Eileen Gentleman

The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro.

Bioactive glasses (BG) which contain strontium have the potential to combine the known bone regenerative properties of BG with the anabolic and anti-catabolic effects of strontium cations. Here we created a BG series (SiO(2)-P(2)O(5)-Na(2)O-CaO) in which 0-100% of the calcium was substituted by strontium and tested their effects on osteoblasts and osteoclasts in vitro. We show that ions released from strontium-substituted BG enhance metabolic activity in osteoblasts. They also inhibit osteoclast activity by both reducing tartrate resistant acid phosphatase activity and inhibiting resorption of calcium phosphate films in a dose-dependent manner. Additionally, osteoblasts cultured in contact with BG show increased proliferation and alkaline phosphatase activity with increasing strontium substitution, while osteoclasts adopt typical resorption morphologies. These results suggest that similarly to the osteoporosis drug strontium ranelate, strontium-substituted BG may promote an anabolic effect on osteoblasts and an anti-catabolic effect on osteoclasts. These effects, when combined with the advantages of BG such as controlled ion release and delivery versatility, may make strontium-substituted BG an effective biomaterial choice for a range of bone regeneration therapies.

Mechanical Characterization of Collagen Fibers and Scaffolds for Tissue Engineering

Engineered tissues must utilize scaffolding biomaterials that support desired cellular functions and possess or can develop appropriate mechanical characteristics. This study assessed properties of collagen as a scaffolding biomaterial for ligament replacements. Mechanical properties of extruded bovine achilles tendon collagen fibers were significantly affected by fiber diameter, with smaller fibers displaying higher tangent moduli and peak stresses. Mechanical properties of 125 micrometer-diameter extruded fibers (tangent modulus of 359.6+/-28.4MPa; peak stress of 36.0+/-5.4MPa) were similar to properties reported for human ligaments. Scaffolds of extruded fibers did not exhibit viscoelastic creep properties similar to natural ligaments. Collagen fibers from rat tail tendon (a well-studied comparison material) displayed characteristic strain-softening behavior, and scaffolds of rat tail fibers demonstrated a non-intuitive relationship between tangent modulus and specimen length. Composite scaffolds (extruded collagen fibers cast within a gel of Type I rat tail tendon collagen) were maintained with and without fibroblasts under standard culture conditions for 25 days; cell-incorporated scaffolds displayed significantly higher tangent moduli and peak stresses than those without cells. Because tissue-engineered products must possess appropriate mechanical as well as biological/chemical properties, data from this study should help enable the development of improved tissue analogues.

The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation

Mineralization is a ubiquitous process in the animal kingdom and is fundamental to human development and health. Dysfunctional or aberrant mineralization leads to a variety of medical problems, and so an understanding of these processes is essential to their mitigation. Osteoblasts create the nano-composite structure of bone by secreting a collagenous extracellular matrix (ECM) on which apatite crystals subsequently form. However, despite their requisite function in building bone and decades of observations describing intracellular calcium phosphate, the precise role osteoblasts play in mediating bone apatite formation remains largely unknown. To better understand the relationship between intracellular and extracellular mineralization, we combined a sample-preparation method that simultaneously preserved mineral, ions, and ECM with nano-analytical electron microscopy techniques to examine osteoblasts in an in vitro model of bone formation. We identified calcium phosphate both within osteoblast mitochondrial granules and intracellular vesicles that transported material to the ECM. Moreover, we observed calcium-containing vesicles conjoining mitochondria, which also contained calcium, suggesting a storage and transport mechanism. Our observations further highlight the important relationship between intracellular calcium phosphate in osteoblasts and their role in mineralizing the ECM. These observations may have important implications in deciphering both how normal bone forms and in understanding pathological mineralization.

Nano-analytical electron microscopy reveals fundamental insights into human cardiovascular tissue calcification

The accumulation of calcified material in cardiovascular tissue is thought to involve cytochemical, extracellular matrix and systemic signals; however, its precise composition and nanoscale architecture remain largely unexplored. Using nano-analytical electron microscopy techniques, we examined valves, aortae and coronary arteries from patients with and without calcific cardiovascular disease and detected spherical calcium phosphate particles, regardless of the presence of calcific lesions. We also examined lesions after sectioning with a focused ion beam and found that the spherical particles are composed of highly crystalline hydroxyapatite that crystallographically and structurally differs from bone mineral. Taken together, these data suggest that mineralized spherical particles may play a fundamental role in calcific lesion formation. Their ubiquitous presence in varied cardiovascular tissues and from patients with a spectrum of diseases further suggests that lesion formation may follow a common process. Indeed, applying materials science techniques to ectopic and orthotopic calcification has great potential to lend critical insights into pathophysiological processes underlying calcific cardiovascular disease.

Bioactive Glass Scaffolds for Bone Regeneration

There is a need for new materials that can stimulate the bodys own regenerative mechanisms and heal tissues. Porous templates (scaffolds) are thought to be required for three-dimensional tissue growth. This article discusses bone regeneration and the specifications of an ideal scaffold and the materials that may be suitable. Bioactive glasses have high potential as scaffold materials as they stimulate bone cells to produce new bone, they are degradable in the body and they bond to bone. The two types of bioactive glasses, their mechanisms for bioactivity and their potential for scaffold production are reviewed. Examples of their current clinical use are highlighted.

