Bioactive glasses beyond bone and teeth: Emerging applications in contact with soft tissues

Accepted Manuscript Review Bioactive glasses beyond bone and teeth: emerging applications in contact with soft tissues Valentina Miguez-Pacheco, Larry L. Hench, Aldo R. Boccaccini PII: DOI: Reference:

S1742-7061(14)00496-6 http://dx.doi.org/10.1016/j.actbio.2014.11.004 ACTBIO 3462

To appear in:

Acta Biomaterialia

Received Date: Revised Date: Accepted Date:

25 July 2014 19 October 2014 4 November 2014

Please cite this article as: Miguez-Pacheco, V., Hench, L.L., Boccaccini, A.R., Bioactive glasses beyond bone and teeth: emerging applications in contact with soft tissues, Acta Biomaterialia (2014), doi: http://dx.doi.org/10.1016/ j.actbio.2014.11.004

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BIOACTIVE GLASSES BEYOND BONE AND TEETH: EMERGING APPLICATIONS IN CONTACT WITH SOFT TISSUES Valentina Miguez-Pacheco1, Larry L. Hench2, Aldo R. Boccaccini1,3 1) Institute of Biomaterials, University of Erlangen-Nuremberg, 91058 Erlangen, Germany 2) Department of Biomedical Engineering, Florida Institute of Technology, Melbourne, Florida, USA 3) Department of Materials, Imperial College London, London SW7 2AZ, UK Corresponding author. Email : [email protected]

Abstract The applications of bioactive glasses (BGs) have been to a great extent related to the regeneration and repair of hard tissues such as bone and teeth and there is an extensive bibliography documenting the role of BGs as a bone replacement material and in bone tissue engineering applications. Interestingly, many of the biochemical reactions arising from the contact of BGs with bodily fluids, in particular the local increase in concentration of various ions at the glass-tissue interface, are relevant also to mechanisms involved in soft tissue regeneration. An increasing number of studies report on the application of BGs in contact with soft tissues, aiming at exploiting the well-known bioactive properties of BGs in soft tissue regeneration and wound healing. This review focuses on research, sometimes involving preliminary in vitro studies but also in vivo evidences, that demonstrates the suitability of BGs in contact with tissues outside the skeletal system, which includes studies investigating vascularization, wound healing and cardiac, lung, nerve, gastrointestinal, urinary tract and laryngeal tissue repair using BGs in various forms of particulates, fibres and nano-particles with and without polymer components. Potentially active mechanisms of interaction of BGs and soft tissues based on the surface bioreactivity of BGs and on biomechanical stimuli affecting the soft tissue-BG collageneous bonding are discussed based on literature results.

1.

Introduction and Historical Background

The role of BGs and glass-ceramics as suitable biomaterials for orthopedic and dental applications as well as in bone tissue engineering (BTE) has been well documented and comprehensive review articles have been published recently (1–6). The important characteristics of BGs as bioactive systems suitable for bone and tooth repair and regeneration, such as a high bioactivity, osteoconduction and osteostimulation, have been well established by numerous investigators in the last 40 years since the seminal report of the discovery of bioactive glass by Hench et al in 1971 (7, 8). Classical applications of BGs involve bioactive coatings on orthopedic implants (9, 10), bone filling materials, dental applications and small bone implants (4, 11, 12). The most widely investigated BG composition is the bioactive silicate “45S5 Bioglass®” with a composition (in wt. %) 45% SiO2, 24.5% each of Na2O and CaO and 6% P2O5 (12), also known as the “grandfather composition” (4). The “13-93” bioactive silicate glass is also receiving increasing attention, this composition has a higher SiO2 content (53wt.%) than 45S5 Bioglass®, and includes K2O and MgO as network modifiers (12 wt% and 5 wt%, respectively) (13, 14). With respect to clinical applications, the composition “S53P4” is the most widely used, with plenty of successful outcomes for the treatment of osteomyelitis (15). Several other compositions have been developed over the years aiming to improve the processability and bioactivity of 45S5 Bioglass® (1, 13, 16), some typical BG compositions are presented in Table 1. From the early days of research on BGs, it has been demonstrated that these materials form a direct bond to bone (7, 8) and a recent comprehensive review has summarized knowledge about the interactions between BGs and collagen in the context of both bone/BG and soft tissue bonding (17). An important body of research has shown that the ionic products of BG dissolution in body fluid stimulate osteoblast proliferation (18–20) and may enhance angiogenesis in certain in-vivo and in-vitro conditions (21). Given the increasing interest in BG applications in medicine and dentistry, a variety of innovative applications are emerging, for example, the use of BGs in the musculoskeletal system has extended to the successful culture of Annulus Pulposus cells onto PLLA/Bioglass® composite films for intervertebral disk tissue repair (22,23). Also, BGs in combination with polymers are being considered for interface tissues (hard-soft tissue interfaces) (24–26). In addition, bioactive glasses have been shown to be effective antibacterial agents against a range of bacteria known to be

associated with post-operative infections (27–29) and with implant failure (30,31). Also, the doping of BGs with trace elements is being increasingly considered to enhance certain characteristics of the glasses, for example: silver has been used to impart an enhanced antibacterial effect in BG (32–34); lithium substituted 45S5 BG was shown to increase cell proliferation and metabolic activity (35); copper-doped BGs have been proposed to induce angiogenesis in bone regeneration strategies (36,37); cobalt-containing mesoporous glasses with controllable ion release were shown to mimic hypoxic conditions and to enhance angiogenesis (38,39) and zinc-containing glasses are being developed to increase the proliferation rate of osteoblasts in culture (40). In addition, Sr-containing BGs are being investigated in the context of strategies to combat osteoporosis (41). The application of BGs containing Fe for applications in cancer treatment by hyperthermia are also being considered (42,43) as well as for embolization therapy (44). Bioactive glasses, especially in their mesoporous form, are being also applied as drug delivery devices for a more controlled and longer lasting delivery after implantation (45–50). For these glasses, the higher the surface area, the higher the amount of therapeutic agents that can be loaded into them (46). Surface modification of BGs has been employed to increase their bioactivity, for example, by calcium surface-enrichment (51) or to increase BG biocompatibility by addition of appropriate biomolecules (52, 53) in order to increase interactions between the BG surface and proteins. The uses of BGs extend also to cosmetic and dental care applications. For example, BGs in particulate form are added to toothpaste (54-56) and as mineralization agents (57) for example in the form of composites containing BG nanoparticles (58–60) or as electrospun cotton-wool like templates (61). In addition, a sodium calcium phosphosilicate glass (Vitryxx®) has been extensively tested to investigate skin reaction in humans to high concentrations of the glass in various cosmetic products ranging from nail to skin care products (62). Promising applications in ophthalmology and ocular surgery have been also reported (63). Perhaps not surprisingly, given the inorganic nature and mechanical rigidity and brittleness of BGs, which exhibit physical characteristics closer to ‘hard’ tissues, much reduced attention has been paid to the role that BGs may have as materials for soft-tissue engineering (STE) applications, even though some of the characteristics that make BGs ideal for bone or dental applications are also key to soft tissue regeneration approaches. The early reports by Wilson et al. (64, 65) documented the stable interactions of bioactive glasses and soft tissues. In

