3.1 Introduction

All biomaterials in use today are a compromise compared to the natural tissues they replace. Mismatches in elastic moduli, breakdown of the tissue-material interface, fatigue, wear, or other factors lead to 15–50 % failures of prostheses over a 15–30-year lifetime. An alternative approach to the present emphasis on replacement of body parts and tissues is now possible: regeneration of tissues. In order to regenerate tissues, it is essential to activate the body’s own repair processes. To stimulate self-repair it is necessary to control both the proliferation and the differentiation of progenitor stem cells towards mature cells capable of generating proper types and quantities of extracellular matrix that are present in nearly all tissues.

Controlled release of ionic dissolution products of soluble Ca and Si ions from bioactive glasses can provide this stimulus for enhanced osteogenesis. This chapter reviews the chronology of the development of bioactive glasses and the evidence of genetic control of bone regeneration that underlies development of a new approach to maintain quality of life in an aging population.

3.2 Development of Bioactive Glasses

For millennia it was accepted that any man-made material in the body would result in a foreign body reaction and formation of nonadherent scar tissue at the interface with the material. Thus, initial emphasis on biomaterials for use in the body was on materials that were as inert as possible when exposed to a physiological environment. Prevention of corrosion of metals or degradation of polymers was the primary design objective of first-generation biomaterials. A second generation of biomaterials to replace tissues was achieved when a special composition of soda-lime-phosphate-silicate glass was made by the author and implanted in the femurs of rats in 1969 [1–3]. The calcium phosphate-containing silicate glass composition had 45 % SiO2, in weight % with network modifiers of 24.5 % Na2O and 24.5 % CaO. In addition, 6 % P2O5 was added to the glass composition to simulate the Ca/P constituents of hydroxyapatite (HA), the inorganic mineral phase of bone, Table 3.1, with a nomenclature of 45S5 Bioglass.

Table 3.1 Composition of bioactive glasses and glass-ceramics used for medical and dental applications
Full size table

The glass implants bonded to the living bone (rat femora) within 6 weeks and could not be removed from their implant site [1–3]. This discovery led to the development of a new class of biomaterials, called bioactive materials, for use in implants or prostheses and repair or replacement of bones, joints, and teeth. Table 3.2 summarizes the chronology of the development of bioactive glasses as a second generation of biomaterials. The seminal paper describes the composition of the glass and the evidence of bonding to bone by use of transmission electron microscopy (TEM) that reveals the bonded interface as a layer of growing Ca-P-based bone mineral that has interdigitated with collagen fibrils generated by osteoblasts growing at the interface [1]. An acellular in vitro model of soaking the glass samples in a 37 °C calcium phosphate-rich solution showed by X-ray diffraction (XRD) the steps of growth of a biologically active hydroxyapatite crystal phase on the surface of the glass that mimics the XRD pattern of the HA phase in living bone. Details of the bone-bonded interface are described in additional papers in 1971–1972 [1, 2].

Table 3.2 Chronology of science and clinical product development of 45S5 Bioglass
Full size table

3.2.1 Compositions of Bioactive Glasses

Bioactive materials, including bioactive glasses [1–3] and glass-ceramics [4–6], are special compositions made typically from the Na2O-CaO-MgO-P2O5-SiO2 system (Table 3.1). All of the compositions in Table 3.1 form a mechanically strong bond with bone within weeks or months. Details are described in references [7–15]. The rate of bone bonding depends upon composition of the material. Glass compositions with lower contents of SiO2 (<52 % by weight) have the fastest rates of bone bonding and also bond to soft tissues [16]. Compositions such as 45S5 Bioglass with high rates of bioactivity produce rapid regeneration of trabecular bone with an amount, architecture, and biomechanical quality of bone that match that originally present in the site [14, 17–19]. The rapid regeneration of bone is due to a combination of processes called osteostimulation and osteoconduction [16–19]. Large differences in rates of in vivo bone regeneration and extent of bone repair indicate that there are two classes of bioactive materials (Table 3.1) [16–19].

