**3. Clinical relevance of doped bioactive glass**

Bioactive glasses are that bone substitutes which posses3. Clinical relevance of doped bioactive glasss the unique property of osteoconduction as well as osteoproduction by stimulating proliferation and differentiation of osteoprogenitor cells through a direct genetic control [24, 227]. The discovery of these new materials led Hench and Wilson to propose the concept of osteostimulation or osteopromotion to define this class of bioactive materials and their effects on the genetic activation of bone cells [228]. Bioactive glasses are surface reactive biomaterials that, when in contact with physiological fluids, release soluble ionic products that have been suggested to stimulate *in vitro* osteogenesis [227, 229]. On critical analysis, Young's modulus of bioactive glass being between 30 and 50 GPa, nearly that of natural bone, is a great advant‐ age. One disadvantage is the low mechanical strength and decreased fracture resistance [230]. This can be easily overcome by altering the composition, using it in low load-bearing areas, and using it for the bioactive stage. Furthermore, bioactive glass manufactured via the sol-gel technique permits the synthesis of material with higher purity and homogeneity at low temperatures [52]. Additives can be easily introduced during the sol-gel process to improve the bioactivity of such glasses. Indeed, improvement of the biological properties of bioactive materials can be achieved by the incorporation of ions (doping) that positively affect osteoblast behaviour and consequently enhance new bone formation [202].

In addition, *in vivo* studies have demonstrated beneficial results from their use in various clinical situations [231–234]. After implantation, interaction with surrounding tissues results in a time-dependent alteration of the material's surface and the formation of a hydroxyl carbonate apatite layer that is very similar to the mineral phase of bone [235]. More recently, a new category of sol-gel glasses has been manufactured with enhanced bioactivity and open pores enclosed in a mesoporous matrix [134, 236]. Furthermore, bioactive glass manufactured via the sol-gel technique permits the synthesis of materials with higher purity and homoge‐ neity at low temperatures [52]. Additives can be easily introduced during the sol-gel process to improve the bioactivity of such glasses. Indeed, improvement of the biological properties of bioactive materials can be achieved by the incorporation of ions that positively affect osteoblast behaviour and consequently enhance *de novo* bone formation.

Metallic ions in body play a crucial role as cofactors of enzymes and excite a chain of reactions related to cell signalling pathways [176]. A number of literatures have been cited on the interaction of metallic ions in various diseases and metabolic disorders such as cancer, CNS disorders, infectious diseases and hormonal disorders [237, 238]. Hence, the effectiveness and selectivity of the beneficial effect of metallic ions can be enhanced by controlling the exact level in the body. Additionally, due to unstable ionic states of certain metallic ions, toxic effects may follow while directly ingested. Hence, wide spread research is underway to develop matrices to control the local release of metallic ions with less systemic toxicity as well as availability of relatively high concentrations of metallic-ion-based drugs to target tissues. The degree of metallic ion loading into matrices for local delivery as well as their controlled and sustained release is of paramount importance for optimal therapeutic use. Common strategy is to load metallic ions into matrices such as hydroxyapatite, bioactive glass, silica and carbon fibres to improve ionic stability and to release for a prolong period of time [148, 239–248]. Due to these superior characteristics, metallic ion doping in biomaterials is an alternative, cost-effective, safe strategy than use of recombinant proteins or genetic engineering approaches [249].

#### **3.1. Doped bioactive glass in bone regeneration**

227]. The discovery of these new materials led Hench and Wilson to propose the concept of osteostimulation or osteopromotion to define this class of bioactive materials and their effects on the genetic activation of bone cells [228]. Bioactive glasses are surface reactive biomaterials that, when in contact with physiological fluids, release soluble ionic products that have been suggested to stimulate *in vitro* osteogenesis [227, 229]. On critical analysis, Young's modulus of bioactive glass being between 30 and 50 GPa, nearly that of natural bone, is a great advant‐ age. One disadvantage is the low mechanical strength and decreased fracture resistance [230]. This can be easily overcome by altering the composition, using it in low load-bearing areas, and using it for the bioactive stage. Furthermore, bioactive glass manufactured via the sol-gel technique permits the synthesis of material with higher purity and homogeneity at low temperatures [52]. Additives can be easily introduced during the sol-gel process to improve the bioactivity of such glasses. Indeed, improvement of the biological properties of bioactive materials can be achieved by the incorporation of ions (doping) that positively affect osteoblast