early pioneering efforts, Wilson et al. (65) carried out numerous in vitro and in vivo experiments to examine the biocompatibility and possible toxicity of Bioglass® in contact with various types of soft tissues. In vitro tests involved seeding diverse cell types from mice, rats, hamsters, chickens and humans onto solid and powdered BG samples with different formulations, including 45S5, 52S4.6 and 45S5F (see Table 1 for compositions). In vivo testing was carried out by implanting powdered and solid disks samples of various BG compositions subcutaneously, intramuscularly and in the peritoneal cavity of various mammals, including canines. The in vivo tests demonstrated healthy tissue growth and adhesion around the implants, even when placed in areas where shear stresses applied to the surface of the implants would have produced particulates in micro-motion had this not been prevented by the strong collagen-BG bonding. Autopsied animals did not show signs of any inflammatory reaction. These early experiments can be considered as initial significant attempts to understand the interaction of bioactive glasses with soft tissues. In fact, the stable soft tissue 45S5 BG implant interface was the basis for the first BG clinical applications of middle ear prostheses and endoosseous ridge maintenance implants, as reviewed recently in detail by Hench and Greenspan (17). 45S5 Bioglass® has also been added to high-density polyethylene (HDPE) for applications in more rigid soft tissues such as cartilage and connective tissue, which tend to exhibit more difficulties with bonding to prostheses (66). In other relevant early studies by Gatti et al. (67), S53P4 bioactive glass (see Table 1 for composition) granules of around 300µm were implanted in the dorsal muscle and under the dorsal skin of rabbits and in defects surgically created in the jaw of sheep to investigate the reactions of this glass in soft and hard tissues. The granules were left in the rabbits for 2 months and for 3 months in the sheep. After excision of the implanted sites and examination of the samples it was revealed that the nature of the reactions did not depend on the type of tissue the glass was implanted in, and reactions between the BG granules and their surroundings developed in roughly the same manner. Similarly, a recent study by Meretoja et al. (68) investigated the effect of subcutaneously implanted polycaprolactone/lactide and S53P4 BG composite scaffolds in rats and found that after 4 weeks of implantation there was no inflammatory reaction and only a mild foreign body reaction occurred while the scaffolds were fully invaded by well-vascularized connective tissue.

The analysis of the literature shows that indeed for many years research efforts focusing on soft tissue/BG interactions were rather sporadic, however great interest in this subject has emerged recently, and an increasing number of studies are being published, which has motivated preparation of this review article to discuss research related to the potential of BGs in innovative approaches towards regeneration of soft tissues of various types and clinical needs. The authors intend that this review will serve to fill a gap in the available (and abundant) bioactive glass literature, which mainly focuses on bone and dental applications, by comprehensively covering the applications of bioactive glasses in areas such as angiogenesis, wound healing, hemostasis and regeneration of soft tissues as diverse as nerve, cardiac, lung and intestines (Figure 1). The review covers mainly silicate bioactive glasses; however, phosphate and borate glass compositions are also discussed where relevant.

2.

Angiogenesis

A crucial aspect for success of scaffolds for tissue engineering and regeneration of vascularized tissues is the need to develop sufficient neovascularization in the affected area in order to ensure suitable transport of nutrients and growth factors into and removal of waste products from the new tissue formation site (73). Angiogenesis is also crucial for the survival of cells seeded into the scaffold prior to implantation and for the maintenance of any cell populations that may be recruited during tissue formation. The mechanism by which new blood vessels are formed from pre-existing ones, angiogenesis, is regulated by many conditions including growth factors, e.g. vascular endothelial growth factor (VEGF), extracellular matrix (ECM) proteins, the amount and kind of cell membrane receptors available and other signaling molecules (74). Although introduction of growth factors and signaling molecules onto of TE scaffolds is a common approach (45,75,76), there are still many concerns over the duration and efficiency of their delivery to surrounding tissues when implanted, given that these factors are delicate and degrade easily and controlled release has proved difficult to achieve. Therefore, the alternative of developing (inorganic) materials that promote angiogenesis by their own degradation mechanisms, e.g. delivery of bioactive stimulating ions, is very appealing (18,37,38,39,77,78). The angiogenic effect of bioactive glass was first reported in 2004 by Day et al. (79), and relevant in vitro and in vivo

investigations were reviewed by Gorustovich et al. (21). Although most studies of vascularization in BG-based materials have been carried out in the context of bone tissue engineering (3,73,80–84), the discovery that specific compositions of BGs can have a stimulatory effect on angiogenesis is applicable to soft tissue engineering approaches. Various types of cells, including fibroblasts, myocytes, hepatocytes and neurons, release VEGF under hypoxic conditions. Amongst them, fibroblasts are known to secrete VEGF as well as fibroblast growth factor (FGF), a protein with a powerful mitogenic effect on both fibroblasts and endothelial cells (74). This growth factor is of importance when investigating angiogenesis (85). Endothelial cells are crucial in the formation of new blood vessels since they migrate and divide to form tubules connecting to other blood vessels and are affected by the aforementioned growth factors in this process. Whilst growth factors are crucial for neovascularization to occur, there is a need for them to be controlled and localized and their long-term release must be tailored to avoid malformations in the vascular network, uncontrolled or misplaced tissue growth and to allow sufficient time for the growth and remodeling of the new vessels (86,87). To investigate how different concentrations of 45S5 Bioglass ® would affect the production of VEGF and bFGF in human fibroblasts, Day et al (79) seeded populations of these cells on polymer surfaces coated with different amounts of Bioglass® and incubated them for up to 72h. In parallel, they prepared polyglycolic acid (PGA) meshes coated with 45S5 Bioglass® particles at different concentrations which were surgically implanted in subcutaneous pockets in rats (Figure 2). The cultured fibroblasts were found to secrete VEGF under all conditions, as seen in Figure 2. However, at the higher concentrations investigated (0.1-10% culture plate cover) the secretion of the growth factor was inhibited, indicating a dose dependent effect, which has also been found by other researchers (21). In a similar experimental setting, Day et al (88) examined the effect of Bioglass® on fibroblasts and found that all populations secreted significantly higher amounts of both VEGF and bFGF compared to control cultures without Bioglass®. This increase in growth factor secretion was found to originate from direct stimulation of cells by the Bioglass® particles and not by an increase in fibroblast population, since the cultures at the concentrations necessary to elicit this phenomenon (0.3125-6.25 mg/cm2) had smaller cell populations compared to the controls. This direct effect of stimulating fibroblasts was confirmed by measuring increased

amounts of VEGF mRNA. Conditioned media from these experiments were used to culture endothelial cells, which caused a 61.5% increase in cell population compared to controls, confirming that small amounts of Bioglass® directly stimulate the secretion of angiogenic growth factors by fibroblasts and significantly increase angiogenesis in vitro. The results of the investigation thus provided evidence that BGs accelerate the angiogenic process by stimulating the secretion of relevant growth factors, such as VEGF, through stimulation of cytokine production in cells (88). In a more recent study by Gerhardt et al. (89), the angiogenic effect of BGs was investigated by comparing plain poly(D,L lactide) (PDLLA) and composite PDLLA-BG scaffolds and it was found that there was a marked increase in the release of VEGF by fibroblasts cultured on the composite scaffolds compared to plain PDLLA films. Similarly, in vivo experiments in a rat model showed that composite scaffolds induced higher vascularization and increased percentages of blood vessel formation, determined by stereological analysis of the vascularized tissues. Another frequently used bioactive silicate glass (S53P4) (see Table 1 for composition) was investigated in a similar model involving human fibroblasts (70). The cells were cultured in contact with granules of the S53P4 BG in three ranges of particle sizes (0.50.8, 1.0-2.0 and 2.0-3.15 mm) and in different concentrations (0, 0.01, 0.1 and 1 wt/vol%). VEGF production was enhanced for particles of 0.5-0.8 mm and 1.0-2.0 mm at all concentrations, but inhibited by particles of 2.0-3.15 mm. The angiogenic effect of BGs has been also investigated in the CAM model by Vargas et al. (90), which found that high concentrations of 45S5 Bioglass® failed to exhibit any pro-angiogenic effects in chick embryos incubated with scaffolds of this material. These cumulative results indicate that an optimum concentration of Bioglass® released at a controlled rate is necessary to exploit its angiogenic capabilities. In a recent study by Handel et al. (91), 45S5 Bioglass® scaffolds prepared by the foam replica method were coated with collagen and bio-functionalized by seeding with human adipose tissue-derived stem cells (hASC) to be tested in the CAM angiogenesis model, opening the alternative of investigating BGs with ion doping (e.g. Cu, Co, etc), which may be effective to enhance angiogenesis (36,38,92). It was found that, compared to plain Bioglass® scaffolds, the bio-functionalized scaffolds showed an increase in vascular tube lengths. However it is

worth noting that for this particular assay, the release of ionic products from Bioglass® samples was found to have negligible effects in angiogenesis. In summary, numerous investigations, some of which were briefly discussed in this section, have established that BGs can stimulate neovascularization, and it is clear that the ionic products arising from the surface reactions of BGs in contact with the extracellular matrix must be carefully controlled given that very specific concentrations are necessary for the optimum production or secretion of the necessary growth factors that govern the formation of blood vessels in living tissue. Clearly, the use of BGs as angiogenic agents in TE approaches is a field in its infancy and an emerging avenue for exciting research efforts. The fact that BGs of certain compositions exhibit angiogenic potential is an essential finding for further considering BGs in soft tissue repair strategies.