3.2.2 Classes of Bioactivity

Class A bioactivity leads to osteoconduction, growth of new bone along the bioactive interface, and osteostimulation, growth of new bone throughout the defect to regenerate the architecture of bone natural to the site, as a consequence of rapid reactions on the bioactive glass surface [16–19]. The surface reactions involve dissolution of critical concentrations of soluble Si and Ca ions that give rise to both intracellular and extracellular responses at the interface of the glass with its physiological environment. The intracellular and extracellular response of osteoprogenitor cells results in rapid formation of osteoid bridges between particles, followed by mineralization to produce mature bone structures. This process is termed osteostimulation or osteoproduction. Rates of osteostimulation of various types of bioactive particulates have been quantified by Oonishi et al. that provide the fundamental in vivo comparisons of Class A versus Class B bioactive materials [14–19].

3.2.2.1 Characterization of Bioactivity Reaction Stages

New surface and interfacial analyses techniques were developed to understand the mechanisms and kinetics of bioactive reactions in vitro and in vivo [1, 13, 20–23]. These methods showed that there is a sequence of 11 reaction stages that occur at the surface of a Class A bioactive glass. Figure 3.1 indicates in the log time axis that the first five stages of surface reactions occur very rapidly and go to completion within 24 h for the bioactive glasses with highest levels of Class A bioactivity, e.g., 45S5 Bioglass. The effect of the surface reactions is rapid release of soluble ionic species from the glass into the interfacial solution. A high surface area composed of hydrated silica and polycrystalline hydroxyl carbonate apatite (HCA) as a bilayer is formed on the glass surface within hours (Stages 1–5) [23]. The reaction layers enhance adsorption and desorption of growth factors (Stage 6) and decrease the length of time macrophages prepare the implant site for tissue repair (Stage 7).

Fig. 3.1
figure 1

Sequence and kinetics of interfacial reactions between bones and a Class A bioactive glass or glass-ceramic (Reprinted with permission from Hench [19], American Ceramic Society)

Full size image

Attachment of stem cells (Stage 8) and synchronized proliferation and differentiation of the progenitor cells (Stage 9) rapidly occur on the surface of Class A bioactive materials [10, 14–16, 24]. Several weeks or months are required for similar cellular events to occur on the surface of Class B bioactive materials. Differentiation of progenitor cells into a mature osteoblast phenotype does not occur on bio-inert materials and is rare on Class B bioactive materials because of the lack of ionic stimuli. In contrast, osteoprogenitor cells colonize the surface of Class A bioactive materials within 24–48 h and begin production of various growth factors which stimulate cell division, mitosis, and production of extracellular matrix proteins (Stage 10). Mineralization of the matrix follows soon thereafter, and mature osteocytes, encased in a collagen-HCA matrix, are the final product by 6–12 days in vitro and in vivo (Stage 11) [14–16, 24–30].

3.3 Genetic Control of Bone Regeneration by Bioactive Glasses

The original discovery of bioactive glasses and emphasis of research were on the mechanisms of interfacial bonding of bone to bioactive glasses [1–3, 7–22]. The seminal step to shift thinking from bioactive bonding to bone regeneration was the reporting by June Wilson et al. for the first time that new bone had colonized the surface of an array of 45S5 Bioglass particulate placed in a surgically created site in the jaw of monkeys that mimicked the loss of bone from periodontal disease [31]. New bone formed around the particles and created a regenerated architecture of bone that bridged the bioactive glass particles (Fig. 3.2). The new phenomenon was designated as osteoproduction, now generally called osteostimulation. The concept of osteostimulation was quantified by Oonishi et al. a few years later [14, 32]. The second key finding to develop a genetic basis for bone regeneration was the finding of Xynos et al. that it was not only the glass but it was the ionic dissolution products released from 45S5 Bioglass that influenced and controlled the cell cycle of osteogenic precursor cells and ultimately controlled the differentiated cell population [26, 27]. Cells that were not capable of achieving a fully differentiated phenotype characteristic of mature osteocytes were eliminated from the in vitro cultures by programmed cell death, apoptosis. The shift in cell population towards mature osteoblasts occurred rapidly, in hours, and led to mineralized bone nodules in culture, without addition of organic bone growth factors, such as bone morphogenetic proteins (BMP). Another key finding from the work of Xynos et al. was that the effective ionic dissolution products released at slow rates from 45S5 Bioglass were biologically active soluble Si and Ca ions. Both ions are necessary for osteostimulation. Osteostimulation occurs only when the ions are present at a particular ratio of ions and at a particular concentration range of 15–30 ppm Si and 60–90 ppm Ca.

