Potential risks for public health? - A systematic review.

BACKGROUND: Civil air travel is increasingly recognized as an important potential source for the rapid spread of infectious diseases that were geographically confined in the past, creating international epidemics with great health and socio-economic impact. OBJECTIVE: The objective of this systematic review is to elucidate the correlations of materials surfaces (composition, structure, properties) and microbial dependences on them in aircraft. METHODS: The review was prepared according to PRISMA guidelines. Based on a systematic search for studies published before 30 June 2018 in English, we selected and reviewed the contamination, tenacity, and transmission of microorganisms related to specific surfaces within the aircraft cabin. We also reviewed the chemical composition and properties of these surface materials applied within aircraft. RESULTS: From a total of 828 records 15 articles were included for further analysis in this systematic review, indicating that the aircraft interior surfaces in seat areas (tray tables, armrests, seat covers) and lavatories (door knob handles, toilet flush buttons) are generally colonized by various types of potentially hazardous microorganisms. CONCLUSIONS: The interior surfaces in seat and lavatory areas could pose higher health risks by causing infections due to their relatively high microbial contamination compared with other interior surfaces. The classification, chemical composition, surface structures and physicochemical properties of materials surfaces have a varied effect on the adhesion, colonization, tenacity and potential transmission of microorganisms within the aircraft cabin. Strategies are proposed for the interruption of surface-related infection chains in the aircraft field.

Critical physiological factors influencing the outcome of antimicrobial testing according to ISO 22196 / JIS Z 2801.

Bactericidal materials gained interest in the health care sector as they are capable of preventing material surfaces from microbial colonization and subsequent spread of infections. However, commercialization of antimicrobial materials requires proof of their efficacy, which is usually done using in vitro methods. The ISO 22196 standard (Japanese test method JIS Z 2801) is a method for measuring the antibacterial activity of daily goods. As it was found reliable for testing the biocidal activity of antimicrobially active materials and surface coatings most of the laboratories participating in this study used this protocol. Therefore, a round robin test for evaluating antimicrobially active biomaterials had to be established. To our knowledge, this is the first report on inaugurating a round robin test for the ISO 22196 / JIS Z 2801. The first round of testing showed that analyses in the different laboratories yielded different results, especially for materials with intermediate antibacterial effects distinctly different efficacies were noted. Scrutinizing the protocols used by the different participants and identifying the factors influencing the test outcomes the approach was unified. Four critical factors influencing the outcome of antibacterial testing were identified in a series of experiments: (1) incubation time, (2) bacteria starting concentration, (3) physiological state of bacteria (stationary or exponential phase of growth), and (4) nutrient concentration. To our knowledge, this is the first time these parameters have been analyzed for their effect on the outcome of testing according to ISO 22196 / JIS Z 2801. In conclusion, to enable assessment of the results obtained it is necessary to evaluate these single parameters in the test protocol carefully. Furthermore, uniform and robust definitions of the terms antibacterial efficacy / activity, bacteriostatic effects, and bactericidal action need to be agreed upon to simplify communication of results and also regulate expectations regarding antimicrobial tests, outcomes, and materials.

Gold nanoparticle contact point density controls microbial adhesion on gold surfaces.

Surface structures in the nanometer range emerge as the next evolutionary breakthrough in the design of biomaterials with antimicrobial properties. However, in order to advance the application of surface nanostructuring strategies in medical implants, the very nature of the microbial repealing mechanism has yet to be understood. Herein, we demonstrate that the random immobilization of gold nanoparticles (AuNPs) on a material's surface generates the possibility to explore microbial adhesion in dependence of contact point densities at the biointerface between the microbe, i.e., Escherichia coli and the material's surface. By optimizing the contact point density defined by individual AuNPs, yet keeping the surface chemistry unchanged as evidenced by X-ray photoelectron spectroscopy, we show that the initial microbial adhesion can be successfully reduced up to 50%, compared to control (unstructured) surfaces. Furthermore, we observed a decrease in the size of microbial cells adhered to nanostructured surfaces. The results show that the spatial distance between the contact points plays a crucial role in regulating microbial adhesion, thus advancing our understanding of the microbial adhesion mechanism on nanostructured surfaces. We suggest that the introduced strategy for nanostructuring materials surfaces opens a research direction for highly microbial-resistant biomaterials.