3.

Cardiac tissue regeneration

Blockage of the coronary arteries that irrigate the heart results in cardiac ischemia and subsequent tissue death and scarring. This phenomenon is known as Myocardial Infarction (MI) and is one of the leading causes of death worldwide. Depending on the effectiveness and immediacy of treatment following an episode of MI, the severity of the ischemic injury can be alleviated and swift medical intervention can considerably reduce cardiac tissue damage and its associated pathophysiology (93). However, in most cases (around 90% of patients) MI causes cardiac arrhythmia, leading to a decreased pumping efficiency which may be life threatening depending on the extent of the injury. Cardiac tissue lacks the ability to regenerate after definitive damage (around 6 hours after MI) and thus tissue engineering using injectable scaffolds or engineered cardiac patches is being increasingly investigated as routes to regenerate this damaged tissue (94,95) Polymeric heart patches, for example, have been developed to provide mechanical support to the left ventricle of the heart and to serve as a cell delivery system to repair the damaged tissue (96–98). With this double purpose in mind, Chen et al (99) developed an elastomeric composite for heart patches combining poly(glycerol sebacate) (PGS), to provide the necessary mechanical flexible support, and BG nanoparticles, which were incorporated to improve the mechanical properties of the patches and to provide anchorage points for cells

to aid their delivery. Nanocomposite samples were prepared at several BG concentrations ranging from 0 to 10 wt%. The patches were placed under culture conditions to evaluate their degradation behavior. PGS undergoes hydrolytic degradation in culture medium and thus the pH decreases rapidly. The addition of BG nanoparticles was found to counteract the acidity caused by the degradation of PGS. In terms of mechanical properties, the Young’s modulus and strain at break of the samples increased with increasing proportion of incorporated BG particles, which confirmed the positive effect of using nano-scale BG in combination with biopolymers, as reviewed elsewhere (59). Mouse fibroblasts were cultured with the extract media of the materials that were cross-linked for 2 days and it was shown that the proliferation of cells was greater on the nanocomposite samples than on pure PGS. Similarly, human ESC-derived cardiomyocytes (hESC-CM) cultured with extract media demonstrated decreased cytotoxicity of nano-BG containing samples compared to pure PGS samples. Similar values of beating rates were measured compared to hESC-CM cultured in standard culture medium. These results show that PGS-nano-BG composites possess suitable mechanical properties and superior biocompatibility, making them a viable option for the development of palliative treatment of congestive heart failure via the cardiac patch strategy. Clearly, more research is required to expand the innovative application of BGs in this highly relevant field. Indeed the use of mesoporous glass particles, which can be loaded with bioactive molecules and exhibit a controlled drug delivery capability (45,100–102), could be considered as a suitable addition to biodegradable polymers to enhance the functionality of the cardiac patches incorporating local drug and growth factor releasing effect. A SEM micrograph of such cardiac patch under development is shown in Fig. 3a while Figure 3b shows an schematic diagram of such flexible polymer matrix composites incorporating mesoporous bioactive glass particles (of typical pore size in the range 2-50 nm and surface area of up to 1000 m2g-1) (101) as local drug releasing vehicles.

4.

Wound healing/dressing

Control of severe hemorrhages could decrease the mortality rate in extreme situations such as the battlefield, natural disaster or accident areas by increasing the time window for the injured to be transported to an adequate medical facility. Simple, palliative wound dressings are often inadequate to treat patients in such cases, and novel biomaterial solutions have

been devised to improve treatment such as QuikClot® (Z-Medica Corporation, Wallingford, CT). However, the results of a variety of wound healing approaches indicate that there is need for improvement, given that the exothermic reaction on application of some dressings causes mild to severe pain in treated patients and has been reported to cause burns on application (103). Thus the search for biomaterials systems for wound healing involving internal and external wounds is a continuous task. Bioactive glasses are being considered for this important application. For example, Ostomel et al (104) produced mesoporous BG microspheres (MBGMs) by the sol-gel method with diameters in the range from 100nm to 1µm for application as hemostatic agents. The increased surface area of these particles resulted in faster deposition of HA on the surface of the MBGMs when immersed in SBF compared to irregularly shaped melt-derived BG particles, and this effect has been confirmed to positively influence bioactivity (105,106). By adding different amounts of MGBMs to sheep blood, the hemostatic effect of the microspheres was examined, and it was found that an increasing amount of the spheres decreased the clotting time, i.e. the rate of coagulation of blood increased in contact with MBGMs (104). Although it is unclear whether the accelerated rate of blood coagulation is due to an increased availability of Ca 2+ ions or due to the increased surface area of the calcium-containing BG, the results indicated that MGBMs are promising hemostatic agents and this field is attracting increasing research interest. For example, mesoporous silver exchanged silica spheres enriched with calcium (AgCaMSS) with diameters in the range of 600µm to 1.2 mm were produced by Dai et al. (107) to investigate them as hemostatic agents with antibacterial properties. In vitro experiments showed that the addition of Ca and Ag ions into the silica network increased the dissolution rate and weight loss in Tris-HCl buffer solution, and that the optimal formulation of AgCaMSS increased blood clotting rates. Also, incubation of the spheres in a bacterial broth containing E. coli and S. aureus demonstrated the antibacterial effect of AgCaMSS. In vivo studies involved application of the silica spheres to open wounds in the femoral arteries and livers of rabbits, and found that animals treated with the AgCaMSS agents had lower mortality than controls, which were only treated with conventional gauze and pressure. The hemostatic agents also exhibited low exothermic effects, thus highlighting them as promising materials for hemorrhage control. In diseases such as diabetes, the wound healing ability of an individual is highly compromised resulting in longer healing times, increased discomfort and risk of infections