In addition, *in vivo* studies have demonstrated beneficial results from their use in various clinical situations [231–234]. After implantation, interaction with surrounding tissues results in a time-dependent alteration of the material's surface and the formation of a hydroxyl carbonate apatite layer that is very similar to the mineral phase of bone [235]. More recently, a new category of sol-gel glasses has been manufactured with enhanced bioactivity and open pores enclosed in a mesoporous matrix [134, 236]. Furthermore, bioactive glass manufactured via the sol-gel technique permits the synthesis of materials with higher purity and homoge‐ neity at low temperatures [52]. Additives can be easily introduced during the sol-gel process to improve the bioactivity of such glasses. Indeed, improvement of the biological properties of bioactive materials can be achieved by the incorporation of ions that positively affect

Metallic ions in body play a crucial role as cofactors of enzymes and excite a chain of reactions related to cell signalling pathways [176]. A number of literatures have been cited on the interaction of metallic ions in various diseases and metabolic disorders such as cancer, CNS disorders, infectious diseases and hormonal disorders [237, 238]. Hence, the effectiveness and selectivity of the beneficial effect of metallic ions can be enhanced by controlling the exact level in the body. Additionally, due to unstable ionic states of certain metallic ions, toxic effects may follow while directly ingested. Hence, wide spread research is underway to develop matrices to control the local release of metallic ions with less systemic toxicity as well as availability of relatively high concentrations of metallic-ion-based drugs to target tissues. The degree of metallic ion loading into matrices for local delivery as well as their controlled and sustained release is of paramount importance for optimal therapeutic use. Common strategy is to load metallic ions into matrices such as hydroxyapatite, bioactive glass, silica and carbon fibres to improve ionic stability and to release for a prolong period of time [148, 239–248]. Due to these superior characteristics, metallic ion doping in biomaterials is an alternative, cost-effective, safe strategy than use of recombinant proteins or genetic engineering approaches [249].

behaviour and consequently enhance new bone formation [202].

298 Advanced Techniques in Bone Regeneration

osteoblast behaviour and consequently enhance *de novo* bone formation.

In bone tissue engineering, bioceramics or bioactive glasses and biodegradable polymers [15], often comprise metallic ions as part of the bioceramic or bioactive glass structural composition. The metal ion is generally released during their degradation *in vitro* or *in vivo* [148, 250]. For instance, when bioactive glass (e.g., 45S5 Bioglass) [26, 251] is used as scaffolds to fill a bone defect, critical concentrations of soluble Si, Ca, P and Na ions are released, with the capacity to generate both intracellular and extracellular effects at the interface between the glass and the cellular environment [124, 133, 148, 227, 252–261]. It has also been observed that released ions from bioactive glasses can induce gene expression which in turn helps in bone metabolism by signal transduction as well as enhance cell differentiation and osteogenesis [27, 124, 227, 254]. Furthermore, the ionic dissolution products of bioactive glasses can also encourage angiogenesis [262]. Other metallic ions which may have significant role in bone tissue engi‐ neering include copper, magnesium, strontium, manganese, iron, zinc and silver owing to their imminent role as cofactors in metabolic processes in bone, articular tissues and immune system functions [149, 263].

The application of chitosan-doped bioactive glass (BG-CH) was assessed in the guided bone regeneration in ovariectomised rats. The histomorphometric analysis showed increased bone/ tissue volume, osteoblast number and osteoblast surface/bone surface and trace elements such as Sr and Fe were detected in the newly formed bone may be responsible for enhanced bone healing and found clinically useful as a therapeutic and implantable material [264].

Zinc being a trace mineral in human body performs a variety of functions in relation to the immune system, cell division, fertility and the body growth and maintenance. Moreover, zinc is also a necessary element for the formation, mineralisation, development and maintenance of healthy bones. These unique properties of zinc evoked the interest of researchers to use it along with silicate-based bioactive glasses for bone tissue engineering and found to have significant ability to enhance antibacterial effects, bioactivity and distinct physical, structural and mechanical properties of bioactive glasses [265]. Zinc also stimulates bone formation and mineralisation by activating aminoacyl-tRNA synthetase in osteoblastic cells, and it stimulates cellular protein synthesis. Zinc plays a role in the preservation of bone mass by inhibiting osteoclast-like cell formation from marrow cells [171]. It also promotes attachment, prolifera‐ tion of osteoblast and increase ALP expression that is responsible for laying down the bone callus. The doping of Zn into bioactive glasses produces higher chemical stability and densi‐ fication of glasses matrices. Zinc doping in bioglass for repair of diaphyseal defect creates a good link of interface between bone and Zn-BG during the first speeds, whereas during the last speeds osseoingration, resorption and degradation of bioactive glass and their replacement by bone cells occurs [266].

