Ex vivo proton spectroscopy (1 H-NMR) analysis of inborn errors of metabolism: Automatic and computer-assisted analyses.

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy (1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism.

Nanoparticle decoration impacts airborne fungal pathobiology.

Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.

Biofabrication of SDF-1 Functionalized 3D-Printed Cell-Free Scaffolds for Bone Tissue Regeneration.

Large segmental bone defects occurring after trauma, bone tumors, infections or revision surgeries are a challenge for surgeons. The aim of our study was to develop a new biomaterial utilizing simple and cheap 3D-printing techniques. A porous polylactide (PLA) cylinder was printed and functionalized with stromal-derived factor 1 (SDF-1) or bone morphogenetic protein 7 (BMP-7) immobilized in collagen type I. Biomechanical testing proved biomechanical stability and the scaffolds were implanted into a 6 mm critical size defect in rat femur. Bone growth was observed via x-ray and after 8 weeks, bone regeneration was analyzed with µCT and histological staining methods. Development of non-unions was detected in the control group with no implant. Implantation of PLA cylinder alone resulted in a slight but not significant osteoconductive effect, which was more pronounced in the group where the PLA cylinder was loaded with collagen type I. Addition of SDF-1 resulted in an osteoinductive effect, with stronger new bone formation. BMP-7 treatment showed the most distinct effect on bone regeneration. However, histological analyses revealed that newly formed bone in the BMP-7 group displayed a holey structure. Our results confirm the osteoinductive character of this 3D-biofabricated cell-free new biomaterial and raise new options for its application in bone tissue regeneration.

Comparison of growth & function of endothelial progenitor cells cultured on deproteinized bovine bone modified with covalently bound fibronectin and bound vascular endothelial growth factor.

OBJECTIVES: The objective of this study was to assess and compare the growth and function of Endothelial Progenitor Cells (EPCs) cultured on covalently bonded Vascular Endothelial Growth Factor (VEGF) and covalently bonded Fibronectin (FN) coating on deproteinized bovine bone (DBB) (test samples), compared to non-modified DBB blocks (control sample). MATERIALS AND METHODS: The test samples were prepared by plasma polymerization of allylamine onto DBB blocks. Group1 of test samples were prepared with VEGF coating (VEGF-DBB) where as the Group2 test samples were coated with FN (FN-DBB). Non-modified DBB blocks served as a Control. EPCs were isolated and cultivated from buffy coats of peripheral blood of healthy volunteers and cultivated in the different samples and examined at time intervals of 24 h, 3 days, and 7 days. Evaluation of growth by cell count and cell morphology was done using Confocal Laser Scanning Electron Microscopy; vitality and function of cells was assessed using MTT assay and RT-PCR and ELISA for eNOS and iNOS respectively. RESULTS: The results of the study show that both VEGF and FN could be successfully immobilized by plasma polymerization onto a complex, porous, three-dimensional structure of DBB. When comparing vital cell coverage, proliferation and function of EPCs, FN-DBB provided more positive values followed by VEGF-DBB as compared to DBB samples. eNOS level were significant higher in VEGF-DBB and FN-DBB when compared to DBB (P = 0.019 and P = 0.002). The difference between VEGF-DBB and FN-DBB was not significant. CONCLUSIONS: Biomimetic coatings of Fibronectin may clinically relate to faster angiogenesis and earlier healing potential.

A New Bone Substitute Developed from 3D-Prints of Polylactide (PLA) Loaded with Collagen I: An In Vitro Study.

Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering.

The influence of various rehydration protocols on biomechanical properties of different acellular tissue matrices.

