Sascha Heinemann

Bioactive silica-collagen composite xerogels modified by calcium phosphate phases with adjustable mechanical properties for bone replacement.

The development of composites has been recognized as a promising strategy to fulfil the complex requirements of biomaterials. The present study reports on the modification of a novel silica-collagen composite material by varying the inorganic/organic mass ratio and introducing calcium phosphate cement (CPC) as a third component. The sol-gel technique is used for processing, followed by xerogel formation under specific temperature and relative humidity conditions. Cylindrical monolithic samples up to 400mm(3) were obtained without any sintering processes. Various hierarchical phases of the organic component were applied, ranging from tropocollagen and collagen fibrils up to collagen fibers, each characterized by atomic force microscopy. Focusing on the application of fibrils, various inorganic/organic mass ratios were used: 100/0, 85/15 and 70/30; their influence on the structure of the composite material was demonstrated by scanning electron microscopy. The composition was extended by the addition of 25wt.% CPC which led to increased bioactivity by accelerating the formation of bone apatite layers in simulated body fluid. Synchrotron microcomputed tomography demonstrated the homogeneous distribution of the cement particles in the silica-collagen matrix. Compressive strength tests showed that the mechanical properties of the brittle pure silica gel are changed significantly due to collagen addition. The highest ultimate strength of about 115MPa at about 18% total strain was registered for the 70/30 silica-collagen composite xerogels. Incorporation of CPC lowered the gels strength. By demonstrating differentiation of human monocytes into osteoclast-like cells, an important feature of the composite material regarding successful bone remodeling is fulfilled.

Novel Textile Chitosan Scaffolds Promote Spreading, Proliferation, and Differentiation of Osteoblasts

Two novel scaffold models made of chitosan fibers were designed, fabricated, and investigated. Raw chitosan fibers were either tightened between plastic rings or were processed into stand-alone scaffolds. Chitosan fiber scaffolds were further modified by coating with a thin layer of fibrillar collagen type I to biologize the surface. Cell culture experiments were carried out using murine osteoblast-like cells (7F2). Confocal laser scanning microscopy (cLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were cultivated for up to 4 weeks on the materials and proliferation as well as osteogenic differentiation was quantitatively analyzed in terms of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity. We found a 14-16-fold increase of cell number and the typical pattern of ALP activity, whereas the collagen coating does not remarkably influence these parameters. The maintenance of osteogenic phenotype on the novel materials was furthermore confirmed by immunostaining of osteocalcin and study of matrix mineralization. The feature of the collagen-coated but also the raw chitosan fiber scaffolds to support the attachment, proliferation, and differentiation of osteoblast-like cells suggest a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.

Nanostructural organization of naturally occurring composites-part II: silica-chitin-based biocomposites

Investigations of the micro-and nanostructures and chemical composition of the sponge skeletons as examples for natural structural biocomposites are of fundamental scientific relevance. Recently, we show that some demosponges (Verongula gigantea, Aplysina sp.) and glass sponges (Farrea occa, Euplectella aspergillum) possess chitin as a component of their skeletons. The main practical approach we used for chitin isolation was based on alkali treatment of corresponding external layers of spicules sponge material with the aim of obtaining alkali-resistant compounds for detailed analysis. Here, we present a detailed study of the structural and physicochemical properties of spicules of the glass sponge Rossella fibulata. The structural similarity of chitin derived from this sponge to invertebrate alpha chitin has been confirmed by us unambiguously using physicochemical and biochemical methods. This is the first report of a silica-chitin composite biomaterial found in Rossella species. Finally, the present work includes a discussion related to strategies for the practical application of silica-chitin-based composites as biomaterials.