Strontium (Sr) is a naturally occurring trace element often acts similarly to Ca in the human body; both have strong bone-seeking properties, and Sr can be substituted with Ca in the apatitic phase of bone mineral [267]. Administration of Sr in moderate doses prevented caries in rats [268]. Among the trace metals present in human bone, Sr was the only that was correlated with bone compression strength [198]. Furthermore, over the past few years, Sr has attracted attention through its beneficial effects on bone healing. Indeed, both *in vitro* and *in* *vivo* studies have demonstrated stimulatory effects of Sr on osteoblasts and an inhibitory effect on osteoclasts, associated with an increase in bone density and resistance [199, 269–271]. Nowadays, strontium ranelate is used as a commercial antiosteoporotic oral drug that has been proven to reduce the incidence of fractures in osteoporotic patients [196, 272]. Addition of strontium-substituted bioactive glass (SrBG) into PCL and fabricating into 3D bioactive composite scaffolds utilising additive manufacturing technology yield higher compressive Young's modulus [273]. Oxidative stress, a pivotal pathological factor inducing bone osteo‐ porosis, can also be reduced by Zn doping of bioglass in overiectomised Wistar rats as Zn significantly enhances superoxide dismutase (SOD), catalase (CAT) and glutathione peroxi‐ dase (GPx) and the Ca/P ratio whereas decreases thiobarbituric acid-reactive substances and thus improves bone mineralisation [274]. The study on effects of the substitution of calcium oxide with Sr on bioactive glass also shows promotion of osteogenesis in a differentiating bone cell culture model using mesenchymal stromal cells obtained from rat bone marrow and proved to be potential for bone tissue regeneration [275]. Sr-doped bioglass implant enhances bone regeneration in patients suffering from osteoporosis [276]. The growing evidence of the beneficial effects of strontium on bone justifies the increasing interest in Sr incorporation into biomaterials for hard tissue repair. Thus, strontium-doped bioactive glasses have been recently developed via a sol-gel method that enables a better control of the reaction kinetics [201, 277].

A multifunctional bioactive scaffold should combine angiogenesis capacity, and osteostimu‐ lation, for regenerating lost bone tissues. To achieve these objectives when copper (Cu) containing mesoporous bioactive glass (Cu-MBG) scaffolds with interconnective large pores are used *in vitro* both Cu-MBG scaffolds and their ionic extracts stimulates hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) expression in human bone marrow stromal cells (hBMSCs). Thus, incorporation of Cu2+ ions into MBG scaffolds increase hypoxia-like tissue reaction which enhance angiogenesis and osteogenesis and has promising scope for the treatment of large bone defect [278]. Controlled delivery of 3 wt% CuO from borate bioactive glass scaffolds implanted in rat calverial defect shows significantly better capacity to stimulate angiogenesis and regenerate bone when compared to the undoped glass scaffolds [279]. It is also evident that copper-doped bioglass scaffold *in vivo* acts on BMSCs ((bone-marrow derived mesenchymal stem cells) to stimulate secretion of VEGF which in turn enhances the angiogenic growth into the scaffolds [280]. Copper (Cu) has the property to stimulate vascularisation/angiogenesis and silicate bioceramics have also stimulatory effects on vascularisation *in vitro* due to the release of silicon (Si) ions. Hence, when combined in bioceramic implant Cu and Si have synergistic effects [281].

