Edited by: Stevo J. Najman, University of Niš, Serbia
Reviewed by: Francesco Baino, Politecnico di Torino, Italy; Hae-Won Kim, Institute of Tissue Regeneration Engineering (ITREN), South Korea
This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Biomaterials are playing an increased role in the regeneration of damaged or absent bone tissue in the context of trauma, non-union, infection or congenital abnormality. Restoration of not only the physical scaffold that bone provides, but also of its homeostatic functions as a calcium store and hematopoietic organ are the gold standards of any regenerative procedure. Bioactive glasses are of interest as they can bond with the host bone and induce further both bone and blood vessel growth. The composition of the bioactive glasses can be manipulated to maximize both osteogenesis and angiogenesis, producing a 3D scaffolds that induce bone growth whilst also providing a structure that resists physiological stresses. As the primary endpoints of studies looking at bioactive glasses are very often the ability to form substantial and healthy tissues, this review will focus on the methods used to study and quantify osteogenesis and angiogenesis in bioactive glass experiments. These methods are manifold, and their accuracy is of great importance in identifying plausible future bioactive glasses for clinical use.
The regeneration of damaged or absent bone tissue in the context of trauma, non-union, infection or congenital abnormality remains a challenging task for medical teams around the world. Restoration of not only the physical scaffold that bone provides, but also of its homeostatic functions as a calcium store and hematopoietic organ are the gold standards of any regenerative procedure.
The incidence of bone defects is increasing due in part to an aging population (
More recently, focus has changed to the development of biomaterials. These are engineered materials designed to induce bone regrowth and regeneration. There is now a dedicated field to developing biomaterials for bony defects using various materials, production methods and scaffold designs with the option to embed biological materials such as cells or growth factors within them.
One such group of materials is the now well-established bioactive glasses. These glasses are bioactive as they bond with the host bone and induce further bone and blood vessel growth. The original product is the now trademarked 45S5 “bioglass®” designed in the late 1960s; a silicon-based glass network with Na2O, CaO, and P2O5 network modifiers (due to trademark, bioglass® refers to the original 45S product only, all other materials are hence known as bioactive glasses) (
Compositional diagram for bone bonding. Class A biomaterials (Region E) are able to recruit cells involved in bone formation while Class B biomaterials only demonstrate bone growth at the implant-bone interface (Region A). SiO2, silicon dioxide; CaO, calcium oxide; Na2O, sodium oxide.
As well as modifications to the composition of the bioactive glasses in an effort to maximize bone and blood vessel growth, there is also vast interest in different manufacturing techniques that allow production of other desirable characteristics, such as porosity, pore interconnectivity and overall strength (
Throughout the literature studying bioactive glasses
Prior to the production of the first bioactive glass (45S5 Bioglass®) in 1969, biomaterials were designed to be as inert as possible so that they replaced the tissue, rather than regenerated it (
Bioglass® was designed to be both biologically inert and able to chemically bond with bone. This lead to the now well-known result that the glass could not be removed from the bone without breaking the bone itself (
The mechanism of bone bonding is discussed later in this article, but relies on the surface of the glass forming an HCA network after dissolution of various glass ions, which also stimulate cells to form collagen networks. Surface area is therefore a key characteristic of the product as it affects dissolution.
Since then, the original Bioglass® composition has found some commercial success, particularly in dentistry. Varying compositions of network modifiers in silicone glasses have been trialed but bar one, none are as bio reactive as the original (
The products produced for clinical use have been developed from monoliths to granulates with sizes as small as 18micrometres and putty forming substances that allow surgeons to inject the biomaterial into the defect and fill it completely (
Despite the bioactivity of 45S5 bioglass, its inherent chemical composition leads to limitations in the structures can be manufactured from it. In particular during the traditional manufacturing method of melt quenching, the glass tends to crystallize during the sintering phase, producing regions of crystal-amorphous transition which are prone to fracture. It is therefore not possible to build modeled scaffolds from this particular bioactive glass (though this has been achieved with newer bioactive compositions). Formation of scaffolds is desirable for both their structural integrity and vastly increased surface area, and is the current focus of much of the field of biomaterials (
More recently, certain quench derived bioactive glasses have been produced which combined with novel manufacturing ideas such as composites and nanoparticle or nanofiber formation aim to produce an ideal mix of structural integrity and bone-induction in a bioactive glass scaffold (
In addition, bioactive glass also has the potential to be used more widely in tissue repair due to its ability to induce angiogenesis. Bioactive glass improved the mechanical properties and scaffold bioactivity when added to poly(glycerol sebacate), an elastomeric polymer studied for cartilage repair (
This summary outlines the current state of play for bioactive glass design. This field has multiple avenues of research and understanding the techniques that allow us to assess osteogenesis and angiogenesis is paramount.