The GDF5 mutant BB-1 enhances the bone formation induced by an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Targeted delivery of osteoinductive bone morphogenetic proteins (eg, GDF5) in bioresorbable calcium phosphate cement (CPC), potentially suitable for vertebroplasty and kyphoplasty of osteoporotic vertebral fractures, may be required to counteract augmented local bone catabolism and to support complete bone regeneration. The biologically optimized GDF5 mutant BB-1 may represent an attractive drug candidate for this purpose. PURPOSE: The aim of the current study was to test an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming CPC containing low-dose BB-1 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and were filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BB-1 (L5; CPC+fibers+BB-1; 5, 100, and 500 µg BB-1; n=6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BB-1) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (BV/TV) (assessed by micro-CT and histomorphometry), trabecular thickness (Tb.Th), and trabecular number (Tb.N); (3) bone formation, that is, osteoid volume/bone volume (OV/BV), osteoid surface/bone surface (OS/BS), osteoid thickness, mineralizing surface/bone surface (MS/BS), mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BB-1 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BB-1, additional significant effects of BB-1 were demonstrated for BMD, bone structure (BV/TV, Tb.Th, and Tb.N), and bone formation (OS/BS and MS/BS). The BB-1 effects on bone formation at 3 and 9 months were dose dependent, with 100 µg as the potentially optimal dosage. CONCLUSIONS: BB-1 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as 100 µg BB-1 was sufficient to augment middle- to long-term bone formation. A CPC containing the novel GDF5 mutant BB-1 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty and kyphoplasty.

GDF5 significantly augments the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Biodegradable calcium phosphate cement (CPC) represents a promising option for the surgical treatment of osteoporotic vertebral fractures. Because of augmented local bone catabolism, however, additional targeted delivery of bone morphogenetic proteins with the CPC may be needed to promote rapid and complete bone regeneration. PURPOSE: In the present study, an injectable, poly(l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing the bone morphogenetic protein GDF5 was tested in a sheep lumbar osteopenia model. STUDY DESIGN/SETTING: This is a prospective experimental animal study. METHODS: Defined bone defects (diameter 5 mm) were placed in aged, osteopenic female sheep. Defects were treated with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of GDF5 (L5; CPC+fibers+GDF5; 1, 5, 100, and 500 µg GDF5; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months postoperation, structural and functional effects of the CPC (±GDF5) were assessed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralized surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), both CPC+fibers (L4) and CPC+fibers+GDF5 (L5) numerically or significantly improved all parameters of bone formation, bone resorption, and bone structure. These significant effects were observed both at 3 and 9 months, but for some parameters they were less pronounced at 9 months. Compared with CPC without GDF5, additional significant effects of CPC with GDF5 were demonstrated for BMD and parameters of bone formation and structure (bone volume/total volume, trabecular thickness, and trabecular number, as well as mineralized surface/bone surface). The GDF5 effects were dose-dependent (predominantly in the 5-100 µg range) at 3 and 9 months. CONCLUSIONS: GDF5 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. The results indicated that a local dose as low as ≤100 µg GDF5 may be sufficient to augment middle to long-term bone formation. The novel CPC+GDF5 combination may thus qualify as an alternative to the bioinert, supraphysiologically stiff poly(methyl methacrylate) cement currently applied for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures.

Low-dose BMP-2 is sufficient to enhance the bone formation induced by an injectable, PLGA fiber-reinforced, brushite-forming cement in a sheep defect model of lumbar osteopenia.