Biomimetically Mineralized Salmon Collagen Scaffolds for Application in Bone Tissue Engineering

Biomimetic mineralization of collagen is an advantageous method to obtain resorbable collagen/hydroxy-apatite composites for application in bone regeneration. In this report, established procedures for mineralization of bovine collagen were adapted to a new promising source of collagen from salmon skin challenged by the low denaturation temperature. Therefore, in the first instance, variation of temperature, collagen concentration, and ionic strength was performed to reveal optimized parameters for fibrillation and simultaneous mineralization of salmon collagen. Porous scaffolds from mineralized salmon collagen were prepared by controlled freeze-drying and chemical cross-linking. FT-IR analysis demonstrated the mineral phase formed during the preparation process to be hydroxyapatite. The scaffolds exhibited interconnecting porosity, were sufficiently stable under cyclic compression, and showed elastic mechanical properties. Human mesenchymal stem cells were able to adhere to the scaffolds, cell number increased during cultivation, and osteogenic differentiation was demonstrated in terms of alkaline phosphatase activity.

In Vitro Evaluation of Textile Chitosan Scaffolds for Tissue Engineering using Human Bone Marrow Stromal Cells

Textile chitosan fiber scaffolds were developed and tested in terms of biocompatibility for human bone marrow stromal cells (hBMSCs). A part of the scaffolds was further modified by coating with fibrillar collagen type I in order to biologize the surface. hBMSCs of two donors were used for cell culture experiments in vitro. Confocal laser scanning microscopy (CLSM) as well as scanning electron microscopy (SEM) revealed fast attachment and morphological adaptation of the cells on both the raw chitosan fibers and the collagen-coated scaffolds. Cells were osteogenically induced after 3 days and cultivated for up to 28 days on the scaffolds. Activity of lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) was analyzed to evaluate proliferation as well as osteogenic differentiation. We found a 3.5-6-fold increase in the cell number, whereas the collagen coating did not noticeably influence these factors. Osteogenic differentiation was confirmed by the course of ALP activity and immunostaining of osteocalcin. The feature of the collagen-coated as well as the raw chitosan fiber scaffolds to support attachment, proliferation, and differentiation of hBMSCs suggests a potential application of chitosan fibers and textile chitosan scaffolds for the tissue engineering of bone.

Calcium phosphate phases integrated in silica/collagen nanocomposite xerogels enhance the bioactivity and ultimately manipulate the osteoblast/osteoclast ratio in a human co-culture model

A human co-culture model of osteoblasts and osteoclasts, derived from bone marrow stromal cells and monocytes respectively, was used to characterize the influence of biomaterial modification on the bioactivity and ultimately the ratio of bone-forming to bone-resorbing cells cultivated directly on the surface. Nanocomposites of silica and collagen have been shown to function as skeletal structures in nature and were reproduced in vitro by using a sol-gel approach. The resulting xerogels exhibit a number of features that make it a valuable system for the development of innovative materials for bone substitution applications. In the present study, the incorporation of different calcium phosphate phases in silica/collagen-based gels was demonstrated to enhance the bioactivity of these samples. This ability of the biomaterial to precipitate calcium phosphate on the surface when incubated in simulated body fluids or cell culture medium is generally considered to an advantageous property for bone substitution materials. By co-cultivating human osteoblasts and osteoclasts up to 42 days on the xerogels, we demonstrate that the long-term ratio of these cell types depends on the level of bioactivity of the substrate samples. Biphasic silica/collagen xerogels exhibited comparably low bioactivity but encouraged proliferation of osteoblasts in comparison to osteoclast formation. A balanced ratio of both cell types was detected for moderately bioactive triphasic xerogels with 5% calcium phosphate. However, enhancing the bioactivity of the xerogel samples by increasing the calcium phosphate phase percentage to 20% resulted in a diminished number of osteoblasts in favor of osteoclast formation. Quantitative evaluation was carried out by biochemical methods (calcium, DNA, ALP, TRAP 5b) as well as RT-PCR (ALP, BSP II, OC, RANKL, TRAP, CALCR, VTNR, CTSK), and was supported by confocal laser scanning microscopy (cell nuclei, actin, CD68, TRAP) as well as scanning electron microscopy.