A key part of the design criteria of bioactive glasses is the ability to bond with bone. As well as this, they can ideally induce its growth not only at the bone-material interface, but also away from the implant site. These are features shown by the original Bioglass® and represent the gold standard. The glass bonds with bone in two basic steps, first, formation of a hydroxyl carbonated apatite (HCA) layer on the glass surface and second, the subsequent cellular responses to this both in the local area and more distally (
Schematic illustration of the formation of HCA layer on the surface of bioactive glass. The stages are as follows:
Following the generation of the HCA layer, proteins and subsequently osteoprogenitor cells are attracted to the bone surface and begin forming extracellular matrix (ECM) (
The mechanism of angiogenesis in these constructs has also been studied, but not as extensively. This small section will focus on the perceived mechanism of growth in glasses not containing additional cellular components. As with bone growth, ionic dissolution products are likely responsible for the angiogenic potential, and there is much interest in the relative concentrations and dissolution rates of each of these.
The proposed mechanisms are complex and not well understood. It is postulated that silicon ions mimic hypoxic conditions, acting both directly on fibroblast and or osteoblasts to increase vascular endothelial growth factor (VEGF) release (
The techniques used fall broadly into the following categories: histological staining and immunohistochemistry, imaging, gene expression and growth factor assays, enzymatic activity assays and spectroscopy techniques. This article will focus on the commonly used techniques.
In the formation of any new tissue, angiogenesis must occur simultaneously to overcome the limitations of diffusion alone. There is evidence to suggest bioactive glasses can induce angiogenesis particularly well when compared to other bioactive materials, these properties are obviously important for both bone and soft tissue repair and have been reviewed extensively (
The method of analysis favored by early groups is the direct examination of bone structure or blood vessel number and diameter by histological examination. The tissue of interest is fixed and subsequently decalcified to allow sections to be cut from the specimen. A stain or antibody is applied to the section which is examined with light microscopy. Although mentioned here, non-IHC staining is seldom used in current studies and IHC tends to be favored.
Histological techniques have the benefit of being able to directly examine the volume and quantity of vessel or bone growth without being hindered by image resolution or contrast perfusion as in radiological techniques. They also permit a direct qualitive analysis of the vascular and bone structures, with analysis of their makeup possible through the various histological features present. This means that tissue morphology can be compared to “normal” sections prepared in the same way.
Plain light microscopy using traditional staining will allow for a structural and numerical comparison of the new bone and vessels across various specimens. To some degree, cell types are able to be recognized, but there are no markers of biological activity in this type of staining.
Valuable information can still be obtained from these techniques when assessing samples for osteogenesis. Comparing the histological appearance of several test groups can provide information on the amount of bone formation, the presence of immature or lamellar bone and amount of bioactive glass degradation. This can be particularly useful when assessing whether changes to a bioactive glass scaffold affects the induction of mature bone at defect sites. The yield of these techniques can be improved by fluorescence labeling. Use of fluorescent dyes such as alizarin red S, calcein and tetracycline localize to areas of high calcium and can also aid in differentiating newly formed bone from existing bone.
Common stains used in assessing osteogenesis and angiogenesis.
