David Eglin

The effect of human osteoblasts on proliferation and neo-vessel formation of human umbilical vein endothelial cells in a long-term 3D co-culture on polyurethane scaffolds.

Angiogenesis is a key element in early wound healing and is considered important for tissue regeneration and for directing inflammatory cells to the wound site. The improvement of vascularization by implementation of endothelial cells or angiogenic growth factors may represent a key solution for engineering bone constructs of large size. In this study, we describe a long-term culture environment that supports the survival, proliferation, and in vitro vasculogenesis of human umbilical vein endothelial cells (HUVEC). This condition can be achieved in a co-culture model of HUVEC and primary human osteoblasts (hOB) employing polyurethane scaffolds and platelet-rich plasma in a static microenvironment. We clearly show that hOB support cell proliferation and spontaneous formation of multiple tube-like structures by HUVEC that were positive for the endothelial cell markers CD31 and vWF. In contrast, in a monoculture, most HUVEC neither proliferated nor formed any apparent vessel-like structures. Immunohistochemistry and quantitative PCR analyses of gene expression revealed that cell differentiation of hOB and HUVEC was stable in long-term co-culture. The three-dimensional, FCS-free co-culture system could provide a new basis for the development of complex tissue engineered constructs with a high regeneration and vascularization capacity.

Physical Stimulation of Chondrogenic Cells In Vitro: A Review

BackgroundMechanical stimuli are of crucial importance for the development and maintenance of articular cartilage. For conditioning of cartilaginous tissues, various bioreactor systems have been developed that have mainly aimed to produce cartilaginous grafts for tissue engineering applications. Emphasis has been on in vitro preconditioning, whereas the same devices could be used to attempt to predict the response of the cells in vivo or as a prescreening method before animal studies. As a result of the complexity of the load and motion patterns within an articulating joint, no bioreactor can completely recreate the in vivo situation.Questions/purposesThis article aims to classify the various loading bioreactors into logical categories, highlight the response of mesenchymal stem cells and chondrocytes to the various stimuli applied, and determine which data could be used within a clinical setting.MethodsWe performed a Medline search using specific search terms, then selectively reviewed relevant research relating to physical stimulation of chondrogenic cells in vitro, focusing on cellular responses to the specific load applied.ResultsThere is much data pertaining to increases in chondrogenic gene expression as a result of controlled loading protocols. Uniaxial loading leads to selective upregulation of genes normally associated with a chondrogenic phenotype, whereas multiaxial loading results in a broader pattern of chondrogenic gene upregulation. The potential for the body to be used as an in vivo bioreactor is being increasingly explored.ConclusionsBioreactors are important tools for understanding the potential response of chondrogenic cells within the joint environment. However, to replicate the natural in vivo situation, more complex motion patterns are required to induce more physiological chondrogenic gene upregulation.

In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane) composite scaffold for bone tissue engineering.

Scaffolds for bone tissue engineering should provide an osteoconductive surface to promote the ingrowth of new bone after implantation into bone defects. This may be achieved by hydroxyapatite loading of distinct scaffold biomaterials. Herein, we analyzed the in vitro and in vivo properties of a novel nanosize hydroxyapatite particles/poly(ester-urethane) (nHA/PU) composite scaffold which was prepared by a salt leaching-phase inverse process. Microtomography, scanning electron microscopy and X-ray spectroscopy analyses demonstrated the capability of the material processing to create a three-dimensional porous PU scaffold with nHA on the surface. Compared to nHA-free PU scaffolds (control), this modified scaffold type induced a significant increase in in vitro adsorption of model proteins. In vivo analysis of the inflammatory and angiogenic host tissue response to implanted nHA/PU scaffolds in the dorsal skinfold chamber model indicated that the incorporation of nHA particles into the scaffold material did not affect biocompatibility and vascularization when compared to control scaffolds. Thus, nHA/PU composite scaffolds represent a promising new type of scaffold for bone tissue engineering, combining the flexible material properties of PU with the advantage of an osteoconductive surface.