OBJECTIVES: This study evaluated the influence of different rehydration media and time periods on biomechanical and structural properties of different acellular collagen matrices (ACMs). MATERIALS AND METHODS: Specimens of three ACMs (mucoderm®, Mucograft®, Dynamatrix®) were rehydrated in saline solution (SS) or human blood for different time periods (5-60 min). ACMs under dry condition served as controls. Biomechanical properties of the ACMs after different rehydration periods were determined by means of tensile testing. ACMs' properties were further characterized using Fourier-transform-infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). RESULTS: At dry conditions, mucoderm® presented the highest tensile strength (TS) and Dynamatrix® showed the maximum elastic modulus (EM; p each ≤0.036). Rehydration in SS and blood resulted in significant TS changes of mucoderm® (p each ≤0.05). Concering EM, mucograft® showed significantly decreased values after rehydration in SS compared to Dynamatrix® and mucoderm® after 10 min (p each ≤0.024). mucoderm® hydrated for 5 min in blood displayed nearly double TS and a significantly increased EM after 60 min (p = 0.043) compared to rehydration in SS. TS and EM values of Dynamatrix® and Mucograft® were not altered following rehydration in blood versus SS (p each ≥0.053). FTIR analysis confirmed the recovery of the graft protein backbone with increased rehydration in all samples. DSC measurements revealed that tissue hydration decreased thermal stability of the investigated ACMs. CONCLUSION: Our findings demonstrated that the rehydration protocol affects the biomechanical properties of ACMs. CLINICAL RELEVANCE: Clinicians should be aware of altered handling and mechanical properties of ACMs following different rehydration protocols.

The influence of bone substitute materials on the bone volume after maxillary sinus augmentation: a microcomputerized tomography study.

OBJECTIVES: This study aims to evaluate the effect of adding bone substitute materials (BSM) to particulated autogenous bone (PAB) on the volume fraction (Vf) of newly formed bone after maxillary sinus augmentation. MATERIALS AND METHODS: Thirty healthy patients undergoing maxillary sinus augmentation were included. PAB (N = 10), mixtures of PAB and beta-tricalciumphosphate (PAB/ß-TCP) (N = 10), as well as PAB and ß-TCP and hydroxyapatite (PAB/HA/ß-TCP) (N = 10) were randomly used for sinus augmentation. A sample of the graft material was maintained from each patient at time of maxillary sinus augmentation, and Vfs of the PAB and/or BSM in the samples were determined by means of microcomputerized tomography (µ-CT). Five months later, samples of the grafted areas were harvested during implantation using a trephine bur. µ-CT analysis of these samples was performed, and the Vf of bone and BSM were compared with the data obtained 5 months earlier from the original material. RESULTS: The mean Vf of the bone showed a statistically significant increase (p < 0.05) in all groups after a healing period of 5 months without statistically significant difference between the groups. CONCLUSIONS: With regard to the increase of bone volume, it is not relevant if PAB is used alone or combined with ß-TCP or HA/ß-TCP. CLINICAL RELEVANCE: The amount of PAB and associated donor site morbidity may be reduced by adding BSM for maxillary sinus augmentation.

Depth profile analysis of non-specific fluorescence and color of tooth tissues after peroxide bleaching.

PURPOSE: To examine laboratory changes of endogenous non-specific fluorescence and color throughout subsurface of tooth structures prior to and following peroxide bleaching. METHODS: Extracted human teeth were cross sectioned and mounted on glass slides. Cross sections were examined for internal color (digital camera) and nonspecific fluorescence (microRaman spectroscopy) throughout the tooth structure at specified locations. Surfaces of sections were then saturation bleached for 70 hours with a gel containing 6% hydrogen peroxide. Cross sections were reexamined for color and non-specific fluorescence changes. RESULTS: Unbleached enamel, dentin-enamel junction and dentin exhibit different CIELab color and non-specific fluorescence properties. Bleaching of teeth produced significant changes in color of internal cross sections and substantial reductions of non-specific fluorescence levels within enamel dentin and DEJ. Enamel and dentin non-specific fluorescence were reduced to common values with bleaching with enamel and the DEJ showing larger reductions than dentin.

Early root surface colonization by human periodontal ligament fibroblasts following treatment with different biomaterials.