Stain | Target | Description |
Haematoxylin | Stains nuclei blue | Stains the chromatin in cell nuclei dark blue. Also stains rough endoplasmic reticulum, ribosomes, collagen, myelin, elastic fibers, and mucins. |
Eosin | Stains cytoplasm pink | Often used as a counterstain with haematoxylin, together known as H&E. Stains cytoplasm pink. |
Masson’s trichrome | Stains collagen blue/green | Variable three color staining depending on the specific application of the stain. Usually produces: blue/green collagen; red keratin and muscle fibers; pink cytoplasm and black nuclei. |
Toluidine blue | Stains proteoglycans and glycosaminoglycans purple | Stain color is produced by metachromasia. High affinity for DNA and RNA which are stained blue. Stains proteoglycans and glycosaminoglycans purple. |
Van Gieson | Stains collagen red | Combination of piric acid and acid fuchsin. Differentiates collagen from other connective tissue. Known as HvG when combined with haematoxylin and collagen will appear pink. |
Alizarin Red S | Stains calcium orange/red | Used to locate tissues with high calcium content such as bone |
Calcein | Binds to calcium ions | Fluoresces green with excitation/emission wavelengths 488 nm/520 nm, respectively. |
Tetracycline | Binds to calcium | Fluoresces yellow with excitation/emission wavelengths 450–490 nm/529 nm. Localizes to sites of active mineralization. |
IHC retains all of the beneficial features of traditional histological stains, but with the added ability to assess biological activity in the cells, and better recognize complex cell lines. This type of analysis is used commonly in assessing angiogenesis in bioactive glass experiments as it overcomes issues that cross-sectional imaging has with imaging vasculature. It is less commonly used to assess osteogenesis where CT imaging prevails.
Common IHC targets used to quantify angiogenesis and osteogenesis.
IHC target | Marker of | Description |
CD31/PECAM-1 | Angiogenesis | Platelet endothelial cell adhesion molecule (PECAM-1), or CD31, is expressed by endothelial cells, platelets and all leucocytes. It is used as a biomarker for the presence of epithelial cells and for angiogenesis. |
CD34 | Angiogenesis | Expressed in haematopoietic stem cells. Used as a biomarker for vascular endothelial cells to assess angiogenesis ( |
α-SMA | Angiogenesis | Alpha-Smooth Muscle actin is highly expressed in vascular smooth muscle cells which facilitates its use as a biomarker of angiogenesis. |
VEGF | Angiogenesis | Vascular endothelial growth factor (VEGF) is a potent simulant of angiogenesis. It would be expected to be upregulated in regions undergoing active angiogenesis. It also has effects on bone remodeling with pro-migratory and pro-proliferative effects on osteoblasts and stimulates osteoclasts via the RANK pathway ( |
Endomucin | Angiogenesis | A marker of endothelial and haematopoietic stem cells. |
ALP | Osteogenesis | ALP is expressed early in bone development and has is involved with the early stages of calcification and mineralization. |
Osteopontin | Osteogenesis | Osteopontin (OPN) is a non-collagenous protein component of extracellular bone and is expressed early in osteogenesis. |
Type I collagen | Osteogenesis | Type I collagen is a mid-to-late target in investigating osteogenesis. It is the predominant protein in osteoid. |
Osteocalcin | Osteogenesis | Produced exclusively by osteoblasts and a major non-collagenous component of bone. Its expression peaks late in osteogenesis. |
Several IHC protein targets are used to evaluate bioactive glass angiogenic and osteogenic activity. For example, having targets that peak in expression during different stages of osteogenesis provides additional information on the scaffold’s suitability for defect repair. An ideal bioglass scaffold will result in adequate stimulation of osteogenesis and will promote progression to mature bone. An appreciation for the temporal difference in expression is needed when timing collection of samples and selection of IHC targets.
Early targets include alkaline phosphatase (ALP) and osteopontin (OPN). ALP is expressed early in bone development and has functions in initiating calcification and mineralization while OPN is an extracellular structural component of bone. It has a multifaceted role during early osteogenesis including playing a key role in influencing MSCs toward an osteogenic lineage (
However, these methods have several disadvantages. Histological analysis means that longitudinal studies are seldom possible on the same subject, which introduces more variation and necessitates more animal subjects. The nature of histological sections means they are a 2D representation of a 3D structure, and only a “snapshot” of the overall structure. This can be overcome in part using serial sections and analyzing them as a cohort, however, as mentioned latterly, truly quantitative analysis of bone or blood vessel growth is not possible.