Cells and biomaterials in cartilage tissue engineering

Cartilage defects are notoriously difficult to repair and owing to the long-term prognosis of osteoarthritis, and a rapidly aging population, a need for new therapies is pressing. Cell-based therapies for cartilage regeneration were introduced into patients in the early 1990s. Since that time the technology has developed from a simple cell suspension to more complex 3D structures. Cells, both chondrocytes and stem cells, have been incorporated into scaffold material with the aim to better recreate the natural environment of the cell, while providing more structural support to withstand the large forces applied on the de novo tissue. This review aims to provide an overview of potential cell sources and different scaffold materials, which are in development for cartilage tissue engineering.

In vivo biocompatibility and vascularization of biodegradable porous polyurethane scaffolds for tissue engineering.

Scaffolds for tissue engineering should be biocompatible and stimulate rapid blood vessel ingrowth. Herein, we analyzed in vivo the biocompatibility and vascularization of three novel types of biodegradable porous polyurethane scaffolds. The polyurethane scaffolds, i.e., PU-S, PU-M and PU-F, were implanted into dorsal skinfold chambers of BALB/c mice. Using intravital fluorescence microscopy we analyzed vascularization of the implants and venular leukocyte-endothelial cell interaction in the surrounding host tissue over a 14 day period. Incorporation of the scaffolds was analyzed by histology, and a WST-1 assay was performed to evaluate their cell biocompatibility in vitro. Our results indicate that none of the polyurethane scaffolds was cytotoxic. Accordingly, rolling and adherent leukocytes in venules of the dorsal skinfold chamber were found in a physiological range after scaffold implantation and did not significantly differ between the groups, indicating a good in vivo biocompatibility. However, the three scaffolds induced a weak angiogenic response with a microvessel density of only approximately 47-60 and approximately 3-10cm/cm(2) in the border and centre zones of the scaffolds at day 14 after implantation. Histology demonstrated that the scaffolds were incorporated in a granulation tissue, which exhibited only a few blood vessels and inflammatory cells. In conclusion, PU-S, PU-M and PU-F scaffolds may be used to generate tissue constructs which do not induce a strong inflammatory reaction after implantation into patients. However, the scaffolds should be further modified or conditioned in order to accelerate and improve the process of vascularization.

A versatile bioink for three-dimensional printing of cellular scaffolds based on thermally and photo-triggered tandem gelation.

Layer-by-layer bioprinting is a logical choice for the fabrication of stratified tissues like articular cartilage. Printing of viable organ replacements, however, is dependent on bioinks with appropriate rheological and cytocompatible properties. In cartilage engineering, photocrosslinkable glycosaminoglycan-based hydrogels are chondrogenic, but alone have generally poor printing properties. By blending the thermoresponsive polymer poly(N-isopropylacrylamide) grafted hyaluronan (HA-pNIPAAM) with methacrylated hyaluronan (HAMA), high-resolution scaffolds with good viability were printed. HA-pNIPAAM provided fast gelation and immediate post-printing structural fidelity, while HAMA ensured long-term mechanical stability upon photocrosslinking. The bioink was evaluated for rheological properties, swelling behavior, printability and biocompatibility of encapsulated bovine chondrocytes. Elution of HA-pNIPAAM from the scaffold was necessary to obtain good viability. HA-pNIPAAM can therefore be used to support extrusion of a range of biopolymers which undergo tandem gelation, thereby facilitating the printing of cell-laden, stratified cartilage constructs with zonally varying composition and stiffness.