OBJECTIVES: The present in-vitro study examined the effects of different biomaterials on early root surface colonization by human periodontal ligament (PDL) fibroblasts using confocal-laser-scanning-microscopy (CLSM). MATERIALS AND METHODS: Fifteen periodontally-diseased teeth were extracted, treated with scaling/root planing and longitudinally cut to obtain 30 root fragments. Fragments were treated either with 24% EDTA following application of enamel matrix derivative (EMD), 24% EDTA or EMD only, nanocrystalline hydroxyapatite (NHA) paste or oily calcium hydroxide suspension (OCHS) for 1 h each. The analogue untreated root specimens served as controls. Root fragments were incubated with human PDL fibroblasts and cellular proliferation and morphology were evaluated after 1, 3, 5 and 8 days using CLSM-visualization and image recognition software. RESULTS: The rate of cellular proliferation was different among treatment modalities examined (p = 0.019). Except treatment with NHA paste all treatment modalities improved cellular proliferation on root surfaces at all different points of time compared with the control specimens. A significant difference between treatment modalities was observed between EMD and NHA paste (p = 0.008). No synergistic effect could be demonstrated comparing root surface conditioning with 24% EDTA and EMD application compared to 24% EDTA or EMD application only. CONCLUSION: The present results suggest that initial root surface colonization by PDL fibroblasts may be enhanced by root surface conditioning with 24% EDTA and application of EMD, application of 24% EDTA or EMD alone and OCHS. The addition of 24% EDTA for root surface conditioning prior to EMD application provided no synergistic effects in terms of early root surface colonization by PDL fibroblasts.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Bone Regeneration In vitro and In vivo.

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I. Two critical size bone defect models were used to analyze bone regeneration. BSP incorporated in collagen type I increased bone regeneration only marginally at one concentration in a calvarial defect model. To induce the mechanical stability, three-dimensional printing was used to produce a stable porous cylinder of polylactide. The cylinder was filled with collagen type I and immobilized BSP and implanted into a femoral defect of critical size in rats. This hybrid material was able to significantly induce bone regeneration. Our study clearly shows the osteogenic effect of BSP when combined with collagen type I as carrier and thereby offers various approaches and options for its use as bioactive molecule in bone substitute materials.

Circumferential spicule growth by pericellular silica deposition in the hexactinellid sponge Monorhaphis chuni.

The giant basal spicule of the hexactinellid sponge Monorhaphis chuni represents the longest natural siliceous structure on Earth. This spicule is composed of concentrically arranged lamellae that are approximately 10 µm thick. In the present study, we investigated the formation of outer lamellae on a cellular level using microscopic and spectroscopic techniques. It is shown that the formation of an outermost lamella begins with the association of cell clusters with the surface of the thickening and/or growing spicule. The cells release silica for controlled formation of a lamella. The pericellular (silica) material fuses to a delimited and textured layer of silica with depressions approximately 20-30 µm in diameter. The newly formed layer initially displays 40 µm wide, well-structured banded ribbons and only attains its plain surface in a final step. The chemical composition in the depressions was studied using energy dispersive X-ray spectroscopy and by staining with Texas Red. The data suggest that those depressions are the nests for the silica-forming cells and that silica formation starts with a direct association of silica-forming cells with the outer surface of the spicule, where they remain and initiate the development of the next lamellae.

Comparative analysis of numerical and experimental data of orthodontic mini-implants.

The purpose of this study was to compare numerical simulation data derived from finite element analysis (FEA) to experimental data on mini-implant loading. Nine finite element (FE) models of mini-implants and surrounding bone were derived from corresponding experimental specimens. The animal bone in the experiment consisted of bovine rib. The experimental groups were based on implant type, length, diameter, and angle of insertion. One experimental specimen was randomly selected from each group and was digitized in a microCT scanner. The FE models consisted of bone pieces containing Aarhus mini-implants with dimensions 1.5 × 7 mm and 1.5 × 9 mm or LOMAS mini-implants (dimensions 1.5 × 7 mm, 1.5 × 9 mm, and 2 × 7 mm). Mini-implants were inserted in two different ways, perpendicular to the bone surface or at 45 degrees to the direction of the applied load. Loading and boundary conditions in the FE models were adjusted to match the experimental situation, with the force applied on the neck of the mini-implants, along the mesio-distal direction up to a maximum of 0.5 N. Displacement and rotation of mini-implants after force application calculated by FEA were compared to previously recorded experimental deflections of the same mini-implants. Analysis of data with the Altman-Bland test and the Youden plot demonstrated good agreement between numerical and experimental findings (P = not significant) for the models selected. This study provides further evidence of the appropriateness of the FEA as an investigational tool in relevant research.