Biomaterial-centred bacterial infection, one of the major reasons for revision surgery [282], led the researchers to explore such material that could control infection as well as promote bone healing. Incorporation of silver oxide (Ag2O) proved its promising future to combat against microbial infection on biomedical materials and devices [241, 242, 283–285]. The introduction of Ag2O into the bioactive glass shows promising bactericidal efficacy against *Escherichia coli, P. aeruginosa* and *S. aureus in vitro* by leaching of Ag+ ions from bioglass matrix [223, 286–288]. Doping of Ag+ ions in 45S5 bioglass based scaffolds even proves to be effective against MRSA (methicillin-resistant *S. aureus*) *in vitro* [289]. Silver-doped bioactive gel-glass Ag-S70C30 has beneficial role as antimicrobial wound healing agent in inflammatory response in a local body compartment such as in acne lesions and in non-healing dermal wounds as it has no cytotox‐ icity against human epidermal keratinocytes [290]. Mesoporous bioactive glasses doped with Ti/Ag have improved hydroxyapatite- (HAP) induced growth and antimicrobial properties and more potency than pure MBGs in bone-tissue regeneration and surgery [291]. Very recently, scaffolds of a borosilicate bioactive glass (composition: 6Na2O, 8K2O, 8MgO, 22CaO, 36B2O3, 18SiO2, 2P2O5; mol%) doped with varying amounts of Ag2O (0.05, 0.5 and 1.0 wt%) is being used for bone defect repair and as well as to control infection caused by *E. coli* and *S. aureus*. Better adhesion, proliferation and alkaline phosphatase activity of murine osteoblastic MC3T3-E1 cells on the Ag2O-doped bioactive glass scaffolds is found than on the undoped scaffolds *in vitro* [292].

*vivo* studies have demonstrated stimulatory effects of Sr on osteoblasts and an inhibitory effect on osteoclasts, associated with an increase in bone density and resistance [199, 269–271]. Nowadays, strontium ranelate is used as a commercial antiosteoporotic oral drug that has been proven to reduce the incidence of fractures in osteoporotic patients [196, 272]. Addition of strontium-substituted bioactive glass (SrBG) into PCL and fabricating into 3D bioactive composite scaffolds utilising additive manufacturing technology yield higher compressive Young's modulus [273]. Oxidative stress, a pivotal pathological factor inducing bone osteo‐ porosis, can also be reduced by Zn doping of bioglass in overiectomised Wistar rats as Zn significantly enhances superoxide dismutase (SOD), catalase (CAT) and glutathione peroxi‐ dase (GPx) and the Ca/P ratio whereas decreases thiobarbituric acid-reactive substances and thus improves bone mineralisation [274]. The study on effects of the substitution of calcium oxide with Sr on bioactive glass also shows promotion of osteogenesis in a differentiating bone cell culture model using mesenchymal stromal cells obtained from rat bone marrow and proved to be potential for bone tissue regeneration [275]. Sr-doped bioglass implant enhances bone regeneration in patients suffering from osteoporosis [276]. The growing evidence of the beneficial effects of strontium on bone justifies the increasing interest in Sr incorporation into biomaterials for hard tissue repair. Thus, strontium-doped bioactive glasses have been recently developed via a sol-gel method that enables a better control of the reaction kinetics [201, 277].

300 Advanced Techniques in Bone Regeneration

A multifunctional bioactive scaffold should combine angiogenesis capacity, and osteostimu‐ lation, for regenerating lost bone tissues. To achieve these objectives when copper (Cu) containing mesoporous bioactive glass (Cu-MBG) scaffolds with interconnective large pores are used *in vitro* both Cu-MBG scaffolds and their ionic extracts stimulates hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) expression in human bone marrow stromal cells (hBMSCs). Thus, incorporation of Cu2+ ions into MBG scaffolds increase hypoxia-like tissue reaction which enhance angiogenesis and osteogenesis and has promising scope for the treatment of large bone defect [278]. Controlled delivery of 3 wt% CuO from borate bioactive glass scaffolds implanted in rat calverial defect shows significantly better capacity to stimulate angiogenesis and regenerate bone when compared to the undoped glass scaffolds [279]. It is also evident that copper-doped bioglass scaffold *in vivo* acts on BMSCs ((bone-marrow derived mesenchymal stem cells) to stimulate secretion of VEGF which in turn enhances the angiogenic growth into the scaffolds [280]. Copper (Cu) has the property to stimulate vascularisation/angiogenesis and silicate bioceramics have also stimulatory effects on vascularisation *in vitro* due to the release of silicon (Si) ions. Hence, when combined in

Biomaterial-centred bacterial infection, one of the major reasons for revision surgery [282], led the researchers to explore such material that could control infection as well as promote bone healing. Incorporation of silver oxide (Ag2O) proved its promising future to combat against microbial infection on biomedical materials and devices [241, 242, 283–285]. The introduction of Ag2O into the bioactive glass shows promising bactericidal efficacy against *Escherichia coli, P. aeruginosa* and *S. aureus in vitro* by leaching of Ag+ ions from bioglass matrix [223, 286–288]. Doping of Ag+ ions in 45S5 bioglass based scaffolds even proves to be effective against MRSA (methicillin-resistant *S. aureus*) *in vitro* [289]. Silver-doped bioactive gel-glass Ag-S70C30 has

bioceramic implant Cu and Si have synergistic effects [281].