Imaging in this field is dominated by microCT due to its high contrast and spatial resolution. In particular, the technique is useful in assessing bone structure, with more recent advances made in its ability to image vascular structures by using contrast media (e.g., most commonly iodine-based agents such as Omnipaque and Isovue). Other radiological techniques such as nuclear imaging (i.e., single photon emission computed tomography and positron emission tomography) have also been used either alone or in combination with CT imaging (
MicroCT finds its origins in the early 1980s as a research tool which afforded higher quality imaging than conventional CT, but at the cost of greater radiation doses. It was not until 1994 that this became a viable option with commercially available scanners, allowing microCT to become the mainstay of bone imaging in animal models. Future developments in this imaging modality are discussed at the end of this section.
In principle a microCT scanner is no different to a conventional CT scanner, using the attenuation of X-ray radiation in multiple 2D sections to produce a 3D image of an object through back projection. It is able to produce voxels of <1 μm, where trabeculae in mice models are around 30 μm at their smallest (
Live specimens are imaged using a scanner that rotates around the animal, and prepared bone specimens are themselves rotated. Live specimens must be anaesthetized to minimize movement artifact and to position them optimally and reliably with respect to the radiation source. This is particularly important when considering image registration which is discussed later (
This modality produces 3D images of the bone microstructure as well as quantified measures of bone morphometry and tissue density. The output morphometric data and their descriptions are illustrated in
Definitions and descriptions of bone morphometric data.
Abbreviation | Variable | Description | Standard unit |
TV | Total volume | Volume of the entire region of interest | mm3 |
BV | Bone volume | Volume of the region segmented as bone | mm3 |
BS | Bone surface area | Surface of the region segmented as bone | mm2 |
Ratio of the segmented bone volume to the total volume of the region of interest | % | ||
BS/TV | Bone surface density | Ratio of the segmented bone surface to the total volume of the region of interest | mm2/mm3 |
BS/BV | Specific bone surface | Ratio of the segmented bone surface to the segmented bone volume | mm2/mm3 |
Conn.D | Connectivity density | A measure of the degree of connectivity of trabeculae normalized by TV | 1/mm3 |
SMI | Structure model index | An indicator of the structure of trabeculae; SMI will be 0 for parallel plates and 3 for cylindrical rods | - |
Measure of the average number of trabeculae per unit length | 1/mm | ||
Mean thickness of trabeculae, assessed using direct 3D methods | mm | ||
Mean distance between trabeculae, assessed using direct 3D methods | mm | ||
Tb.Th.SD | Standard deviation of trabecular thickness | Measure of the homogeneity of trabecular thickness, assessed using direct 3D methods | mm |
Tb.Sp.SD | Standard deviation of trabecular separation | Measure of the homogeneity of trabecular separation, assessed using direct 3D methods | mm |
DA | Degree of anisotropy | 1 = isotropic, >1 = anisotropic by definition; DA = length of longest divided by shortest mean intercept length vector | – |
MIL | Mean intercept length | Measurements of structural anisotropy | – |
The images can be used for interpretation much like a histological section, in comparing the structure to the known structure of bone. However, there are a number of advantages micro-CT has over other methods beyond conserving the specimen. It is significantly quicker and is less labor intensive than many other methods. A larger continuous region of interest can be examined than is possible using histological methods, where there is loss of sample with the preparation of each section. This results in a greater likelihood of capturing transition zones, such as the interface between material and old bone, to assess if there has been substantial integration of the new bone-scaffold construct and the existing bone. Furthermore, the 3D microarchitecture, as well as measurement such as volume and thickness, can be more faithfully represented by micro-CT as it does not rely on stereological methods to model these characteristics (
MicroCT scanning can make use of intravenous contrast media to visualize and measure the degree of angiogenesis. Usually, blood vessels have too low an x-ray attenuation to be visualized, which necessitates the use of contrast, of which there are two commonly used (MV-122—silicon rubber and BaSO4/gelatin). These allow for direct visualization of the vasculature and the calculation of various parameters from this data, though these are less well defined than in osteogenesis (
This technique is currently only used in post-mortem specimens as discussed below. It involves euthanasia of the specimen, followed by a multistep process of injecting the contrast medium and curing agent intracardially before a fixing period of several hours (