Fig. 3.2
figure 2

Bioactive glass BG particulate used to enhance bone [B] formation around the tooth [T] and thereby restores its function following treatment of periodontal, gum, disease in a patas monkey periodontal defect model. Note the mineralized collagen that surrounds the bioactive glass particles forming a regenerated bone structure. Also note the periodontal ligament labeled PDM that is stable above the regenerated bone. (Magnification 20x). (Photo courtesy of Dr. June Wilson)

Full size image

These findings provide an understanding of the clinical success of use of 45S5 Bioglass particles, NovaBone and PerioGlas, in a wide range of dental and orthopedic applications in use for nearly 20 years, as discussed in [17–19]. When used as a synthetic bone graft, the bioactive glass particles dissolve slowly and release the critical concentrations of Si and Ca over many weeks, as needed for progressive regeneration of bone to fill the defect. The initial effect is proliferation of osteogenic precursor cells at the periphery of the particles. These cells undergo mitosis and lead to an expanded population of mature osteoblasts that generate extracellular matrix proteins, especially Type I collagen that mineralizes to form regenerated bone (Fig. 3.3).

Fig. 3.3
figure 3

Higher magnification of collagen fibrils that have been formed and adherent to bioactive glass particles in a regenerated periodontal defect model equivalent to that shown in Fig. 3.2. (Magnification 100x). (Photo courtesy of June Wilson)

Full size image

Oonishi et al. used a critical size defect model in a rabbit femoral condyle model to quantify the histological sequence of osteostimulation by 45S5 Bioglass particulate [14, 32]. The studies show that there is both more rapid bone formation in the presence of the osteostimulation particles and regeneration of a more highly mineralized quality of bone in the defect, in comparison with synthetic hydroxyapatite (HA) particles or bioactive A/W glass-ceramic particles. The rate of bone regeneration in the Oonishi model is related to the rate of release of the soluble Si and Ca ions from the particulates tested. A rapid rate of release, as depicted in Fig. 3.1 Stages 1–5, is necessary for rapid bone regeneration and optimal fill of the defect by regenerated trabecular bone.

The third critical step in developing a genetic basis for tissue regeneration was the discovery that critical concentrations of ionic dissolution products (soluble Si and Ca ions) activate or upregulate seven families of genes in osteogenic precursor cells [27–30]. The genes encode transcription of numerous proteins that control the cell cycle, proliferation, and ultimately the differentiation of the cells towards the mature osteoblastic phenotype. The seminal paper leading to identification of the genetic response to bioactive dissolution products was published by Xynos et al. in 2001 resulting from a collaboration at Imperial College London of biomaterials investigators (the author’s group) and the cell and molecular biology groups of Professor Dame Julia Polak [27].

3.3.1 Confirmation of Genetic Control of Bone Regeneration

Numerous studies have confirmed the results of the early Xynos et al. research and extended the generality to include several types of precursor cells and differing sources of ionic stimuli. Gene array analyses of five different in vitro models using five different sources of inorganic ions provide the experimental evidence for a genetic theory of osteogenic stimulation [26–30, 33–36]. The cell and organ culture models are summarized in Table 3.3. Sources of the ionic stimuli are given in Table 3.4.