Wnt pathway has been found to play a central role in controlling embryonic bone development and bone mass [293] during the past decade. In the developing skeletogenesis, Wnt signalling is required for limb bud initiation, early limb patterning, and, finally, late limb morphogenesis events. It has been reported that Wnt-3a and Wnt-7a are expressed in the limb bud and have roles in skeletal pattern determination [294], and that Wnt-14 is involved in joint formation [295]. In addition, Wnt-3a, Wnt-4, Wnt-5a and Wnt-7a all influence cartilage development [295]. Wnt are 39-46 kDa cysteine-rich, secreted glycoproteins that have been identified in organisms ranging from hydra to humans [296]. Recently, it has been suggested that canonical Wnt signalling plays an important role in fracture healing [297]. Lithium (Li) is an element known to mimic the Wnt signalling pathway, which plays a central role in osteoblast prolif‐ eration and differentiation [298]. Expression of various Wnts has been reported to be upregu‐ lated during fracture repair, and increased β-catenin signalling by lithium administration has been shown to improve fracture healing [299]. Edgington et al. reported that lithium-based dopants to β-TCP induced an effect on the cell-material interaction of osteoblast cells as well as the study exhibited increased proliferative activity at the lower concentration of Li-doping, while the higher concentration showed a decrease in activity, indicating a toxic effect of Li at elevated doses *in vitro* [300]. Lithium activates β-catenin signalling by inhibiting GSK3β [301– 303]. It is also reported that lithium enhances bone formation and improves bone mass in mice [304]. Bioactive glasses with Li-containing composition (55% SiO2-36% CaO-4% P2O5-5% Li2O) synthesised through a quick alkali sol-gel process stimulate apatite formation after immersion in SBF. Furthermore, addition of Li enhances chemical durability and antibacterial activity against *Enterococcus faecalis*. Li-doped bioglass has excellent antibacterial property against tooth infections for the treatment of root canal, other dental applications [305]. Researches reveal that different concentrations of Li2O (0-12 wt%) substitution for Na2O in 45S5 bioglass causes *in vitro* more apatite formation and osteoblastic cell responses than non-substituted 45S5 bioglass thus prove its efficacy for bone defect filler [211]. Another study shows that Li doping in therapeutic range (<8.3 ppm) in 45S5 Bioglass causes more HA deposition than nondoped bioglass *in vitro* [306].

There are even some more ions or materials, doping of which positively improve the quality, bioactivity or bone regeneration. Study with boron modified bioactive glass particle shows significantly more thickness of osseointegrated tissue and more area of neoformed bone tissue than non-doped 45S5 glass along with increase in the Ca:P ratio. Boron modification enhances bone formation more than 45S5 glass when implanted into the intramedullary canal of rat tibiae [307]. Modification of bioactive glass by substitution of Na2O with doping of fluorides, such as CaF2 and MgF2 or B2O3 increases its mechanical property [308]. Nickel and cobalt both stimulate the hypoxia-inducible factor-1 (HIF-1a), which significantly improving blood vessel formation in tissue engineering applications. No significant structural differences or dissolu‐ tion rate occur when nickel and cobalt are doped in bioactive glasses [309]. Magnesium-doped melt-derived glasses in the system SiO2-CaO-Na2O-P2O5 influences the formation and the evolution of the newly formed layers, promotes the dissolution of the silica network, increases the thickness of the silica gel layer as well as slows down the crystallisation of the apatite layer [310]. Silica- and phosphate-based bioactive glass nanoparticles (58SiO2-33CaO-9P2O5) doped with neem (*Azadirachta indica*) leaf powder and silver nanoparticles show good antimicrobial activity against *S. aureus* and *E. coli* and less bioactivity compared with silver-doped glass particles [311].