(108). Also, reduction of scarification and faster healing times are desirable features for a wound healing accelerating device, and certainly there are studies where inorganic materials have been investigated. For example, Majeed et al. (109) applied HA intra-incisionally in a rabbit wound model and found that it promoted faster proliferation of connective tissue and thus enhanced wound healing; in another study, Kawai et al. (110) administered calciumbased nanoparticles to wounded mice and found that the open wound size decreased dramatically in treated animals. The authors speculate that the nanoparticles increased calcium uptake in fibroblasts and caused contracture of collagenous tissues populated with these cells. However, the application of BGs remains an attractive option to be explored in the field of wound healing given the huge versatility they present in the range of ions that can be incorporated in the glass matrix to elicit specific cellular responses (18). To examine the effects of particulate (<20 µm) BG in full thickness skin wounds, Gillette et al (111) applied BG into open wounds surgically made in 9 dogs. Bilateral wounds were made so that one side could be treated with BG and the other would serve as a control. Before suturing, particulate BG was mixed with a small amount of blood to produce a slurry that was applied to the wound. Most of this mixture stayed in the subcutaneous area of the wound with a smaller amount resting between the wound edges. Laser Doppler Perfusion Imaging (LDPI) was used to examine changes in blood perfusion around the wound areas and it was shown that there was no significant difference between treated and control wounds at days 3 and 5. At day 5 postoperatively, the skin and subcutaneous tissue around the wounded areas were excised and placed in a tensiometer to determine the breaking strength of the healed tissue. Whilst no significant difference was found in the breaking strength of the healed skin in all samples, there was an increase in the breaking strength of healed subcutaneous and cutaneous trunci in bioactive glass treated wounds compared to control wounds. It was also found that BG does not cause an increased inflammatory reaction in wounds and could potentially be used to increase the strength of wounded soft tissue. Application of nano-scale bioactive glasses in wound healing applications has also been explored. For example, nano-porous bioactive glass containing silver (n-BGS) was fabricated by Hu et al. (112) to evaluate their performance as hemostatic and antibacterial dressings compared to bioactive glass without nano-pores (BGS). The n-BGS and BGS samples were

prepared by the sol-gel method with silver contents ranging from 0.01 to 0.04 wt%. These particles were shown to have an average surface area of about 450 and 91 m 2/g, respectively, with the nano-pore samples having a mean pore diameter of 6nm. As a result of the increased surface area, water absorption efficacy of n-BGS was much higher than that of the normal BGS. In terms of silver release, the nano-pore samples were found to release Ag ions faster, although the concentration of silver in solution was the same after 24h incubation in PBS. E. coli bacteria were cultured with n-BGS to test the material antibacterial properties. While samples at all concentrations exhibited antibacterial activity, n-BGS at 0.02 wt% silver concentration was found to have an antibacterial rate of 99% after an incubation time of 12h. This glass was chosen for further in vivo experiments since higher Ag concentrations were not considered relevant as they would cause cytotoxicity. The hemostatic performance of the n-BGS samples was measured by both prothrombin time (PT) and activated partial thromboplastin time (APTT) in vitro and also by applying them on fatal injuries in skin and the femoral arteries and veins of male New Zealand white rabbits. As an example of the results obtained, Figure 4 shows hemorrhage control in a rabbit skin injury model treated with n-BGS and BGS particles (110). In all experiments, the n-BGS outperformed the BGS samples, with significantly lower clotting times in the PT and APTT assays and bleeding times of rabbit injuries. The findings also demonstrate that nano-porous silver doped BGS treatments accelerate clotting, facilitate hemorrhage control and exhibit a satisfactory bactericidal effect. In a study by Jebahi et al. (113), ovarectomized rats had their dermal and muscular wounds treated with both plain 46S6 (BG) (see table 1 for composition (72)) and strontium-containing 46S6 (BG-Sr) and it was found that, compared to untreated control groups, the treated animals exhibited regenerated and well vascularized muscle tissue and fully epithelialized skin. The extent of the healed wounds was more evident in the rats treated with the BG-Sr, due to a protective effect against reactive oxygen species from the implants, which the authors speculate was due to ion release in their surroundings. With the aim of accelerating the healing of full-thickness skin wounds, Lin et al. (114) produced Vaseline-based ointments with 18 wt% of sol-gel bioactive glass 58S (SGBG-58S), nanoscale bioactive glass (NBG-58S) and melt-derived 45S5 bioactive glass powders, which were applied to superficial injuries in healthy and diabetic rats. The healing rates of the wounds treated with the different ointments were compared. After creating full-thickness skin wounds in diabetic and healthy rat groups, the bioactive glass-containing ointments and

Vaseline for the control group were applied directly on the wound site, covered with sterilized elastic bands and re-applied every other day. Wound healing was studied by calculating the healed area as a percentage of the total wound every 2 days up to 16 days and it was found that for both diabetic and healthy rats, healing was accelerated in the presence of the bioactive glasses, most notably with the SGBG-58S, with almost complete wound healing at day 16. Figure 5 shows the evolution of treated and untreated fullthickness skin wounds in rats from day 0 to day 16. The control groups had significantly longer healing times and the wounds were still open by day 16 and, as expected, the diabetic rats had longer healing times than the healthy rats. Histological examination of wound samples demonstrated an increased proliferation of fibroblasts, increased formation of granulation tissue and development of new capillaries in animals treated with the BGcontaining ointments, whilst immune-histochemical assays showed that VEGF was present in all tissue samples at day 7. No inflammatory reaction was observed in the animals treated with the bioactive-glass ointments. These results demonstrate that bioactive glasses can accelerate wound healing in both normal and diabetic rats. The sol-gel derived glasses led to quicker and more effective wound healing than the melt-derived 45S5 Bioglass®, which was ascribed to the higher surface area and surface nano-scale topography of sol-gel glasses. Similarly, Mao et al. (115) produced Vaseline ointments containing 16 wt% 45S5 Bioglass® (particle size <53µm) and different amounts of Yunnan baiyao, a traditional Chinese medicine of herbal origin, and applied them to full-thickness skin wounds on diabetic rats. It was found that the combination of 45S5 Bioglass® and 5wt% Yunnan baiyao powder significantly accelerated the wound healing rates, and the authors estimated that this phenomenon is due to the combined anti-inflammatory effect of Yunnan baiyao and increased release of relevant growth factors, namely bFGF and VEGF, due to the presence of 45S5 Bioglass®. It is speculated that these actions resulted in increased fibroblast proliferation and activity, vascularization and granulation tissue growth thus supporting enhanced healing rates in diabetic rats. The process of wound healing is complex and involves several stages, starting with an inflammatory reaction that is mediated by several chemical cues, including growth factors and cytokines (116). It is thought that the ionic products of the reaction of bio-degradable BG particles with their surrounding medium induces a reduction of this initial inflammatory

reaction, which can increase wound healing rates resulting in more satisfactory healing (17). This effect was evidenced in a study by Grotheer et al. (117), where ortho-silicic acidreleasing silica gel fiber fleeces (SIFIB) were implanted in full-thickness skin wounds in pigs. Wounds treated with the fleeces exhibited significantly faster healing rates compared to the controls (the wound was simply covered with a gauze and plasters). Dramatic results have been obtained for repair of chronic wounds in a small-scale human study of “worst case patients” reported recently. Bioactive 13-93B3 (borate) glass nanofibres of composition (wt%): 5.5% Na2O, 11.1% K2O, 4.6% MgO, 18.5% CaO, 3.7% P2O5 and 56.6% B2O3 with diameters ranging from 300nm to 5µm were used to treat chronic wounds in diabetic patients (118,119). It was found that wounds healed at an accelerated rate, which could be otherwise achieved, for example, by applying a vacuum-assisted closure system (VAC) which is a relatively expensive and cumbersome process. In addition, there was a marked decrease in scar tissue formation compared to conventionally treated wounds, thus offering a possibility for the treatment of full-thickness skin wounds without lengthy hospital stays or expensive equipment. The success of the limited trials has led to expanded use of the bioactive borate glass fiber wools in patients that otherwise would be candidates for amputation (119). Given the hemostatic properties of BGs, discussed above, Rai et al (120) produced poly(3hydroxyoctanoate) (P3HO) composite films containing nano-sized bioactive glass (nBG) to be used as multifunctional wound dressings. Composite films were prepared from 5 and 10 wt% polymer solutions containing different nBGs concentrations. An increasing proportion of glass nanoparticles imparted the films with increased surface roughness, enhanced the wettability of the polymer and decreased the clotting time of citrated whole blood. In vitro cultures of human keratinocytes on the nano-composite films showed increased cell proliferation compared to plain polymer films, which the authors attributed to the increase of attachment sites for cells provided by the nBG particles. When there is extensive tissue loss due to injury, the healing process is delayed given that tissue replacement takes place simultaneously to repair mechanisms. To aid progress of these parallel processes, Keshaw et al. (121) produced polyglycolic acid (PLGA) and BG composite micro-porous spheres with an average diameter of 1.82 ± 0.01 mm by a thermally induced phase separation (TIPS) technique. This approach exploited the bioactivity and