BACKGROUND CONTEXT: Bioresorbable calcium phosphate cement (CPC) may be suitable for vertebroplasty/kyphoplasty of osteoporotic vertebral fractures. However, additional targeted delivery of osteoinductive bone morphogenetic proteins (BMPs) in the CPC may be required to counteract the augmented local bone catabolism and support complete bone regeneration. PURPOSE: This study aimed at testing an injectable, poly (l-lactide-co-glycolide) acid (PLGA) fiber-reinforced, brushite-forming cement (CPC) containing low-dose bone morphogenetic protein BMP-2 in a sheep lumbar osteopenia model. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter 5 mm) were generated in aged, osteopenic female sheep and filled with fiber-reinforced CPC alone (L4; CPC+fibers) or with CPC containing different dosages of BMP-2 (L5; CPC+fibers+BMP-2; 1, 5, 100, and 500 µg BMP-2; n=5 or 6 each). The results were compared with those of untouched controls (L1). Three and 9 months after the operation, structural and functional effects of the CPC (±BMP-2) were analyzed ex vivo by measuring (1) bone mineral density (BMD); (2) bone structure, that is, bone volume/total volume (assessed by micro-computed tomography [micro-CT] and histomorphometry), trabecular thickness, and trabecular number; (3) bone formation, that is, osteoid volume/bone volume, osteoid surface/bone surface, osteoid thickness, mineralizing surface/bone surface, mineral apposition rate, and bone formation rate/bone surface; (4) bone resorption, that is, eroded surface/bone surface; and (5) compressive strength. RESULTS: Compared with untouched controls (L1), CPC+fibers (L4) and/or CPC+fibers+BMP-2 (L5) significantly improved all parameters of bone formation, bone resorption, and bone structure. These effects were observed at 3 and 9 months, but were less pronounced for some parameters at 9 months. Compared with CPC without BMP-2, additional significant effects of BMP-2 were demonstrated for bone structure (bone volume/total volume, trabecular thickness, trabecular number) and formation (osteoid surface/bone surface and mineralizing surface/bone surface), as well as for the compressive strength. The BMP-2 effects on bone formation at 3 and 9 months were dose-dependent, with 5-100 µg as the optimal dosage. CONCLUSIONS: BMP-2 significantly enhanced the bone formation induced by a PLGA fiber-reinforced CPC in sheep lumbar osteopenia. A single local dose as low as ≤100 µg BMP-2 was sufficient to augment middle to long-term bone formation. The novel CPC+BMP-2 may thus represent an alternative to the bioinert, supraphysiologically stiff polymethylmethacrylate cement presently used to treat osteoporotic vertebral fractures by vertebroplasty/kyphoplasty.

Presence of Calcium Lowers the Expansion of Bacillus subtilis Colony Biofilms.

Robust colony formation by Bacillus subtilis is recognized as one of the sessile, multicellular lifestyles of this bacterium. Numerous pathways and genes are responsible for the architecturally complex colony structure development. Cells in the biofilm colony secrete extracellular polysaccharides (EPS) and protein components (TasA and the hydrophobin BslA) that hold them together and provide a protective hydrophobic shield. Cells also secrete surfactin with antimicrobial as well as surface tension reducing properties that aid cells to colonize the solid surface. Depending on the environmental conditions, these secreted components of the colony biofilm can also promote the flagellum-independent surface spreading of B. subtilis, called sliding. In this study, we emphasize the influence of Ca2+ in the medium on colony expansion of B. subtilis. Interestingly, the availability of Ca2+ has no major impact on the induction of complex colony morphology. However, in the absence of this divalent ion, peripheral cells of the colony expand radially at later stages of development, causing colony size to increase. We demonstrate that the secreted extracellular compounds, EPS, BslA, and surfactin facilitate colony expansion after biofilm maturation. We propose that Ca2+ hinders biofilm colony expansion by modifying the amphiphilic properties of surfactin.

Enhanced bone formation in sheep vertebral bodies after minimally invasive treatment with a novel, PLGA fiber-reinforced brushite cement.

BACKGROUND CONTEXT: Injectable, brushite-forming calcium phosphate cements (CPC) show potential for bone replacement, but they exhibit low mechanical strength. This study tested a CPC reinforced with poly(l-lactide-co-glycolide) acid (PLGA) fibers in a minimally invasive, sheep lumbar vertebroplasty model. PURPOSE: The study aimed to test the in vivo biocompatibility and osteogenic potential of a PLGA fiber-reinforced, brushite-forming CPC in a sheep large animal model. STUDY DESIGN/SETTING: This is a prospective experimental animal study. METHODS: Bone defects (diameter: 5 mm) were placed in aged, osteopenic female sheep, and left empty (L2) or injected with pure CPC (L3) or PLGA fiber-reinforced CPC (L4; fiber diameter: 25 µm; length: 1 mm; 10% [wt/wt]). Three and 9 months postoperation (n=20 each), the structural and functional CPC effects on bone regeneration were documented ex vivo by osteodensitometry, histomorphometry, micro-computed tomography (micro-CT), and biomechanical testing. RESULTS: Addition of PLGA fibers enhanced CPC osteoconductivity and augmented bone formation. This was demonstrated by (1) significantly enhanced structural (bone volume/total volume, shown by micro-CT and histomorphometry; 3 or 9 months) and bone formation parameters (osteoid volume and osteoid surface; 9 months); (2) numerically enhanced bone mineral density (3 and 9 months) and biomechanical compression strength (9 months); and (3) numerically decreased bone erosion (eroded surface; 3 and 9 months). CONCLUSIONS: The PLGA fiber-reinforced CPC is highly biocompatible and its PLGA fiber component enhanced bone formation. Also, PLGA fibers improve the mechanical properties of brittle CPC, with potential applicability in load-bearing areas.

pH-Dependent Ordered Fibrinogen Adsorption on Polyethylene Single Crystals.