Table 3.3 Cell and organ culture models used to establish genetic basis for osteostimulation by bioactive glass ionic dissolution products [26–30, 33–36]
Full size table
Table 3.4 Source of ionic dissolution products used in studies of osteostimulation by gene activation [26–30, 33–36]
Full size table

In Table 3.4 the composition of the melt-derived 45S5 bioactive glass culture discs (A) and particulate (B) was 45 % (by weight) SiO2, 24.5 % CaO, 24.5 % Na20, and 6 % P2O5 [1]. Samples of (A) were obtained from US Biomaterials Corp., Alachua, FL, from a certified batch. Commercial powders of (B) with a particle size of 90–710 pm were obtained from NovaBone Products, Alachua, FL. The 58S sol-gel-derived particulate (C) composition (58 % SiO2, 36 % CaO, 6 % P205) and the 70/30 sol-gel sample (D) composition (70 % SiO2, 30 % CaO) were made by the Dept. of Materials, Imperial College London [27, 37]. Sample (E) and the ionic dissolution products of (B), (C), and (D) were obtained by immersing particulates of (B), (C), and (D) in simulated body fluid solution at 37 °C for various times to achieve concentrations of 15–30 ppm of soluble Si ions and 60–90 ppm of soluble Ca ions [38–40].

A study of dose dependence of ionic dissolution products showed this range of concentrations led to enhanced proliferation of osteoblasts [33–41]. Human primary osteoblasts (Table 3.3 Model #1) were obtained from excised femoral heads of total hip arthroplasty patients aged 50–70 years [24]. The first cell cycle and gene array experiments compared samples (A) with Thermanox plastic controls; the 2nd experiment compared ionic dissolution products of (B) with Thermanox controls; and experiment 3 used PCR methods to confirm effects of the ionic dissolution products of (B) on expression of specific genes from osteoblasts obtained from excised femoral heads of five individual patients [26]. Student’s t tests were used to determine statistical significance of the results. The 4th and 5th experiments tested the effects of sample (E) on fHOBS and hES cells. The 6th and 7th experiments confirmed the findings of experiments 1–5 by comparing dosage effects of samples A and E on murine fetal metatarsals grown for 4 days in organ culture post-day 14 gestation [28] and growth of primary hOBs within three-dimensional scaffolds (Source D) [24].

All seven experiments showed enhanced proliferation and differentiation of osteoblasts towards a mature, mineralizing phenotype without the presence of any added bone growth proteins, such as dexamethasone or BMP. Shifts in osteoblast cell cycles were observed as early as 6 h, with elimination (by apoptosis) of cells incapable of differentiation. The remaining cells exhibited enhanced synthesis and mitosis. The cells quickly committed to generation of extracellular matrix (ECM) proteins and mineralization of the matrix. Gene array analyses at 48 h showed early upregulation or activation of seven families of genes that favored both proliferation and differentiation of the mature osteoblast phenotypes, including transcription factors and cell cycle regulators (six with increases of 200–500 %); apoptosis regulators (three at 160–450 % increases); DNA synthesis, repair, and recombination (four at 200–300 %); growth factors (four at 200–300 %) including IGF-I1 and VEG F); cell surface antigens and receptors (four at 200–700 %, especially CD44); signal transduction molecules (three at 200–600 %); and ECM compounds (five at 200–370 %). A summary of the seven families of genes activated or upregulated from the experiments listed above is given in Table 3.5.

Table 3.5 Families of genes in primary human osteoblasts activated or upregulated by ionic dissolution products of bioactive glasses [27]
Full size table

3.3.2 Mechanisms of Genetic Control

There are very few cells in the bones of older people that are capable of dividing and forming new bone. The few osteoprogenitor cells that are present must receive the correct chemical stimuli from their local environment that instruct them to enter the active segments of the cell cycle leading to cell division (mitosis). Resting cells are in the G0 phase, and unless they are stimulated to enter into active phases of the cell cycle, they will not lead to bone regeneration. A new cell cycle begins after a cell has completed mitosis. Regenerative repair of bone requires:

  1. 1.

    Control the population of cells that are capable of entering into active phases of the cell cycle

  2. 2.

    Complete mitosis of osteoprogenitor cells

  3. 3.

    Differentiation into a phenotype capable of synthesizing a full complement of extracellular proteins that constitutes a mature osteocyte

Such osteoblast cell cycle control is achieved by the controlled release of ionic dissolution products from 45S5 bioactive glass, as discussed above [24–36]. Cells colonize the surface of the bioactive glass due to the presence of the biologically active HCA layer formed by surface reaction Stages 1–5, described above and shown in Fig. 3.1. However, the concentration of soluble Si and Ca ions at the cell-solution interface is critical for controlling the cell cycle. Controlled rates of dissolution of the glass provide the critical concentration of the biologically active ions to the cells via the interfacial solution.