angiogenic potential of BGs (discussed above) (21,79,122) and the suitable mechanical properties of PLGA combined with the advantage of the geometry of microspheres. The dimensions and shape of these composite scaffolds make them particularly attractive as fillers for treatment of extensive wounds, as irregular volumes can be packed with these composite microspheres, aiding new tissue growth. In vitro testing showed that composite spheres containing 10 wt% BG decreased in size by 20% in 16 weeks as well as induced increased VEGF secretion by human fibroblasts, adjudicated to the presence of BG (as discussed above). In vivo implantation into rats showed good tissue growth and vascularization around and into the scaffolds. In a similar setting, Day et al. (79) produced meshes from polymer fibres coated with Bioglass® particles that could be applied in a wide variety of soft tissues to aid repair and regeneration. Meshes implanted subcutaneously into rats and left for up to 42 days were removed and examined to show complete tissue infiltration and neovascularization, demonstrating the potential of these scaffolds in wound healing applications. Chen et al. (123) produced nanofibrous gelatin/bioactive glass sponges coated with hyaluronic acid and chitosan (NF-GEL/BHA-CS) to treat chronic non-healing wounds, and found that they possessed superior thermal and structural stability than plain NF-GEL/BG hybrid sponges whilst maintaining bioactivity, as evidenced by the inorganic ion release when immersed into buffer medium. Thus BG containing hybrid sponges are an attractive option to accelerate wound healing. Clearly, with the increasing need for advanced material systems for effective and rapid wound healing, further investigations involving novel chemical compositions and morphologies of BGs are required, including mesoporous BGs (37, 38, 46), to achieve optimal rates of soft tissue regeneration. Research in this field is likely to increase in the near future, with BGs representing a valid alternative to conventional approaches as a consequence of stimulation of multiple routes of tissue repair while providing a biomechanically attractive delivery system.

5.

Nerve regeneration

Peripheral neuropathies resulting from severe injuries cannot always be treated by suturing, and even in cases where suturing is appropriate, they may result in limited nerve function and sensory deprivation of the affected areas. The gold standard for the treatment of

damaged nerves that present a long gap (>4mm) (124) in these conditions is the use of autografts, however due to limited availability and possible secondary effects including donor site morbidity, the need for alternative routes for nerve repair is evident. Alternative routes for bridging nerve gaps include the use of tubular conducts made from natural or synthetic materials. These biomaterial-based approaches have so far yielded less satisfactory responses than autografts and more research efforts are required. The application of bioactive glasses in nerve regeneration approaches requires in principle the availability of flexible bioactive glass fibres. In view of this, Bunting et al. (125) produced a nerve regeneration scaffold by entubulating 45S5 Bioglass® fibres within silastic conduits and tested them by bridging 0.5cm sciatic nerve gaps surgically created in adult rats. Male Wistar rats were divided into four groups: group A rats had conduits filled with Bioglass® fibres implanted between the nerve stumps; group B had empty conduits; group C had a nerve autograft and in group D, the nerve was severed but left untreated. Groups B to D were used as controls. In groups A, B and D there was a tissue bridge between the nerve stumps and regenerating axons grew in mini-fascicles. Transmission electron micrographs of 10µm tissue sections showed that in group A the mini-fascicles were surrounded by connective tissue and were well irrigated by blood vessels. Immune labeling of S-100 positive Schwann cells and neuro-filament positive axons was used to quantify the axonal regrowth between the nerve stumps, which was found to be greater in groups A and C with comparable results between these two groups. Re-innervation was found to be significantly higher in the conduits containing BG (group A) compared to groups B and D (125). Another approach to produce neuronal guidance constructs was explored by Kim et al (126) by rolling phosphate glass microfibers (PGfs) into compressed collagen. The efficacy of these conduits was tested in vitro by seeding dorsal root ganglion (DRG) cells harvested from adult Sprague-Dawley rats on the conduits and in vivo by bridging the surgically severed sciatic nerve in these animals. Compared to the controls, the nerve conduits containing PGfs showed increased neurite growth, both in length and number, and faster recovery of motor control in vivo. Histological examination of the composite constructs implanted into the severed sciatic nerve showed axonal extension along the scaffold, whereas the control group did not. Another relevant study Novajra et al. (127) involved phosphate glass hollow fibres filled with a genipin-crosslinked agar/gelatin hydrogel and a neurotrophic factor solution.

The release profile under culture conditions and the effects that dissolution products of the release system had on neonatal olfactory bulb ensheathing cell line (NOBEC) were investigated. Results showed that there were no deleterious effects on cell growth and expression of pro- and anti-apoptotic proteins in in vitro tests and the use of the hydrogel significantly reduced the release rate of the neurotrophic factor from the hollow glass tubes, which translates into an effective and tailorable system for the delivery of growth factors suitable for tissue engineering applications. More recently, chick DRG cells were cultured with 13-93B3 bioactive borate glass (see Table 1 for composition) microfibers embedded into fibrin scaffolds (128). It was found that the BG microfibers guided axonal growth, evidenced by neurite extensions along the fiber axis. It was also observed that the growth rate was comparable to that of pure fibrin scaffolds. Although experiments conducted with glass rods alone showed decreased cell viability due to a possible cytotoxic effect of glass degradation products, possibly related to pH increase in in vitro cultures with a mix of cells (neurons, glia and fibroblasts), it was found that the proportion of live neurons was significantly higher at the end of the experiment in comparison to control dishes. In a study by Koudehi et al. (129) composite nano-bioactive glass/gelatin (BGGC) conduits for peripheral nerve regeneration were developed and implanted in rats with severed sciatic nerves. The contractility of the gastrocnemius muscle was examined at 1, 2 and 3 months post-operatively and compared to normal rats. The BGGC group exhibited statistically equivalent nerve regeneration rates. To compare the adhesion, proliferation and differentiation of SKNBE neuronal cells on BGs, Sabbatini et al. (130) cultured these cells on Zn-doped BGs at concentrations of 5, 10 and 20% and compared them to plain 45S5 Bioglass®. The results showed that there was an increase in adhesion and proliferation of undifferentiated SKNBE neuronal cells on the low concentration Zn-doped BGs, whilst at the highest concentration proliferation was inhibited but genes responsible for the phenotype of differentiated SKNBE cells were up-regulated. The importance of the chemical composition of the BG used was highlighted, which indicates that different bioactive glass compositions may be required depending on whether de-cellularized or seeded scaffolds are used. In this context, the evaluation of Zn2+ and Ca2+ ion release of Si-Na-Ca-Zn-Ce glasses for potential application in nerve guidance conduits has been also carried out (131).

The emerging results discussed here suggest that there is potential for the use of BG fibres in nerve regeneration and that guided axonal growth can be achieved by employing these fibres in nerve conduits. However, improvements must be made with regards to the material choice for the tubular conduct that houses these fibers and the amount and chemical composition of the BG employed. Another important goal is to compare the performance and cellular mechanisms of nerve response to silicate, phosphate and borate glasses in different constructs, e. g. combined or not with a biopolymer, in their ability to regenerate nerves in controlled in-vivo conditions.

6.