Nanostructured surfaces have the potential to influence the assembly as well as the orientation of adsorbed proteins and may, thus, strongly influence the biomaterials' performance. For the class of polymeric (bio)materials a reproducible and well-characterized nanostructure is the ordered chain folded surface of a polyethylene single crystal (PE-SC). We tested the hypothesis that the trinodal-rod-shaped protein human plasma fibrinogen (HPF) adsorbs on the (001) surface of PE-SCs along specific crystallographic directions. The PE-SC samples were prepared by isothermal crystallization in dilute solution and characterized by atomic force microscopy (AFM) before as well as after HPF adsorption at different concentrations and pH values. At a physiological pH of 7.4, connected HPF molecules, or e.g., fibrils, fibril networks, or sponge-like structures, were observed on PE-SC surfaces that featured no preferential orientation. The observation of these nonoriented multiprotein assemblies was explained by predominant protein-protein interactions and limited surface diffusion. However, at an increased pH of 9.2, single HPF molecules, e.g., spherical-shaped and trinodal-rod-shaped HPF molecules as well as agglomerates, were observed on the PE-SC surface. The presence of single HPF molecules at increased pH was explained by decreased protein-protein interactions. These single trinodal-rod-shaped HPF molecules oriented preferentially along crystallographic [100] and [010] directions on the PE-SC surface which was explained by an increased amount of intermolecular bonds along these crystallographic directions with increased surface atom density. The study established that HPF molecules can align on chemically homogeneous surface topographies one order of magnitude smaller than the dimension of the protein. This advances the understanding of how to control the assembly and orientation of proteins on nanostructured polymer surfaces. Controlled protein adsorption is a crucial key to improve the surface functionality of future implants and biosensors.

Decreased extrusion of calcium phosphate cement versus high viscosity PMMA cement into spongious bone marrow-an ex vivo and in vivo study in sheep vertebrae.

BACKGROUND CONTEXT: Vertebroplasty or kyphoplasty of osteoporotic vertebral fractures bears the risk of pulmonary cement embolism (3.5%-23%) caused by leakage of commonly applied acrylic polymethylmethacrylate (PMMA) cement to spongious bone marrow or outside of the vertebrae. Ultraviscous cement and specific augmentation systems have been developed to reduce such adverse effects. Rapidly setting, resorbable, physiological calcium phosphate cement (CPC) may also represent a suitable alternative. PURPOSE: This study aimed to compare the intravertebral extrusion of CPC and PMMA cement in an ex vivo and in vivo study in sheep. STUDY DESIGN/SETTING: A prospective experimental animal study was carried out. METHODS: Defects (diameter 5 mm; 15 mm depth) were created by a ventrolateral percutaneous approach in lumbar vertebrae of female Merino sheep (2-4 years) either ex vivo (n=17) or in vivo (n=6), and injected with: (1) CPC (L3); (2) CPC reinforced with 10% poly(l-lactide-co-glycolide) (PLGA) fibers (L4); or (3) PMMA cement (L5; Kyphon HV-R). Controls were untouched (L1) or empty defects (L2). The effects of the cement injections were assessed in vivo by blood gas analysis and ex vivo by computed tomography (CT), micro-CT (voxel size: 67 µm), histology, and biomechanical testing. RESULTS: Following ex vivo injection, micro-CT documented significantly increased extrusion of PMMA cement in comparison to CPC (+/- fibers) starting at a distance of 1 mm from the edge of the defect (confirmed by histology); this was also demonstrated by micro-CT following in vivo cement injection. In addition, blood gas analysis showed consistently significantly lower values for the fraction of oxygenized hemoglobin/total hemoglobin (FO2Hb) in the arterial blood until 25 minutes following injection of the PMMA cement (p ≤ .05 vs. CPC; 7, 15 minutes). Biomechanical testing following ex vivo injection showed significantly lower compressive strength and Young modulus than untouched controls for the empty defect (40% and 34% reduction, respectively) and all three cement-injected defects (21%-27% and 29%-32% reduction, respectively), without significant differences among the cements. CONCLUSIONS: Because of comparable compressive strength, but significantly lower cement extrusion into spongious bone marrow than PMMA cement, physiological CPC (+/- PLGA fibers) may represent an attractive alternative to PMMA for vertebroplasty or kyphoplasty of osteoporotic vertebral fractures to reduce the frequency or severity of adverse effects.