During step 1 in the cell cycle, called the G1 phase, the cell grows and carries out its normal metabolism. During the G1 phase osteoblasts are synthesizing phenotypic-specific cellular products. Production of numerous proteins is required for full differentiation. For example, a differentiated, fully functional osteoblast also produces osteocalcin and tropocollagen macromolecules, which self-assemble into Type I collagen, the predominant collagenous molecule present in the bone matrix and numerous other extracellular matrix proteins, listed in Table 3.5. It is especially important that osteocalcin is being produced by osteoblasts grown on the bioactive material. Osteocalcin is a bone extracellular matrix noncollagenous protein produced by mature osteoblasts, and its synthesis correlates with the onset of mineralization, the critical feature of new bone formation. Production of all these extracellular proteins is enhanced in the presence of the ionic dissolution products of bioactive glass.

In order for cell proliferation and repair to occur, there must be a critical period of growth in the G1 phase. Following that growth the cell enters the S (synthesis) phase, when DNA synthesis begins. The S phase eventually leads to duplication of all the chromosomes in the nucleus. Completion of the S phase requires synthesizing a complete genomic sequence of DNA and RNA. The chemical environment of the cell must be suitable to pass through the G1-S checkpoint to initiate the transcription of the host of proteins and nucleic acids required for duplication of the cell. Following DNA replication the cell must prepare to undergo mitosis with a second phase of growth termed the G2 phase. During the G2 phase, as the cell prepares to undergo division, synthesis of the additional proteins required for mitosis occurs.

Also, prior to mitosis, replication accuracy is checked using DNA repair enzymes. A critical increase in cell mass is required, and synthesis and activation of various growth factors are necessary for the G2-M transition. Details of the feedback controls and cell cycle checkpoints are reviewed in other publications [28, 29]. If the local chemical environment does not lead to the full completion of the G1 phase or the G2 phase, then the cell proceeds to programmed cell death, apoptosis. Apoptosis is essential to prevent proliferation of cells that are an incorrect phenotype for bone repair. The chemical environment surrounding bio-inert implants does not stimulate apoptosis. Instead, on bio-inert materials there is rapid proliferation of cell types that are characteristic of nonadherent and non-mineralizing scar tissues. Bio-inert materials or Class B bioactive materials seldom enable the few osteoprogenitor cells present at their interface to pass through these cell cycle checkpoints and become fully differentiated osteoblasts. Only Class A bioactive materials that provide the biologically active ionic stimuli give rise to growth of mineralized bone nodules in vitro and rapid new bone formation in vivo. Details of differences in surface chemistry and cellular interactions between Class A and Class B bioactive materials are given elsewhere [17, 25]. Scanning electron microscopy (SEM) analysis of the human osteoblast cultures showed in the Xynos et al. studies that osteoblasts growing on the Class A bioactive substrate as early as 6 days had already organized, in a process called self-assembly, into a three-dimensional structure composed of cells and mineralized extracellular matrix [24]. This three-dimensional structure is called a bone nodule with an organizational complexity similar to natural bone grown in vivo, although without a blood supply. The time for formation of collagen on bioactive substrates in vitro is similar to the kinetics of collagen formation in vivo, as discussed elsewhere [42]. The rate of formation of mineralized bone nodules in vitro is also similar to the kinetics of bone growth in vivo, as reported by Oonishi et al. using a critical size defect model in the rabbit femoral condyle [14, 32].