Gastrointestinal regeneration

Gastric ulcers are caused by a range of factors including bacterial infection and excessive stomach acid production and are commonly treated with drugs such as Omeprazole and Hydrotalcite (132,133). However, given the long recovery time necessary for healing gastric ulcers, there is an increased risk of side effects and toxicity arising from prolonged drug exposure. Very recently, 45S5 Bioglass® has been considered in the context of gastric ulcer healing. To investigate the protective effects of 45S5 Bioglass®, Ma et al. (134) carried out a series of experiments in rats. In the first study, a single oral dose of 45S5 Bioglass® powder (particle size average of 20µm), Omeprazole and Hydrotalcyte was administered to the animals prior to subjecting them to water immersion stress for 15 hours, which produces gastric erosions. These single dosages proved to have a protective effect against gastric ulcers since all treated groups showed inhibited appearance of these lesions in a dose dependent manner. Chronic ulcers induced in another group were treated with 45S5 Bioglass®, Omeprazole and Hydrotalcyte and it was found that single-daily and multiple-daily doses increased gastric ulcer healing and protected rats from recurring ulcer flare-ups, also in a dose dependent manner. To test whether the Bioglass® was orally absorbed, rats were fed 3mg/kg/day for 7 days and plasma from blood samples from days 1 and 7 was measured for Si content. There were no significant changes to the amount of Si present in circulation after ingestion for 7 days. This result indicated that the Bioglass® particles are not orally absorbable and provided similar ulcer healing rates as two commonly used drugs with the important advantage of providing a protective effect even after a single dose.

The application of bioactive glass in intestinal tissue regeneration has been also considered (135). The intestinal lumen is a highly dynamic environment, where due to transit of food and chemical erosion from substances involved in digestion there is a rapid turnover of epithelial cells (136). When the superficial layer of the intestine is damaged, leading to superficial wounds, the recovery rate depends on the migration of intestinal epithelial cells to cover the wounded area and a fast cell migration rate reduces the chances of further damage to the tissue. Moosvi et al. (137) tested the effect of 45S5 Bioglass® (mean particle size of 2µm) in epithelial cell restitution by carrying out a series of intestinal epithelial cell and myofibroblast cultures and co-cultures in the presence of Bioglass® particles or conditioned media. A wound model was investigated where a monolayer of intestinal epithelial cells was damaged using a razor blade and the cell migration across the wound bed was observed by measuring the area re-covered by cells after 24h. It was found that whilst culture of wounded epithelial cells in medium conditioned with 0.01 and 0.1 w/v% Bioglass® showed no significant increase in cell migration, 1 w/v% conditioned medium increased cell coverage across the wound area by around 50%. This effect was potentiated when the medium was conditioned by Bioglass® and myofibroblasts and it was most evident with a glass concentration of 0.1 w/v%. In addition, the direct co-culture of myofibroblasts and intestinal epithelial cells in the presence of Bioglass® particles at this concentration yielded the highest wound area recovery rate. Total cell number assays demonstrated that there were no significant changes in the number of viable epithelial cells cultured with medium conditioned by myofibroblast and Bioglass® particles, and thus the increased wound cover was attributed to increased cell migration rate and not to proliferation. The study provided evidence that 45S5 Bioglass® can increase epithelial cell migration in vitro, and thus potentially it could help accelerate intestinal epithelium restitution. The application of 45S5 Bioglass® particles added to PLGA matrices in the construction of tubular scaffolds for intestinal regeneration has been also investigated (135). It was found that the scaffolds possessed satisfactory mechanical properties for this application. Pre-conditioned medium obtained from incubation of tubes with 1 wt% Bioglass® particles in cell culture medium (DMEM supplemented with 2 mM glutamine and 1% penicillin and streptomycin) was used to culture mouse fibroblast cells. There was a considerable decrease in cell population compared to controls, on average 32%. However, the morphology of the tubes, with diameters in the range from 1.5 to 3 mm and interconnected porosity was reported to make

them interesting options not only for gastrointestinal applications, but also other applications where tissue-like tubular structures are required. The experimental results indicated that critical combination of bioactive glass particles and biopolymer matrices with a tubular shape could be used for regeneration of tubular-shaped organs. A biodegradable mesoporous structure of BG particles to deliver biomolecules (101,138) would be an ideal, medium for incorporating a local drug delivery function to the scaffold (Fig. 3). Such highly biodegradable, multi-functional 3-D mesoporous BG architectures could be used to ‘tune’ the composite scaffold degradation behavior to synchronize it with the specific cellular changes taking place in soft tissue regeneration.

7.

Urinary tract

Urinary incontinence and vesico-urethral reflux result from insufficient urethral resistance and are a major health complication in aged patients leading to a very poor quality of life and great expense in later years. Some approaches to tackle these conditions have used injection of diverse materials into the bladder neck to increase continence and to avoid the risks associated with open surgery (139). Polytetrafluoroethylene (Teflon) or collagen are commonly used materials in these cases, but there are risks associated with particle migration and loss of volume with time which compromises safety and effectiveness. Stimulated by the seminal discovery of Wilson et al. (65) of strong, stable bonding of soft connective tissues to bulk and powder 45S5 BG samples, Walker et al. (140) began investigating BGs as alternative to the non-adherent bio-inert particle suspensions for treatment of incontinence. Suspensions of 45S5 and 45S5F (composition (wt%): 45% SiO2 5.5% Na2O, 12.25% CaO, 6% P2O5 and 12.25% CaF2) bioactive glasses in sodium hyaluronate were tested as urethral bulking agents in two experiments. The first experiment involved injecting 0.1ml of the BG suspensions in the bladder dome of 12 rabbits and analyzing tissue samples from 2 to 12 weeks for inflammatory reaction or particle migration. Histological examination of bladder samples at the injection site showed that the particulates were surrounded by collagen fibers and connective tissue. No inflammatory response was detected for any of the BGs used. Importantly, chemical analysis did not detect any increase in calcium or silicon content in lung, liver, kidney and lymph nodes. The second Walker and Wilson experiment involved injecting 1ml of 45S5 Bioglass® suspension into each of the 4

quadrants of the bladder neck of 4 pigs and measuring the urethral resistance pre and post operatively with urethral pressure profiles. Three months after the injection, there was an increased maximal urethral pressure indicating that there was an increase in urethral resistance in all animals; however, the significance of the experiments could not be tested given the small size of the test group. Histological examination of injection site samples indicated that all BG particles were well integrated into the surrounding connective tissue and there was no evidence of an inflammatory reaction. The need for a safe and injectable material that is bioactive with strong, stable bonds to the surrounding tissues is evident, and the study (140) showed that BGs can fulfill both these criteria. Further research in this area is required, which should involve in vivo tests in a functional animal model with a sizeable group for statistically significant assessment of the efficacy of this mode of treatment.

8.

Lung tissue engineering

Adult lung tissue exhibits poor regenerative capabilities and in cases where there is extensive damage, for example with diseases like cystic fibrosis or COPD, the only option for patients is a full heart-lung transplant (141), a costly and complicated solution with relatively low survivability rates. Tissue engineering of the lung is being explored using various biomaterials (142–144). BGs have been also considered in this application. Verrier et al. (145) produced composite foams based on poly(DL-lactic acid) (PDLLA) and 45S5 Bioglass® (with a mean particle size <5µm) exhibiting interconnected pores. The foams were seeded with human lung-derived immortal cells to assess their suitability as tissue engineering matrices. In vitro tests indicated that cell adhesion and proliferation were strongly dependent on the Bioglass® content of the foams. The highest cell proliferation occurred in scaffolds containing 5 wt% Bioglass® and cell adhesion increased with an increase in Bioglass® particle content. All scaffolds studied showed full cell penetration, even in the absence of Bioglass® particles. In a study by Tan et al. (146), sol-gel derived BG foams with surface modifications to include amine or mercaptan groups and/or coated with laminin were manufactured and placed in culture with murine lung epithelial cells (MLE-12) to determine the best conditions to promote growth and proliferation. Based on histologic examination of the cell cultures, there was full penetration of the foams by the lung cells and it was shown that the laminin

coated, amine modified foams were most effective in promoting cell growth and attachment. These results seem to indicate the possibility to use BGs in lung tissue engineering approaches although extensive work, including testing with the different cell types found in this complex tissue, are necessary for further advancements. At present the role of the bioactive phase in promoting lung cell proliferation and/or differentiation is as yet unknown. Basic cell and molecular biology studies of potential for bioactive stimulation of type 2 pneumocytes for lung tissue regeneration are still to be done and considerable research efforts selecting appropriate glass compositions and morphologies are needed in order to realistically consider bioactive glasses in this particular field.