Extracellular matrix-mediated osteogenic differentiation of murine embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent and have the ability to differentiate into mineralising cells in vitro. The use of pluripotent cells in engineered bone substitutes will benefit from the development of bioactive scaffolds which encourage cell differentiation and tissue development. Extracellular matrix (ECM) may be a suitable candidate for use in such scaffolds since it plays an active role in cellular differentiation. Here, we test the hypothesis that tissue-specific ECM influences the differentiation of murine ESCs. We induced murine ESCs to differentiate by embryoid body formation, followed by dissociation and culture on ECM prepared by decellularisation of either osteogenic cell (MC3T3-E1) or non-osteogenic cell (A549) cultures, or on defined collagen type I matrix. We assessed osteogenic differentiation by formation of mineralised tissue and osteogenic gene expression, and found it to be significantly greater on MC3T3-E1 matrices than on any other matrix. The osteogenic effect of MC3T3-E1 matrix was reduced by heat treatment and abolished by trypsin, suggesting a bioactive proteinaceous component. These results demonstrate that decellularised bone-specific ECM promotes the osteogenic differentiation of ESCs. Our results are of fundamental interest and may help in tailoring scaffolds for tissue engineering applications which both incorporate tissue-specific ECM signals and stimulate stem-cell differentiation.

Scaffolds for stem cells

Today, most people know at least something about stem cells. Embryonic stem cells enjoy regular mentions in news programs and magazines, probably because of the controversial way in which they are generated but also because of their huge potential in medicine. Here we distinguish between and define types of stem cells, discuss techniques used so far to create various cells and tissues from stem cells, and discuss how three-dimensional supports and stem cells have been and should be used to encourage the development of functional replacement tissue.

Materials characterisation and cytotoxic assessment of strontium-substituted bioactive glasses for bone regeneration

The regeneration of bone lost due to disease and trauma as well as the bonding of bone to biomedical implants present significant challenges to the field of biomaterials. Increasing evidence for the benefit of strontium as a potent anti-osteoporosis therapy suggests that biomaterials which can release strontium to the surrounding environment may find a range of uses in orthopaedic, spinal and dental surgery to regenerate hard tissue. Here we report on the structure and physical properties of a series of ten glasses based on 45S5 Bioglass®, where 0 to 100% of the calcium was substituted with strontium on a molar basis. Characteristic temperatures were determined using differential thermal analysis. We also analysed the structure of the glasses with Raman spectroscopy and assessed their cytotoxicity using a human osteosarcoma cell line. All characteristic temperatures (except crystallisation onset, Tx) decreased with strontium addition to the glass. Glass molecular structure did not change across the series and the silicate network was predominantly composed of linear chains (Q2) and a small amount of (Q3) with isolated orthophosphate units (Q0). The phosphate was present as [PO4]3− orthophosphate units (Q0) in all glasses. Immersion of the Sr-glasses in simulated body fluid (SBF) showed formation of biomimetic apatite in 1 week or less. Cell proliferation assays demonstrated that all the glass compositions supported normal cell attachment and proliferation. In summary, we show that strontium can be added to bioactive glasses in place of and in combination with calcium with minimal alteration to glass physical properties. These strontium-substituted bioactive glasses will be useful for a range of applications in orthopaedic regenerative medicine.

Development of Ligament-Like Structural Organization and Properties in Cell-Seeded Collagen Scaffolds in vitro

Acute anterior cruciate ligament (ACL) injuries lead to poor joint function, instability, and eventually osteoarthritis if left untreated. Current surgical treatment options are not ideal; however, tissue engineering may provide mechanically sound, biocompatible reconstructions. Collagen fiber scaffolds were combined with fibroblast-seeded collagen gels and maintained in culture for up to 20 days. The tensile and viscoelastic behavior of the constructs closely mimicked that of natural ligament. Constructs’ mechanical and viscoelastic properties did not degrade over time in culture, and peak stress was significantly higher for constructs with embedded fibroblasts. Immunocytochemical and histological analyses demonstrated cell proliferation and ligament-like organization. We have created an engineered tissue that closely approaches key mechanical and viscoelastic properties of the ACL, does not degrade after 20 days in culture, and is histologically similar to the native tissue. This study should aid in developing effective treatments for ACL injury.

The role of surface free energy in osteoblast–biomaterial interactions

Abstract The clinical success of many orthopaedic implants relies on good integration between the implant and adjacent bone. As stabilising bone grows not only to the implant, but from it, the quick adhesion of bone forming cells called osteoblasts, their appropriate differentiation and ability to form mineralised bone are vital to achieve a good clinical outcome. Surface free energy can be thought of as a measure of the ‘unsatisfied bond energy’ resulting from ‘dangling bonds’ exposed at a materials surface. This unsatisfied bond energy affects protein adsorption and cell attachment, and thus controls the early stages of cell–biomaterial interactions and ultimately implant fixation. When water, proteins, or cells approach a surface, their surface domains align to minimise the overall surface free energy of the interface. Determining these interactions, however, is not simple. While contact angle measurements on flat surfaces can predict some surface free energy-related interactions, this is not the case when surface topography is modified. Here, the authors review how surface free energy can be altered on self-assembled monolayers, polymers, metals and ceramics and clarify the differences between measurements of surface free energy and wettability. The authors also review how surface free energy affects protein interactions and osteoblast behaviour. The result is a clearer understanding of the effect of surface free energy on cell behaviour and an unambiguous need for further studies that isolate such effects.