Confirmation of the three-dimensional structure of the bone nodules was obtained by Xynos et al. using confocal scanning laser microscopy [24]. The three-dimensional structure of the nodule was mapped to show the presence and organization of the Type I collagenous matrix and calcium deposition within the bone nodules. The results confirm that human osteoblasts growing in culture in the presence of a bioactive glass self-assemble into a three-dimensional architecture and create a mineralized matrix that is characteristic of mature osteocytes in living bone. When the checkpoints in the osteoblast cell cycle described above have been satisfied, cell mitosis and formation of two daughter cells occur. The nuclei of both daughter cells each receive a complete and equivalent complement of genetic material. However, the checkpoints in the cell cycle also result in fewer and fewer progenitor cells that can enter into the M phase unscathed. The built-in protective mechanism from multiplication of damaged genes means that fewer osteoprogenitor cells are available to replace diseased, damaged, or dying bone cells of older people. The cumulative effect is a progressive decrease in bone density with age. Bone regeneration is much slower. In order for bone regeneration to occur at all, it is also necessary for a large fraction of the daughter cells to undergo differentiation into the mature osteoblast phenotype capable of undergoing mineralization and formation of osteocytes. These findings have been confirmed and extended to include other progenitor cell types by Bielby, Christodoulou, and the authors, as summarized in the experiments listed in Tables 3.3 and 3.4.

As discussed above, activation and completion of the osteoblast cell cycle do not merely provide the framework for cell proliferation but also determine to some extent cell commitment and differentiation. Bone cells cover a broad spectrum of phenotypes that include predominately the osteoblast, a cell capable of proliferating and synthesizing bone cell-specific products such as Type I collagen. However, in order for bone to be regenerated and repaired, there must be a vital cellular population consisting of osteocytes. Osteocytes are terminally differentiated osteoblasts that are usually postmitotic and are not capable of cell division. Osteocytes are capable of synthesizing and maintaining the mineralized bone matrix wherein they reside but subsequently do not divide. Thus, osteocytes represent the cell population responsible for extracellular matrix production and mineralization, the final step in bone development and probably the most crucial one given the importance of collagen-hydroxycarbonate-apatite (HCA) bonding in determining the biomechanical properties of bone. Therefore, it is important to observe that the end result of the cell cycle activated by the ionic products of bioactive glass dissolution was the upregulation of numerous genes that express growth factors and cytokines and extracellular matrix components (Table 3.5). An important finding was the 700 % increase in the expression of CD44 (Table 3.5), a specific phenotypic marker of osteocytic differentiation.

The cDNA microarray analysis showed that expression of the potent osteoblast mitogenic growth factor, insulin-like growth factor II (IGF-II), was increased to 320 % by exposure of the osteoblasts to the bioactive glass stimuli (Table 3.5). This is also an important finding because IGF-II is the most abundant growth factor in bone and is a known inducer of osteoblast proliferation in vitro. These results demonstrate that biogenic stimulation of IGF-II by the ionic dissolution products is a key factor in enhanced osteogenesis.

Xynos et al. confirmed the IGF-II mRNA upregulation using quantitative real-time PCR and also showed that the unbound IGF-II protein concentration was increased [26]. The results indicate that the ionic dissolution products of Bioglass 45S5 may increase IGF-II availability in osteoblasts by inducing the transcription of the growth factor as well as its carrier protein and also by regulating the dissociation of this factor from its binding protein. Bioactive induction of the transcription of extracellular matrix components and their secretion and self-organization into a mineralized matrix appears to be responsible for the rapid formation and growth of bone nodules and differentiation of the mature osteocyte phenotype.

3.4 Tissue Engineering of Bone Regeneration

Use of sol-gel processing of bioactive glasses to achieve additional control of the rates of ionic release of biologically active stimuli has led to production of an ideal scaffold for tissue engineering of bone. Compositions and textures of sol-gel-derived glasses can be varied over wide ranges and thereby be used to control the rates and concentrations of soluble Si and Ca released into the physiological solutions. Details of sol-gel processing of bioactive gel-glasses, textural analyses, and bioactivity studies are presented in an extensive series of papers [37–59]. Sol-gel processing makes it possible to produce hierarchical microstructures with nanometer-scale pores in the solid webs of three-dimensional scaffolds while creating an interconnected pore network with greater than 100 μm passages between macropores of 100–300 μm in diameter [41, 46].