9. Laryngeal repair Patients with problems secondary to paralysis or surgery of a cancerous vocal cord have a week, breathy voice. Most important, such patients may have problems due to inefficient air use and potential for liquid aspiration. Effective treatment is movement of the paralyzed cord so it approaches the normal cord sufficiently to improve speech and prevent aspiration down the airwaves. Previous clinical solutions have used an injectable form of PTFE or surgical insertion of silicone rubber. Serious concerns with these treatments include deterioration of the voice and potential release of dangerous emboli. The complication of extrusion into the airway is a particular concern. A material which does not extrude into the airway and which permits some degree of customization of the repair and long term stability is needed. Bioactive glass particulate (45S5 composition) was investigated for vocal cord medialization because of its proven ability to bond to soft tissues and absence of migration or adverse effects due to strong stable bonding to collagen, as reviewed recently by Hench and Greenspan (17). Their review presented an investigation of 45S5 Bioglass® particulate in a canine model conducted by the team of June Wilson, WH Slattery and M. Crary (University of Florida). The implantation of bioactive glass particulate improved bark characteristics over those identified for the paralyzed vocal cord. The characteristics of the dog bark post implantation and repair are similar to the normal larynx suggesting increased laryngeal valving during phonation. The histological analysis showed that the particulate was in place

between the thyroid cartilage and the vocal cord. Most of the particulate was contained in adherent soft tissue. In several areas bony metaplasia, which is characteristic of the normal repair of this type of cartilage, has enclosed bioactive glass in bone and cartilage as well. The conclusion from this study is that implanted bioactive glass powder can be used to produce sufficient medialization of a paralyzed vocal cord to produce improvement in phonation. The tissue augmentation which produces this effect is the combined effect of the immobilization of bioactive glass by the combination of hard and soft tissue in the bony metaplasia which is part of the normal cartilaginous repair process. The study provided an example of the importance of collagen-bioactive glass adherence and the long-term stability of such a bonded interface in a functioning animal model (17). Optimization of the bioactive glass particulate delivery system to ensure accurate placement of the ideal quantity and size of particulate is still to be studied.

10. Stabilization of Percutaneous Devices Catheters are widely used in healthcare, especially for aged patients, such as peritoneal dialysis catheters and indwelling catheters for treatment of urinary incontinence. Catheters typically develop a thin, non-adherent fibrous capsule around the device and thereby lack a mechanical barrier to periluminal bacterial migration and need cuffs for anchorage. The space between the device and the interposed fibrous layer can provide a pathway for spread of infections from the percutaneous site with serious consequence to the patient. Continual monitoring and maintenance of catheters is a growing burden to the healthcare community and innovative approaches to solving this problem are greatly needed. Prior technology developed by Marotta et al. (147), showed that it is possible to produce stable, adherent bioactive glass coatings on silicone catheters and other silicone medical devices and achieve collagen bonding to the bioactive surface that can stabilize the devices in soft tissues. Feasibility for use of the bioactive coatings on silicone for tissue adhesion of peritoneal dialysis catheters was reported by Ross et al in 2003 (148). Segments of 45S5 bioactive glass coated silicone tubes, 9 French size, 2.5 cm long, were implanted in rats with un-coated tubes as controls. Histology of the tissue-implant interfaces was conducted at 2, 4 and 6 weeks using special stains and electron microscopy. Results showed that “un-coated tubes had no adherence to the surrounding tissues and had a physical separation of approximately

50 micrometers width comprised primarily of acellular collagen. In contrast, the bioactive glass coated tubes were palpably fixed to the soft tissues” (148). The authors summarized that the BG coatings improved tissue retention of silicone tubing as they promoted adhesion by collagen and cell proliferation thus suggesting that BG coatings are promising for applications in peritoneal dialysis catheters. This is an area of soft tissue engineering that should be pursued aggressively in the future to achieve rapid and stable interfacial bonding of catheters due to the increasing clinical need. 11. Discussion 11.1 Bonding mechanism between BGs and soft tissues The mechanism of bonding between bioactive glasses and tissues is thought to be governed by the time dependent dissolution and precipitation reactions that occur at the implant interface upon contact with physiological fluids. These reactions cause a local increase in the concentration of various ions at the BG surface, resulting in an overall increase in pH and the formation of a carbonated HA (HCA) layer. This crystalline layer provides the bonding interface between the implant and the aforementioned tissues and, as mentioned in the Introduction, has been the key aspect investigated over the years to rationalize the bone bonding ability of bioactive glasses [4, 8, 12]. It is now well accepted that there are two kinds of interactions that occur in the body upon implantation of BGs [19]: 1. Extracellular interactions that are determined by the specific surface characteristics of the material which in turn govern protein adsorption and subsequent cell attachment. 2. Intracellular interactions between the protein ligands formed on the implant surface and cell receptors that determine the degree of cellular adhesion, differentiation and proliferation. These two types of interaction are affected by the availability of different ions. For example, HA is known to attract macrophages in wounded tissues, evidenced in a study of wound healing in a rabbit model (109); and local delivery of calcium can enhance tissue regeneration in wounded mice (110). In addition, it is thought that the presence of silicon improves regenerated tissue quality, as evidenced by reduced formation of exhuberant

granulation tissue in a wound healing study in a horse model by Ducharme-Dejarlais et al. (149). Additionally, it has been shown that the collagenous constituents of soft tissues are able to attach to the surface of BGs, although it is worth noting that the degree of bonding and rate of regeneration are dependent on the progenitor cell populations available in the surrounding tissues (150), this being precisely the area requiring further research efforts due to the patchy current knowledge. Nevertheless the available data suggest that BGs enhance tissue regeneration and stimulate tissue attachment to the implant surface and that this effect is highly dependent on the glass ion releasing functionality and on biomechanical effects. 11.2 Bio-mechanical factors in soft tissue engineering Some of the clinical applications of bioactive glasses in contact with soft tissues have two primary requirements for success: 1) Rapid formation of an interfacial bond with collagen, and 2) Stable long term interfacial bonding that prevents micro-motion at the interface and onset of an inflammatory response. In addition, long term stability of the implant bonded interface has another requirement, namely: 3) A gradient in stress transfer across the interface that avoids cell signaling to resorb either tissues or the implant. The excellent long term clinical success of 45S5 bioactive glass devices for replacement of the bones of the middle ear (MEPs) and implants for maintenance of edentulous jaws (ERMIs), reviewed by Hench and Greenspan (17), are attributed to satisfying all three of the above requirements for both bone as well as soft connective tissues. Rapid formation of the interfacial bonding layers, within weeks, and fast regeneration of new bone at the implantbone interface makes BG implants stable and ensures proper stress transfer to the stapes footplate or alveolar bone to prevent onset of bone resorption. Stable bonding of the implant to soft tissues prevents extrusion of the ossicular replacements through the tympanic membrane or exfoliation through gingival tissues. A study in dogs conducted by Wilson et al. (151) using the same ERMI implants, burs and protocol as for humans, made it