Long-term response of osteogenic cells on micron and submicron-scale-structured hydrophilic titanium surfaces: sequence of cell proliferation and cell differentiation.

OBJECTIVE: Modifications of surface topography and surface chemistry are key factors for guiding target cells during dental implant healing. Recent in vitro studies confirmed promotion of early osteogenic cell differentiation on submicron scaled surfaces in particular when hydrophilized. However, no long-term observations on both osteogenic cell proliferation as well as on cell maturation have been reported for respectively modified surfaces. Aim of this study was to monitor osteogenic cell proliferation and expression of specific osteogenic cell differentiation markers on a protein level over an extended period of 3 weeks with respect to surface modifications. MATERIAL AND METHODS: Modified titanium (Ti) disks were obtained from Institute Straumann, representing the following surfaces: smooth pretreatment (PT), sandblasted/acid etched (SLA), and hydrophilized (modSLA). Surface topography was analyzed by scanning electron microscopy, surface elemental composition was assessed by X-Ray Photoelectronic Spectroscopy (XPS). Tissue culture polystyrene (TCPS) served as a control substrate. Human osteogenic cells (HOB-c) were cultivated on the respective substrates. After 24 hrs, 48 hrs, 72 hrs, 7 d, 14 d and 21 d, cell count was assessed as well as osteogenic cell differentiation utilizing cellular Quantitative Immuno-Cytochemistry (QIC) assay for collagen type I (COL), alkaline phosphatase (AP), osteopontin (OPN) and osteocalcin (OC). Data were normalized with respect to internal controls. RESULTS: In contrast to the other modified Ti disks, modSLA stands out due to low surface carbon contamination. TCPS and PT surfaces preserved a rather immature, mitotic active osteogenic phenotype (high proliferation rates, no increase of OC production), SLA and especially modSLA surfaces promoted the maturation of osteogenic precursors into post-mitotic osteoblasts. In detail, modSLA resulted in lowest cell proliferation rates, but exhibited highest expression rates of OC. DISCUSSION: Our results, which confirm previous studies, reveal long-term promotion of osteogenic cell maturation by topography (micron and submicron scale roughness) and surface hydrophilicity.

Three-dimensional analysis of bone formation after maxillary sinus augmentation by means of microcomputed tomography: a pilot study.

PURPOSE: Although many studies have analyzed the suitability of different grafting materials for maxillary sinus augmentation by means of histomorphometry in conventional histologic strains, the three-dimensional (3D) structure and remodeling of these grafts after healing beneath the sinus membrane remain unclear. The aim of the present study was to determine whether microcomputed tomography is a suitable method to evaluate the 3D structure and remodeling of grafts after sinus floor augmentation. MATERIALS AND METHODS: Sinus floor augmentation was performed in five patients using autogenous bone (AB) alone, AB and beta-tricalcium phosphate (b-TCP, Cerasorb), AB and b TCP/hydroxyapatite (HA) (BoneCeramic), AB and calcium carbonate (Algipore), and AB and HA (PepGen). Specimens from the grafted sites were harvested by means of a trephine bur 5 to 16 months after maxillary sinus augmentation. Microcomputed tomography of these specimens was performed with a nominal isotropic resolution of 6 x 6 x 6 µm² voxel size. After segmentation, 3D images were reconstructed, and the distribution of bone and substitute material was evaluated by means of volumetric and density measurements. RESULTS: In all images, both bone and substitute material could clearly be identified. The connectivity of trabeculae surrounding the substitute material was visible in the 3D reconstructions. Volumetric evaluation such as total bone volume, volume of substitute material, and trabecular thickness and spacing revealed differences between the different grafting materials. CONCLUSION: Microcomputed tomography is a promising method to evaluate the 3D structure of grafts after sinus floor augmentation with autogenous bone and bone substitute materials.