There are many reasons why microCT has become the commonest technique in evaluating bone morphology in this field. Being non-destructive, unlike histological approaches, allows serial scanning of the same specimen
In terms of the quality of data obtained, microCT provides comparable numerical results to traditional histomorphological techniques in both human and animal models. The benefit of microCT over these earlier methods in terms of accuracy is that microCT analyses a greater volume of interest (VOI), and the measurements are direct rather than inferred from single slices with assumptions made about structure by stereologic models. Furthermore, the vast number of output variables allows many parameters to be calculated from one scan. Although imaging requires some preparation of the specimen or anesthesia of the animal, it remains a quicker and more time-efficient technique than histological techniques or immunohistochemistry (
When microCT imaging is compared to 2D histomorphic sections of the same specimen, measurements show high degrees of correlation, suggesting it is an accurate method for measuring various indices (
There are some downsides to microCT imaging in this context, such as the exposure of the animal to ionizing radiation. The exact significance of this is unclear but is more important with serial scanning of the same animal; with a potential to reduce bone growth by damaging stem cells as shown in one study (
The molecular mechanisms of osteogenesis and angiogenesis can be interrogated via measuring changes in the expression levels of related genes. The most popular method is quantitative reverse transcription polymerase chain reaction (RT-qPCR) where gene expression is measured against a reference gene. Real-time PCR measures PCR amplification as it occurs (using fluorogenic-labeled probes), allowing the real-time detection of specific amplification products and calculation of the starting concentration of nucleic acid. This contrasts with traditional PCR, where results are collected only after the reaction is complete, making it impossible to determine the starting concentration of nucleic acid. Two methods which are commonly used to quantify the results are the standard curve methods and the comparative threshold method. In the former, a standard reference curve is constructed from RNA of known concentration, which is then used to extrapolate quantitative information for mRNA targets of unknown concentrations. In the comparative threshold method, the Ct value of the sample is compared with a control and normalized to an appropriate endogenous housekeeping gene (where expression is assumed to be constant). This is also referred to as the 2–ΔΔCt method (
The genes of interest vary widely as the complex regulatory systems by which bone and blood vessel synthesis is controlled remains incompletely defined. In the case of bone formation, several intracellular signaling pathways are likely to be activated within progenitor cells, which modulate the levels of transcription factors involved in the osteoblast phenotype, which in turn regulate the genes that code for bone matrix proteins.
Osteogenesis-related genes can therefore include any implicated gene from regulators of the osteoblast phenotype, to various transcription factors and matrix proteins. Signaling cascades which promote osteogenic differentiation generally converge on two key transcription factors: proliferator-activated receptor-γ (PPARγ) and Runt-related transcription factor 2 (Runx2). PPARγ has well-described anti-osteoblastogenic effects while Runx2 is implicated in osteogenic regulation. Several cell signaling cascades are implicated (in multiple biological processes not exclusive to osteogenesis), such as β-catenin dependent Wnt signaling (as well as β-catenin independent signaling) (
The increasing burden of bone defects on an aging population necessitates further research into alternatives to conventional treatments. New bioactive glass compositions are constantly being developed and their properties can be altered by incorporating different elements into the glass composition. For example,
As infection represents another significant cause of bone defects, further studies to investigate the effectiveness of these biomaterials should be tested under an infective burden. This research continue alongside the ongoing studies into the drug-releasing kinetics of bioactive glasses.
Bioactive glasses are also produced in forms ranging from powders to 3D scaffolds and research in ongoing in developing optimal constructs for tissue defects (
With the expanding research and proposed applications for bioglasses, it is important to be able to consistently assess their capacity for osteogenesis and angiogenesis. This review article summarizes some of the common methods for assessing osteogenesis and angiogenesis and highlights the importance of these techniques in identifying plausible future bioactive glasses for clinical use.
WK and KS: conceptualization, funding acquisition, supervision, methodology. JC, AH, KS, and WK: investigation, writing. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.