First-time systematic postoperative clinical assessment of a minimally invasive approach for lumbar ventrolateral vertebroplasty in the large animal model sheep.

BACKGROUND CONTEXT: Large animal models are highly recommended for meaningful preclinical studies, including the optimization of cement augmentation for vertebral body defects by vertebroplasty/kyphoplasty. PURPOSE: The aim of this study was to perform a systematic characterization of a strictly minimally invasive in vivo large animal model for lumbar ventrolateral vertebroplasty. STUDY DESIGN/ SETTING: This is a prospective experimental animal study. METHODS: Lumbar defects (diameter 5 mm; depth approximately 14 mm) were created by a ventrolateral percutaneous approach in aged, osteopenic, female sheep (40 Merino sheep; 6-9 years; 68-110 kg). L1 remained untouched, L2 was left with an empty defect, and L3 carried a defect injected with a brushite-forming calcium phosphate cement (CPC). Trauma/functional impairment, surgical techniques (including drill sleeve and working canula with stop), reproducibility, bone defects, cement filling, and functional cement augmentation were documented by intraoperative incision-to-suture time and X-ray, postoperative trauma/impairment scores, and ex vivo osteodensitometry, microcomputed tomography (CT), histology, static/fluorescence histomorphometry, and biomechanical testing. RESULTS: Minimally invasive vertebroplasty resulted in short operation times (28±2 minutes; mean±standard error of the mean) and X-ray exposure (1.59±0.12 minutes), very limited local trauma (score 0.00±0.00 at 24 hours), short postoperative recovery (2.95±0.29 hours), and rapid decrease of the postoperative impairment score to 0 (3.28±0.36 hours). Reproducible defect creation and cement filling were documented by intraoperative X-ray and ex vivo conventional/micro-CT. Vertebral cement augmentation and osteoconductivity of the CPC was verified by osteodensitometry (CPC>control), micro-CT (CPC>control and empty defect), histology/static histomorphometry (CPC>control and empty defect), fluorescence histomorphometry (CPC>control; all p<.05 for 3 and 9 months), and compressive strength measurements (CPC numerically higher than control; 102% for 3 months and 110% for 9 months). CONCLUSIONS: This first-time systematic clinical assessment of a minimally invasive, ventrolateral, lumbar vertebroplasty model in aged, osteopenic sheep resulted in short operation times, rapid postoperative recovery, and high experimental reproducibility. This model represents an optimal basis for standardized evaluation of future studies on vertebral augmentation with resorbable and osteoconductive CPC.

Nanorough titanium surfaces reduce adhesion of Escherichia coli and Staphylococcus aureus via nano adhesion points.

Microbial adhesion to natural and synthetic materials surfaces is a key issue e.g. in food industry, sewage treatment and most importantly in the biomedical field. The current development and progress in nanoscale structuring of materials surfaces to control microbial adhesion requires an advanced understanding of the microbe-material-interaction. This study aimed to investigate the nanostructure of the microbe-material-interface and link it to microbial adhesion kinetics as function of titanium surface nanoroughness to gain new insight into controlling microbial adhesion via materials' surface nanoroughness. Adhesion of Escherichia coli and Staphylococcus aureus was statistically significantly reduced (p≤0.05) by 55.6 % and 40.5 %, respectively, on physical vapor deposited titanium thin films with a nanoroughness of 6nm and the lowest surface peak density compared to 2nm with the highest surface peak density. Cross-sectioning of the microbial cells with a focused ion beam (FIB) and SEM imaging provided for the first time direct insight into the titanium-microbe-interface. High resolution SEM micrographs gave evidence that the surface peaks are the loci of initial contact between the microbial cells and the material's surface. In a qualitative model we propose that the initial microbial adhesion on nanorough surfaces is controlled by the titanium surface peak density via nano adhesion points. This new understanding will help towards the design of materials surfaces for controlling microbial adhesion.