#### **3.2. Doped bioactive glass as coating of orthopaedic implants**

Since the discovery of bioglass it had mainly been used for coating of metallic implant which are bioinert in nature, i.e. bonding ability to bone tissue is poor [312]. On the other hand, bioglass being an excellent osteogenic agent it has also some inherent disadvantages such as poor mechanical properties leading to its limited application in load-bearing implants where metallic alloys are still the materials of choice. Hence, coatings have drawn attention of researchers as a method to improve adherence of bone tissue to metallic alloy to be used as load-bearing implant in orthopaedic surgery. For this purpose, coating material should have thermal coefficient similar to that have bioglass, as well as, has some other properties such as firing cycle during preparation of coating should not degrade the quality of metal and optimum adherence should be achieved with hydroxyapatite formation in contact with body fluid.

To achieve the goal researchers embedded bioglass or hydroxyapatite particles on coating of Ti6Al4V by a simple enamelling technique to enhance their bioactivity and found excellent glass/metal adhesion with well-attached bioactive particles on the surface that can withstand substantial chemical and mechanical stresses [313]. Another family of glasses in the SiO2-Na2O-K2O-CaO-MgO-P2O5 system has been synthesised for coatings on Ti-based and Co-Cr alloys by the scientists, where desired achievement were observed to alloys by formation of 100–200 nm thick interfacial layers (Ti5Si3 on Ti-based alloys and CrO*x* on Co-Cr) and commercially Ti alloy-based dental implants were fabricated with 100 μm thick glass coatings successfully [314]. Surgical suture materials such as absorbable polyglactin 910 and non-resorbable Mersilk when coated with silver-doped bioactive glass powder (AgBG) and tested *in vitro*, after 3 days of immersion in SBF, hydroxyapatite forms on the coated suture surfaces and thus their bioactive behaviour is enhanced as a result their use in body wall repair and wound healing property is also enhanced [243] it also limits bacterial attachment [315]. *In vivo* histologic and histomorphometric study on osteointegration of gradient coatings composed of bioactive glass and nanohydroxyapatite (BG-nHA) on titanium-alloy orthopaedic implants and surrounding bone tissue. Fluorescence micrograph shows better osteointegration of orthopaedic implant in BG-nHA than uncoated implant [316].

than non-doped 45S5 glass along with increase in the Ca:P ratio. Boron modification enhances bone formation more than 45S5 glass when implanted into the intramedullary canal of rat tibiae [307]. Modification of bioactive glass by substitution of Na2O with doping of fluorides, such as CaF2 and MgF2 or B2O3 increases its mechanical property [308]. Nickel and cobalt both stimulate the hypoxia-inducible factor-1 (HIF-1a), which significantly improving blood vessel formation in tissue engineering applications. No significant structural differences or dissolu‐ tion rate occur when nickel and cobalt are doped in bioactive glasses [309]. Magnesium-doped melt-derived glasses in the system SiO2-CaO-Na2O-P2O5 influences the formation and the evolution of the newly formed layers, promotes the dissolution of the silica network, increases the thickness of the silica gel layer as well as slows down the crystallisation of the apatite layer [310]. Silica- and phosphate-based bioactive glass nanoparticles (58SiO2-33CaO-9P2O5) doped with neem (*Azadirachta indica*) leaf powder and silver nanoparticles show good antimicrobial activity against *S. aureus* and *E. coli* and less bioactivity compared with silver-doped glass

Since the discovery of bioglass it had mainly been used for coating of metallic implant which are bioinert in nature, i.e. bonding ability to bone tissue is poor [312]. On the other hand, bioglass being an excellent osteogenic agent it has also some inherent disadvantages such as poor mechanical properties leading to its limited application in load-bearing implants where metallic alloys are still the materials of choice. Hence, coatings have drawn attention of researchers as a method to improve adherence of bone tissue to metallic alloy to be used as load-bearing implant in orthopaedic surgery. For this purpose, coating material should have thermal coefficient similar to that have bioglass, as well as, has some other properties such as firing cycle during preparation of coating should not degrade the quality of metal and optimum adherence should be achieved with hydroxyapatite formation in contact with body