A new metaphyseal bone defect model in osteoporotic rats to study biomaterials for the enhancement of bone healing in osteoporotic fractures

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.

Biological functionalization of dental implants with collagen and glycosaminoglycans—A comparative study

Biological implant surface coatings are an emerging technology to increase bone formation. Such an approach is of special interest in anatomical regions like the maxilla. In the present study, we hypothesized that the coating of titanium implants with components of the organic extracellular matrix increases bone formation and implant stability compared to an uncoated reference. The implants were coated using collagen-I with either two different concentrations of chondroitin sulfate (CS) or two differentially sulfated hyaluronans. Implant coatings were characterized biochemically and with atomic force microscopy. Histomorphometry was used to assess bone-implant contact (BIC) and bone-volume density (BVD) after 4 and 8 weeks of submerged healing in the maxilla of 20 minipigs. Further, implant stability was measured by resonance frequency analysis (RFA). Implants containing the lower CS concentration had significantly more BIC, compared to the uncoated reference at both times of interest. No significant increase was measured from week 4 to 8. Differences in BVD and RFA were statistically not significant. A higher concentration of CS and the application of sulfated hyaluronans showed no comparable increase in BIC. This study demonstrates a positive effect of a specific collagen-glycosaminoglycan combination on early bone formation in vivo.

Preparation and characterization of marine sponge collagen nanoparticles and employment for the transdermal delivery of 17β-estradiol-hemihydrate

Background: Transdermal administration of estradiol offers advantages over oral estrogens for hormone replacement therapy regarding side effects by bypassing the hepatic presystemic metabolism. Aim: The objective of this study was to develop nanoparticles of Chondrosia reniformis sponge collagen as penetration enhancers for the transdermal drug delivery of 17β-estradiol-hemihydrate in hormone replacement therapy. Method: Collagen nanoparticles were prepared by controlled alkaline hydrolysis and characterized using atomic force microscopy and photon correlation spectroscopy. Estradiol-hemihydrate was loaded to the nanoparticles by adsorption to their surface, whereupon a drug loading up to 13.1% of sponge collagen particle mass was found. After incorporation of drug-loaded nanoparticles in a hydrogel, the estradiol transdermal delivery from the gel was compared with that from a commercial gel that did not contain nanoparticles. Results: Saliva samples in postmenopausal patients showed significantly higher estradiol levels after application of the gel with nanoparticles. The area under the curve (AUC) for estradiol time–concentration curves over 24 hours was 2.3- to 3.4-fold higher and estradiol levels 24 hours after administration of estradiol were at least twofold higher with the nanoparticle gel. Conclusions: The hydrogel with estradiol-loaded collagen nanoparticles enabled a prolonged estradiol release compared to a commercial gel and yielded a considerably enhanced estradiol absorption. Consequently, sponge collagen nanoparticles represent promising carriers for transdermal drug delivery.

Properties of injectable ready-to-use calcium phosphate cement based on water-immiscible liquid.

Calcium phosphate cements (CPCs) are highly valuable materials for filling bone defects and bone augmentation by minimal invasive application via percutaneous injection. In the present study some key features were significantly improved by developing a novel injectable ready-to-use calcium phosphate cement based on water-immiscible carrier liquids. A combination of two surfactants was identified to facilitate the targeted discontinuous exchange of the liquid for water after contact with aqueous solutions, enabling the setting reaction to take place at distinct ratios of cement components to water. This prolonged the shelf life of the pre-mixed paste and enhanced reproducibility during application and setting reactions. The developed paste technology is applicable for different CPC formulations. Evaluations were performed for the formulation of an α-TCP-based CPC as a representative example for the preparation of injectable pastes with a powder-to-carrier liquid ratio of up to 85:15. We demonstrate that the resulting material retains the desirable properties of conventional CPC counterparts for fast setting, mechanical strength and biocompatibility, shows improved cohesion and will most probably show a similar degree of resorbability due to identical mineral structure of the set products.