Biological responses to individualized small titanium implants for the treatment of focal full-thickness knee cartilage defects in a sheep model.

BACKGROUND: The present study aimed to evaluate the functional, radiological and histological outcome of a customized focal implant for the treatment of focal full-thickness cartilage defects in sheep. METHODS: The study used magnetic resonance imaging data as the basis for construction of the titanium implant using a three-dimensional printing technique. This was then placed on the medial condyle of the knee joint in eight sheep and left in place in vivo over a period of six months. Following euthanasia, the local biological response was analyzed using micro-computed tomography, light microscopy and histological evaluation (International Cartilage Repair Society (ICRS) score). The variables were analyzed using a generalized linear mixed model. Odds ratios were given with 95% confidence intervals. RESULTS: The osseointegration rate was 62.1% (SD 3.9%). All implants were prone to the neighboring cartilage bed (4.4-1096.1 µm). Using the IRCS score, the elements 'surface', 'matrix', 'cell distribution' and 'cell population' all showed pathological changes on the operated side, although these did not correlate with implant elevation. On average, a difference of 0.7 mm (±2 mm) was found between the digitally planned implant and the real implant. CONCLUSIONS: As a result of imprecise segmentation and difficult preparation conditions at the prosthesis bed, as well as changes at the surface of the implant over the operational lifetime of the prosthesis, it must be stated that the approach implemented here of using a customized implant for the treatment of focal full-thickness cartilage defects at the knee did not meet our expectations.

Effect of bone sialoprotein coating on progression of bone formation in a femoral defect model in rats.

PURPOSE: In orthopedic and trauma surgery, calcium phosphate cement (CPC) scaffolds are widely used as substitute for autologous bone grafts. The purpose of this study was to evaluate bone formation in a femoral condyle defect model in rats after scaffold-coating with bioactive bone sialoprotein (BSP). Our hypothesis was that BSP-coating results in additional bone formation. METHODS: In 20 Wistar rats, defects of 3.0 mm diameter were drilled into the lateral femoral condyles of both legs. BSP-coated scaffolds or uncoated control scaffolds were implanted into the defects. After 4 and 8 weeks, five rats of each group were euthanized, respectively. µCT scans and histological analyses were performed. The ratio of bone volume-total volume (BV/TV) was analyzed and histological sections were evaluated. RESULTS: At week four, bone fraction reached 5.2 ± 1.7% in BSP-coated scaffolds and 4.5 ± 3.2% in the control (p = 0.06). While bone fraction of the BSP-group did not change much between week four and eight [week eight: 5.4 ± 3.8% (p = 0.53)], there was a tendency towards an increase in the control [week eight: 7.0 ± 2.2% (p = 0.08)]. No significant difference in bone fraction were observable between BSP-coated and uncoated scaffolds at week eight (p = 0.08). CONCLUSIONS: A significant superiority of BSP-coated scaffolds over uncoated scaffolds could not be proven. However, BSP-coating showed a tendency towards improving bone ingrowth in the scaffolds 4 weeks after implantation. This effect was only short-lived: bone growth in the control scaffolds tended to outpace that of the BSP-group at week eight.

Axial growth of hexactinellid spicules: formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis.

The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3m and diameters of 8.5mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS-PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30-60nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules.

Evaluation of Bone Sialoprotein Coating of Three-Dimensional Printed Calcium Phosphate Scaffolds in a Calvarial Defect Model in Mice.