Local drug delivery for enhancing fracture healing in osteoporotic bone

Fragility fractures can cause significant morbidity and mortality in patients with osteoporosis and inflict a considerable medical and socioeconomic burden. Moreover, treatment of an osteoporotic fracture is challenging due to the decreased strength of the surrounding bone and suboptimal healing capacity, predisposing both to fixation failure and non-union. Whereas a systemic osteoporosis treatment acts slowly, local release of osteogenic agents in osteoporotic fracture would act rapidly to increase bone strength and quality, as well as to reduce the bone healing period and prevent development of a problematic non-union. The identification of agents with potential to stimulate bone formation and improve implant fixation strength in osteoporotic bone has raised hope for the fast augmentation of osteoporotic fractures. Stimulation of bone formation by local delivery of growth factors is an approach already in clinical use for the treatment of non-unions, and could be utilized for osteoporotic fractures as well. Small molecules have also gained ground as stable and inexpensive compounds to enhance bone formation and tackle osteoporosis. The aim of this paper is to present the state of the art on local drug delivery in osteoporotic fractures. Advantages, disadvantages and underlying molecular mechanisms of different active species for local bone healing in osteoporotic bone are discussed. This review also identifies promising new candidate molecules and innovative approaches for the local drug delivery in osteoporotic bone.

The silicomolybdic acid spectrophotometric method and its application to silicate/biopolymer interaction studies

The possibility to understand natural strategies to build‒up silica SiO2 networks relies on the investigations of the kinetics of silica precursors polymerization in the presence of biopolymers. The silicomolybdic acid spectrophotometric method allows to study this process at the molecular level, provided that reliable procedures are available. Moreover, special care must be taken to avoid interfering processes induced by the inorganic and/or bio-organic species found in solution. In order to illustrate the possibilities and limitations of this method, different sets of data are presented and discussed in the context of silica formation control by natural systems.

Antimicrobial delivery systems for local infection prophylaxis in orthopedic- and trauma surgery

Infectious complications occur in a minor but significant portion of the patients undergoing joint replacement surgery or fracture fixation, particularly those with severe open fractures, those undergoing revision arthroplasty or those at elevated risk because of poor health status. Once established, infections are difficult to eradicate, especially in the case of bacterial biofilm formation on implanted hardware. Local antibiotic carriers offer the prospect of controlled delivery of antibiotics directly in target tissues and implant, without inducing toxicity in non-target organs. Polymeric carriers have been developed to optimize the release and targeting of antibiotics. Passive polymeric carriers release antibiotics by diffusion and/or upon degradation, while active polymeric carriers release their antibiotics upon stimuli provided by bacterial pathogens. Additionally, some polymeric carriers gelate in-situ in response to physiological stimuli to form a depot for antibiotic release. As antibiotic resistance has become a major issue, also other anti-infectives such as silver and antimicrobial peptides have been incorporated in research. Currently, several antibiotic loaded biomaterials for local infection prophylaxis are available for use in the clinic. Here we review their advantages and limitations and provide an overview of new materials emerging that may overcome these limitations.

Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds

Adipose-derived mesenchymal stem cells (adMSCs) exhibit a high angiogenic activity. Accordingly, their incorporation into tissue constructs represents a promising vascularization strategy in tissue engineering. In the present study, we analyzed whether the efficacy of this approach can be improved by seeding adMSCs as three-dimensional spheroids onto porous scaffolds. Green fluorescent protein (GFP)-positive adMSCs expressing CD13, CD73, CD90 and CD117 were isolated from C57BL/6-TgN(ACTB-EGFP)1Osb/J mice for the generation of spheroids using the liquid overlay technique. Porous polyurethane scaffolds were seeded with these spheroids or a comparable number of individual adMSCs and implanted into the dorsal skinfold chamber of C57BL/6 wild-type mice. The vascularization of the implants was analyzed and compared to non-seeded scaffolds by means of intravital fluorescence microscopy and immunohistochemistry. The adMSC spheroids exhibited a homogeneous diameter of ~270μm and could easily be incorporated into the scaffolds by dynamic seeding. After implantation, they induced a strong angiogenic host tissue response, resulting in an improved scaffold vascularization with a significantly higher functional microvessel density when compared to non-seeded scaffolds and scaffolds seeded with individual adMSCs. Immunohistochemical analyses revealed that a high fraction of ~40% of all microvessels within the center of spheroid-seeded scaffolds developed from GFP-positive adMSCs. These vessels inosculated with ingrowing GFP-negative vessels of the host. This indicates that adMSC spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside implanted tissue constructs. Thus, adMSC spheroids may be used to increase the efficacy of MSC-based vascularization strategies in future tissue engineering applications.