Jones et al. demonstrated that such bioactive three-dimensional scaffolds support osteoblast growth and induced differentiation of the cells without use of supplementary organic growth factors [38–41]. Primary human osteoblasts (HOBs) were grown on 70S/30 (70 mol % SiO2, 30 mol % CaO) foam scaffolds made by the sol-gel process [43]. The scaffolds had a modal interconnected pore diameter of 120 μm and a total porosity of 91 %. Prior studies showed that these unique materials resulted in a controlled release of soluble Si and Ca ions when exposed to simulated body fluids at 37 °C. Jones et al. monitored cell viability and growth over a 3-week time period, and the osteoblast marker of alkaline phosphatase enzymatic activity was measured at 4, 7, 14, and 21 days. Production of collagen Type I, the extracellular matrix protein of fully differentiated osteoblasts, was measured at 7 and 14 days using an ELISA technique. The results showed that the bioactive scaffolds stimulated formation of mineralized bone nodules within 2 weeks of in vitro culture of the primary HOBs without the presence of supplementary growth factors in the medium. Evidence of the complete sequence of bone formation occurs by growth of the osteoblasts on the bioactive three-dimensional scaffolds, including cell attachment, cell growth, cell differentiation, extracellular matrix formation, and matrix mineralization. This study shows that the cells completed differentiation into the mature osteoblast phenotype and proceeded towards self-organization of bone architecture without the need of external organic supplements. All of these investigations [15–18, 21, 24] show that the sol-gel-derived bioactive gel-glasses provide controlled release of the ionic stimuli needed to control both proliferation and differentiation of cells of the osteoblast lineage [38–59].

The Bielby et al. study [33, 34] was especially significant because the cell source was embryonic stem (ES) cells. Soluble Si and Ca ions released from 58S sol-gel-derived glasses stimulated gene expression in the murine ES cells characteristic of a mature phenotype in primary osteoblasts. Differentiation of the ES cells into osteogenic cells was characterized by alkaline phosphatase (ALP) activity and the formation of multilayered, mineralized bone nodules. The nodules contained cells expressing the transcription factor runx2/cbfa-1. Deposition of osteocalcin in the extracellular matrix was detected by use of immunostaining. The osteogenic effect of the bioactive gel-glass extracts was dose dependent. The conclusion was that the bioactive gel-glass material was capable of stimulating differentiation of ES cells towards a lineage with therapeutic potential in tissue engineering. This conclusion extends the implications of the therapeutic use of the genetic findings of the studies of Xynos et al. described above (Table 3.5) where the cells were primary human osteoblasts from older people.

The study by Christodoulou et al. [35, 36] expanded even further the scientific basis for understanding the genetic effect of the dissolution products of bioactive gel-glasses on osteogenesis (Table 3.5). The material studied was 58S bioactive gel-glass [47–51]. The soluble Si and Ca dissolution products from the gel-glass were added to cultures of primary osteoblasts derived from human fetal long bone explants cultures (hFOBs). U133A human GeneChip oligonucleotide arrays were used to examine 22,283 transcripts and variants, which represent over 18,000 well-substantiated human genes. A 24-h treatment with a single dosage of ionic products induced the differential expression of a number of genes important to differentiation of the osteoblast phenotype, including IL-6 signal transducer/gp130, ISGF-3/STAQT1, HF-1-responsive RTP801, ERK1 p44 MAPK (MAPK3), MAPKAPK2, IGF-I, and IGFBP-5. The over 200 % upregulation of gp130 and MAPK3 and downregulation of IGF-1 were confirmed by real-time RT-PCR analysis. These data suggest that 58S ionic dissolution products, Ca and Si, possibly mediate the bioactive effect of the gel-glass through components of the IGF system and MAPK signaling pathways. The results from human fetal osteoblasts also confirm many of the findings reviewed above (Table 3.5) using primary human osteoblast cultures derived from excised femoral heads of elderly patients and thereby demonstrate the generality of the findings of genetic stimulation by the ionic dissolution products of bioactive glasses and gel-glasses. The findings are also consistent with prior investigations of the role of ionic dissolution products in stimulation of growth and especially mineralization of fetal long bones, mouse fetal metatarsals, as reported by Maroothynaden and Hench [60].

The implication of the above studies is that it is now feasible to design the dissolution rates and architecture of bioactive, resorbable inorganic scaffolds to achieve specific biological effects in vivo that synchronize with the progenitor cell population present in situ, as discussed previously by the author [61]. This offers for the first time the potential to design biomaterials for specific patients and their clinical needs.