To achieve the goal researchers embedded bioglass or hydroxyapatite particles on coating of Ti6Al4V by a simple enamelling technique to enhance their bioactivity and found excellent glass/metal adhesion with well-attached bioactive particles on the surface that can withstand substantial chemical and mechanical stresses [313]. Another family of glasses in the SiO2-Na2O-K2O-CaO-MgO-P2O5 system has been synthesised for coatings on Ti-based and Co-Cr alloys by the scientists, where desired achievement were observed to alloys by formation of 100–200 nm thick interfacial layers (Ti5Si3 on Ti-based alloys and CrO*x* on Co-Cr) and commercially Ti alloy-based dental implants were fabricated with 100 μm thick glass coatings successfully [314]. Surgical suture materials such as absorbable polyglactin 910 and non-resorbable Mersilk when coated with silver-doped bioactive glass powder (AgBG) and tested *in vitro*, after 3 days of immersion in SBF, hydroxyapatite forms on the coated suture surfaces and thus their bioactive behaviour is enhanced as a result their use in body wall repair and wound healing property is also enhanced [243] it also limits bacterial attachment [315]. *In vivo* histologic and histomorphometric study on osteointegration of gradient coatings composed of bioactive glass and nanohydroxyapatite (BG-nHA) on titanium-alloy orthopaedic implants and surrounding

**3.2. Doped bioactive glass as coating of orthopaedic implants**

particles [311].

302 Advanced Techniques in Bone Regeneration

fluid.

Mesoporous bioactive glass coatings immobilised with L-ascorbic acid phosphate magnesi‐ um salt *n*-hydrate (AsMg) on stainless steel plate causes osteoblast MC3T3-E1 cells stimula‐ tion by the MBG with enhanced cell attachment, proliferation, differentiation and better developed cytoskeleton as well as, enhanced fibroblast NIH3T3 proliferation *in vitro* [317]. To compare the behaviour of hydroxyapatite and the bioactive glass coated titanium dental implants different clinical and radiological parameters were studied for 12 months in 31 patients. The study revealed equal potency of bioglass as hydroxyapatite to achieve osteoin‐ tegration in dental implants [318]. Similarly, nanoparticulate bioactive glass coating on the porous titanium implants promotes better osteointegration and stimulates the formation of bone within the pores than non-coated implants [319]. Incorporation of nanosized HAP into ZnO containing bioglass coating on Ti-6Al-4V substrate improves mechanical properties of the coating but do not hamper *in vitro* bioactivity [320]. Composite orthopaedic coatings with antibacterial capability containing chitosan, Bioglass particles (9.8 μm) and silver nanoparti‐ cles (Ag-np) were coated in stainless steel 316 substrate and studied *in vitro* in SBF. Result showed low released concentration of Ag ions (<2.5 ppm) was efficiently antibacterial against *S. aureus* up to 10 days and coating enhanced proliferation of MG-63 osteoblast-like cells up to 7 days in culture and it was also found that high concentration of Ag-np (342 μg) have cytotoxic effect [321]. 45S5 bioglass-silica coatings on 316L stainless steel also causes good osteointegration as well as prevents the metallic implant from corrosion in presence of body fluid [322].

#### **3.3. Doped bioactive glass for delivery of growth factors in bone healing**

Growth factors are proteins secreted by cells, act on the appropriate target cell or cells to carry out specific action and thereby there over expression have also been shown in different stages of fracture healing. This phenomenon has led the researchers to study their role as well as the potential to be used as therapeutic agent to accelerate fracture healing. Hence, growth factors are also incorporated into bioactive glass implant, scaffold or coating materials to enhance osteogenic property. Incorporation of bioactive glass and fibroblasts into alginate beads stimulates VEGF as a result potentially it can be used for therapeutic angiogenesis [323]. Combination of prolonged localised VEGF presentation from a matrix coated with a bioac‐ tive glass enhances bone regeneration as VEGF has beneficial role in osteogenesis [324]. The combination of novel MBG/silk fibrin scaffold and BMP7 and/or PDGF-B adenovirus synerg‐ istically promotes wound healing in acute buccal periodontal defects and osteoporosis related fracture by recruitment of recruitment of mesenchymal progenitor cells [325, 326]. Borate bioactive glass microfibres doped with 0-3.0 wt% CuO has remarkable ability to stimulate angiogenesis which help to heal full-thickness skin defects in rodents and promotes human umbilical vein endothelial cells (HUVEC) migration, tubule formation and secretion of vascular endothelial growth factor, as well as the expression of angiogenic-related genes of the fibroblasts *in vitro* [327].