possible to achieve a quantitative histo-morphometric analysis of the hard and soft tissue bonding interfaces of the ERMIs. Within 3 months the bonding had stabilized for both hard and soft tissues. The soft tissue was bonded by collagen fibers interdigitated within a 150 to 400 micrometers thick bonding gel layer composed of biological hydroxyl carbonate apatite (HCA) and an underlying silica-rich gel layer that began to form on the implants within minutes of implantation. An important finding in the Wilson et al. study was that the thickness of the soft tissue bonding layer (Fig. 6A) was nearly 50% thicker than the bonding zone between bone and the implant (Fig. 6B). The thickness of both bone and soft tissue interfaces was stable after about 7 months, as shown in Fig. 6 (151). Finite element modelling of the bioactive glass tissue interfaces by Weinstein et al. (152) provided important insight as to the effect of elastic modulus mismatch on the success of implants bonded to either hard of soft tissues. Figure 6A, upper right, plots the gradient of stiffness (Young’s modulus) between a soft tissue interface and a bulk bioactive glass implant. The difference in modulus is spread over a substantial thickness of interface due to the elastically compliant hydrated silica-gel, hydroxyl-carbonate apatite (HCA) layer formed on the bioactive glass that is several hundreds of micrometers thick. The rapid formation of the bonding layer as well as the elastic compliance (low stiffness) of this bonding zone and the favorable stress transfer resulting from the bonding of collagen fibrils is concluded to be responsible for both short and long term success of the 45S5 Bioglass ERMIs (151,152). In contrast, synthetic bioceramic HA implants bond much more slowly and form a bond only to bone with a thickness of bone-implant bond of less than 1 micrometer. This thin bonding interface of HA implants results in a much greater, factor of >400X, gradient of elastic modulus between the living and non-living materials. The unnatural gradient in elastic modulus of HA implants results in large stress gradients to the bone and stimulates resorption and failures of the alveolar ridge or maxillofacial and cranial bones, as described in the literature (17). The bio-mechanical behavior of clinical applications that involve interfacial bonding of bioactive glass particulates or fibres to soft tissues or in situ regeneration of soft tissues, as reviewed above, is unlikely to be as important as for bulk bioactive implants. However, it is important that a systematic study be conducted to investigate the effect of various volume fractions, size and shape of bioactive glass particles and fibres on elastic properties of

augmented and regenerated soft tissues that contain bioactive phases. Long term cyclic fatigue effects that can lead to particle-tissue interfacial breakdown must be avoided for long term survivability of such clinical applications.

12. Conclusions The range of applications for BGs in contact with soft tissues is vast, and the specific biochemistry of BGs in relevant environments both in-vitro and in-vivo has been shown to stimulate regenerative processes such as wound healing and angiogenesis. Further investigations of the specific molecular biological mechanisms involved in the regulation of these processes and how they are affected by the ionic products that result from the dissolution of BGs when implanted into soft tissue are necessary to fully unlock the potential that these inorganic materials offer to regenerative approaches in tissues other than hard (bone, teeth) tissues. Fundamental knowledge of the mechanisms involved should also lead to strategies in which the addition of BGs will complement the application of growth factors or other biomolecules, possibly reducing the reliance on such molecules in tissue engineering. It is clear from the results of the investigations discussed in this review that there is a huge potential for applications of BGs in soft tissue engineering and wound healing, however there are significant research needs to be carried out in order to establish the specific mechanisms involved in the interaction of BGs and the wide range of cells that compose different soft tissues in the body, just as it has been carried out extensively for ‘hard’ tissue applications. It is crucial to develop constructs intended for tissue engineering applications in a manner that they will support cell attachment and proliferation, present suitable mechanical properties and have degradation rates that match recovery rates of the host tissue, as well as being available in a manner that appeals to surgeons and other clinical staff directly involved in their possible use. Research in this area needs to move beyond current investigations involving co-culture of relevant cells with BG particles and towards incorporation of these materials into 3D matrices suited to the function and morphology of specific soft tissues, considering the hierarchical structure and complexity of tissues and their nano-topography and cell cytokine-bioactive material signaling. Biomechanical aspects specifically related to the bonding mechanisms of BGs to soft tissue which should lead to

improved interface behavior (e.g. via collagen graded structures) must be further quantified by considering additional in-vivo tests and to quantify the effect of ion dissolution involving different chemistries (most studies in this area have been conducted using the 45S5 BG composition). Addition of biochemical cues such as doping ions, growth factors or antibiotics that may potentiate and support tissue renewal will be also relevant for new regeneration strategies. The addition of BGs in different forms, namely micro- and nanoparticles, fibres, mesoporous structures and hybrid inorganic-organic matrices is bound to expand in a number of applications, from vascularization to wound healing and potentially even organ regeneration and repair. These approaches will help move towards a soft tissue engineering construct that takes full advantage of the ideal physiological conditions in the body whilst maximizing the regenerative potential of soft tissues.

ACKNOWLEDGEMENT V.M. P. and A. R. B. acknowledge financial support from EU ITN Project BIOBONE.

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FIGURE CAPTIONS Figure 1: Schematic diagram indicating emerging BG applications in soft tissue regeneration Figure 2: Schematic diagram depicting the experiment discussed in ref. [79] including the image of PGA/45S5 Bioglass®-composite scaffold 42 days after implantation in rat. The arrows denote newly-formed vasculature. Reproduced with permission of Elsevier (TBC). Figure 3: a) SEM image of a PGS-BG composite developed for cardiac tissue engineering (Courtesy Dr R. Rai, Erlangen); b) schematic diagram showing biopolymer matrix incorporating drug loaded mesoporous bioactive glass particles with dual therapeutic ion and drug delivery ability. Figure 4: Treatment of a skin injury in a male New Zealand rabbit with n-BGS (A) and BGS (B) showing the haemostatic action of BGs (112). Reproduced with permission, TBC) Figure 5: BG treated and untreated wounds from day 0-16 according to Lin et al. (114). Wounds were treated with different types of BGs, as indicated. It can be appreciated that SGBG-58S led to fastest healing rates (114) (Reproduced with permission, TBC) Figure 6: Development of reaction layers on Bioglass® ERMI ridge maintenance implants for (A) soft tissue interface and (B) bone interface. Inserts are the calculated elastic modulus gradients of the (A) implant–soft tissue interface and (B) bone–implant interface. The figure is based upon results of Wilson et al. (151) and Weinstein et al. (152) (Reproduced with permission of J. Austr. Ceram. Soc.)

Table1 – Examples of some BG compositions investigated in laboratories worldwide (1. 4. 11-15) Composition (wt.%) 45S5 13-93 13-93B3

58S

Na2O

0.00 23.00

24.50

S53P4 52S4.6 45S5F S53P4 46S6

6.00

5.50

K2O

0.00 12.00

11.10

0.00

0.00

0.00

0.00

0.00

0.00

MgO

0.00

4.60

0.00

0.00

0.00

0.00

0.00

0.00

5.00

21.61 24.50 23.00 24.00

CaO

24.50 20.00

18.50 32.60 20.00

21.64 12.25 20.00 24.00

SiO2

45.00 53.00

0.00 58.20 53.00

50.76 45.00 53.00 46.00

P2O5

6.00

4.00

3.70

9.20

4.00

5.99

6.00

4.00

6.00

B2O3

0.00

0.00

56.60

0.00

0.00

0.00

0.00

0.00

0.00

CaF₂

0.00

0.00

0.00

0.00

0.00

0.00 12.25

0.00

0.00

FIGURE 1

Laryngeal repair Cardiac tissue regeneration

Lung tissue repair

Nerve regeneration

Gastrointestinal regeneration

Urinary tract repair

Vascularization

Wound healing/ dressing

FIGURE 2

PGA mesh

BG particles (in slurry)

Coated mesh

FIGURE 3

Polymeric matrix

Bioactive mesoporous glass granule

Biologically active molecules

Ions

Mesopore

FIGURE 4

Wound

Wound BGS

n-BGS

Fur

Fur

FIGURE 5

Figure 6

Graphical Abstract

Laryngeal repair Cardiac tissue regeneration

Lung tissue repair

Nerve regeneration

Gastrointestinal regeneration

Urinary tract repair

Vascularization

Wound healing/ dressing