3.4.1 Control of Vascularization of Tissues by Bioactive Glass Ionic Dissolution Products

Clinical use of bioactive glass particulate for dental, maxillofacial, and orthopedic applications has been successful in part because fully vascularized bone is regenerated in situ (Fig. 3.4). The gene array studies summarized above show that VEGF (vascular endothelial growth factor) is one of the important growth factors upregulated (Table 3.5). Healing of wounds in soft tissues and soft tissue engineering also require establishment of a viable blood supply, i.e., vascularization. Recent studies show that ionic dissolution products released from 45S5 Bioglass particulate are effective in promoting angiogenesis in numerous in vitro and in vivo models [62–75]. The experiments confirm that there is upregulation of VEGF production from human microvascular endothelial cells (HMVEC) and other cells involved in the repair and maintenance of the circulatory system. The stimulation of angiogenesis depends upon the concentration of ions present in the cultures which can be controlled by using differing quantities of 45S5 Bioglass in collagen sponges or other multiphase delivery systems. The findings are that when there are too few ions, there is no angiogenic stimulatory effect; too many ions also have no effect. Leu and Leach showed that larger concentrations of the ionic dissolution products led to osteogenesis [66], as described in the seven osteogenic experiments reviewed above. Thus, controlled release of a critical concentration of Si and Ca ions is required for the proangiogenic effect.

Fig. 3.4
figure 4

The X-radiograph shows the use of bioactive glass, 45S5 Bioglass, after repair of bone following removal of a large unicameral cyst in the ischium of a patient prior to revision of hip arthroplasty due to implant loosening. The existing hardware was removed and replaced, and the cyst was debrided. The residual cavity (denoted by the arrows) then was grafted with a 50:50 mixture of autogenous bone and bioactive glass (NovaBone®). Three years after implantation, the graft site is stable with no recurrence of the cyst, the site being filled with dense bony tissue. There has been no recurrence of the cyst (Photo courtesy of Dr. Dave Gaisser, NovaBone Products, Alachua, FL, USA)

Full size image

There are important implications from these findings. At present most treatment modalities for damage to the small intestine or many soft tissue defects, including chronic wounds, are at best palliative. There is great need for bioactive wound dressings that can counter the negative stimuli that prevent healing of chronic wounds. Combining the anti-inflammatory characteristics of 45S5 Bioglass particles with their proangiogenic potential, described above, offers great promise for design of wound dressings that stimulate keratinogenesis and angiogenesis required to achieve a rapid regeneration of the skin.

3.5 Conclusions

The molecular biological mechanisms involved in the behavior of bioactive glasses are now understood with sufficient confidence that the results can be used to design a new generation of bioactive materials for tissue regeneration and tissue engineering. The bioactive response appears to be under genetic control. Bioactive glasses that are osteostimulative enhance osteogenesis through a direct control over genes that regulate cell cycle induction and progression towards a mature osteoblast phenotype. This process is termed osteostimulation. Cells that are not capable of forming new bone are eliminated from the cell population, a characteristic that is missing when osteoblasts are exposed to bio-inert or Class B bioactive materials. The biological consequence of genetic control of the cell cycle of osteoblast progenitor cells is the rapid proliferation and differentiation of osteoblasts. The result is rapid regeneration of bone. The clinical consequence is rapid fill of bone defects with regenerated bone that is structurally and mechanically equivalent to normal, healthy bone. The use of bioactive glass particulate to release smaller concentrations of ionic dissolution products from a polymer sponge offers the possibility of designing a new generation of wound-care dressings and soft tissue engineering constructs.

Perhaps of even more importance in the long term is the possibility that bioactive ionic dissolution products can be used to activate genes in a preventative treatment to maintain the health of our bones as we age. Only a few years ago, this concept of using bioactive materials for preventative therapeutics would have seemed to be impossible. We need to remember that it was only 40 years ago that the concept of a material that would not be rejected by living tissues was considered to be impossible. If we can activate genes by use of glasses to grow bone and stimulate repair of soft connective tissues, it is certainly possible that we may one day be able to use glasses to control genes to prevent the loss of tissues.