The bioactive coating of calcium phosphate cement (CPC) is a promising approach to enhance the bone-healing properties of bone substitutes. The purpose of this study was to evaluate whether coating CPCs with bone sialoprotein (BSP) results in increased bone formation. Forty-five female C57BL/6NRj mice with an average age of six weeks were divided into three groups. Either a BSP-coated or an uncoated three-dimensional plotted scaffold was implanted into a drilled 2.7-mm diameter calvarial defect, or the defect was left empty (control group; no CPC). Histological analyses revealed that BSP-coated scaffolds were better integrated into the local bone stock eight weeks after implantation. Bone volume/total volume (BV/TV) ratios and bone thickness at the bone⁻implant contact were analyzed via micro computed tomography (µCT) after eight weeks. BSP-coated scaffolds and uncoated CPC scaffolds increased bone thickness in comparison to the control (CPC + BSP: 691.1 ± 253.5 µm, CPC: 603.1 ± 164.4 µm, no CPC: 261.7 ± 37.8 µm, p < 0.01). Accordingly, BV/TV was enhanced in both scaffold groups (CPC + BSP: 1.3 ± 0.5%, CPC: 0.9 ± 0.5%, no CPC: 0.2 ± 0.3%, p < 0.01). The BSP coating showed a tendency towards an increased bone thickness (p = 0.18) and BV/TV (p = 0.18) in comparison to uncoated CPC scaffolds. However, a significant increase in bone formation through BSP coating was not found.

Effect of bone sialoprotein coated three-dimensional printed calcium phosphate scaffolds on primary human osteoblasts.

The combination of the two techniques of rapid prototyping 3D-plotting and bioactive surface functionalization is presented, with emphasis on the in vitro effect of Bone Sialoprotein (BSP) on primary human osteoblasts (hOBs). Our primary objective was to demonstrate the BSP influence on the expression of distinctive osteoblast markers in hOBs. Secondary objectives included examinations of the scaffolds' surface and the stability of BSP-coating as well as investigations of cell viability and proliferation. 3D-plotted calcium phosphate cement (CPC) scaffolds were coated with BSP via physisorption. hOBs were seeded on the coated scaffolds, followed by cell viability measurements, gene expression analysis and visualization. Physisorption is an effective method for BSP-coating. Coating with higher BSP concentrations leads to enhanced BSP release. Two BSP concentrations (50 and 200 µg/mL) were examined in this study. The lower BSP concentration (50 µg/mL) decreased ALP and SPARC expression, whereas the higher BSP concentration (200 µg/mL) did not change gene marker expression. Enhanced cell viability was observed on BSP-coated scaffolds on day 3. hOBs developed a polygonal shape and connected in an intercellular network under BSP influence. Quantitative cell morphology analyses demonstrated for BSP-coated CPCs an enhanced cell area and reduced circularity. The strength of the above-mentioned effects of BSP-coated scaffolds in vivo is unknown, and future work is focusing on bone ingrowth and vascularization in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2565-2575, 2018.

CD34+ cells seeded in collagen scaffolds promote bone formation in a mouse calvarial defect model.

Bone tissue engineering (BTE) holds promise for managing the clinical problem of large bone defects. However, clinical adoption of BTE is limited due to limited vascularization of constructs, which could be circumvented by pre-cultivation of osteogenic and endothelial derived cells in natural-based polymer scaffolds. However, until now not many studies compared the effect of mono- and cocultures pre-seeded in collagen before implantation. We utilized a mouse calvarial defect model and compared five groups of collagen scaffolds: a negative control of a collagen scaffold alone, a positive control treated with BMP-7, monocultures of either human osteoblasts (hOBs) or CD34+ cells, and a coculture of hOB and CD34+ cells. Each pre-seeded collagen scaffold was implanted in mice. After 6 weeks mice were sacrificed and their skulls prepared for volumetric and histologic analysis. We found that a monoculture of CD34+ cells and a coculture of hOB and CD34+ cells pre-cultured in the collagen scaffold increased bone regeneration to a similar extend. In these groups, greater amounts of new bone were found compared with hOB monocultures. Interestingly, monoculture of CD34+ cells demonstrated better fracture healing than monoculture of hOBs, emphasizing the possible role of angiogenesis. Our results are promising regarding a cellular based collagen BTE construct, but more work is needed to understand the complex interaction between the osteogenic and endothelial cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1505-1516, 2018.