Present Strategies for Critical Bone Defects Regeneration

A critical size bone defect may arise due to severe trauma or tumors where a large portion of the bone is removed. In some instances, autografts cannot be used for filling such large defects. Allografts may be used to reconstruct large bone defects, but these grafts may not incorporate in the healing response. Consequently, it is still a challenge for reconstructive surgery to reconstruct large bone defects. A variety of treatment strategies have been progressed to promote the healing response and close the bone defects. Micro and nano particles (MNPs) technology is a newer option than traditional grafts, which may defeat many limitations of the bone graft usage. However, there are still no well approved treatment strategies to override all the expected requirements. Due to the existence of variety strategies for treatment of critical size bone defects, this impartial review, highlights on the techniques and strategies that have been accomplished to anatomize the complicated treatment problems of large bone defect healing, the limitations of therapeutic relevant biodegradable materials, and service the regeneration of large bone defects. keywords: Scaffolds; Composite materials; Critical bone defects; Bone healing; Nanomaterials; Neovascularization


Introduction
A critical size bone defect is a large void in a bone whereby the bone cannot heal itself naturally [1]. This may arise due to severe trauma or tumors where a large portion of the bone is removed, or it may occur due to bone irradiation. The critical size defect appears to be equal to or greater than 20% of the length of a long bone [2]. Bone defect healing passes in several phases, these include hematoma formation, inflammation, soft cartilaginous callus formation, neovascularization, soft callus mineralization, hard callus formation and osteoclastic remodeling of the hard callus to create the lamellar bone [3]. Bone healing is not sufficient in large bone defects and may be complicated. Under these circumstances, the autografts are the most popular method for bone replacement [4][5][6]. In some instances, autografts cannot be used for filling such large defects. Allografts may be used to reconstruct large bone defects, but these grafts may not incorporate in the healing response; hence, it may be absorbed by the rejection. Xenografts are more popular than auto and allografts, but the healing process outcomes are poorly understood [7]. Also, considering the large number of local complications and the unpredictable nature of the radiological and histological outcome xenografting should be discontinued [8]. Consequently, it is still a challenge for reconstructive surgery to reconstruct large bone defects. A variety of treatment strategies have been progressed to promote the healing response and close the bone defects. Micro and Nano particles (MNPs) technology is a newer option than traditional grafts, which may defeat many limitations of the bone graft usage. However, there are still no well approved treatment strategies to override all the expected requirements. Simvastatin seems to play an important role in bone regeneration by participating in osteoblast activation (increasing BMP2 expression) and in osteoclast inhibition, also by stimulating neovascularization [9]. Local delivery of simvastatin from carriers appears to be an attractive solution to the problem of maintaining therapeutic doses to treat large bone defects and to minimize undesired side effects [10]. Generally, controlled release of drugs can be triggered by various external or internal stimuli. The idiom "smart" has been applied to MNPs that can react in an expectant and certain course to external and internal stimuli. Light, ultrasound, electromagnetic fields, and temperature, are known as external stimuli, while pH, redox, enzyme activity and temperature, are internal stimuli [11,12]. Thus, controlled drug delivery approaches based on micro/ nano particles could be a promising approach for sustained-localized delivery of simvastatin. Due to the existence of variety strategies for treatment of critical size bone defects, this impartial review, highlights on the techniques and strategies that have been accomplished to anatomize the complicated treatment problems of large bone defect healing, the limitations of therapeutic relevant biodegradable materials, and service the regeneration of large bone defects. collagen fibrils, osteopontin and osteocalcin. Within the bone extracellular matrix, osteopontin is known to promote cell attachment through covalent binding with fibronectin and type I collagen. Both osteopontin and osteocalcin have an alliance with calcium and may support the nucleation of calcium phosphate during mineralization [14]. The inorganic components of the matrix are calcium, carbonate, and phosphate ions, arranged in a crystalline-like structure. Matrix mineralization starts with nucleation of calcium phosphate crystals, and followed by crystal growth [15]. Non-collagenous proteins can be nucleation points for crystallization [16]. There are three types of bone cells in bone tissue: osteoblasts, osteoclasts, and osteocytes. Osteoblasts are responsible for bone formation through the synthesis and secretion of an organic extracellular matrix, also synthesize a variety of growth factors including transforming growth factor-β (TGF-β) and bone morphogenic proteins (BMPs) that can aid in both the recruitment and differentiation of stem cells. When matrix is no longer actively being formed, the osteoblasts become embedded within the extracellular matrix and become osteocytes. Osteoclasts are responsible for bone resorption. Communication between the three types of bone cells regulate the formation and resorption of bone [10].

Enhancement and Limitations of Bone Defects Healing
Four elements are needed for bone grafts healing, osteoconduction, osteoinduction, osteointegration, and osteogenesis. Osteoconduction is the ability to support bone growth on a surgical site, during which pores, channels, and blood-vessels are formed within the bone. Osteoblasts from the margin of the defect that is being grafted utilize the bone graft material as a framework upon which to spread and generate new bone. Osteoinduction involves the stimulation of osteoprogenitor cells to differentiate into osteoblasts that then begin new bone formation. Osteointegration is the direct contact of living bone to graft material. Finally, osteogenesis is the formation of new bone by osteoblasts within the graft material [17]. It is important to link these elements together by assessing all techniques, materials and information available for bone regeneration. Different treatment strategies have been designed to increase the effectiveness, rate and quality of bone defect healing. Each modality has its own limitations, therefore has not been suggested as a perfect modality to enhance the healing of bone defects. One of the alternative clinical techniques is Masquelet technique. This technique is divided into two stages, during 1st stage, biological membrane is applied on a cement spacer, this acts as chamber for insertion of non-vascularized autograft at the 2nd stage [18]. Another technique used to enhance bone defects regeneration is the distraction using intramedullary devices [19]. However, this technique needs long time (1 mm per day) and may be associated with complications [20]. Autografts provide necessary components dependent on each other to promote bone regeneration. Autograft contains collagen and bone minerals so it forms, a scaffold for osteoconduction, also, it includes non-collagenous bone matrix proteins which regulate activities of bone cells for osteoinduction, and it takes in progenitor cells for osteogenesis [5,21]. Autograft can be harvested as a tricortical graft for structural support [22], or as a vascularized bone graft for restoration of large bone defects [23]. The anterior and posterior iliac crests are the commonly used donor sites. Nevertheless, harvesting requires an additional surgical procedure [24][25][26]. Although, autografts do not stimulate immune response, and can integrate into their new site, they are associated with morbidity, infection, and pain at the donor site. Allografts are available in many combinations, such as demineralized bone matrix (DBM), morcellised cancellous chips, cortico-cancellous and cortical grafts. All lack cellular component and osteoinductive properties because they are devitalized by irradiation or freeze-drying processes [27]. Allografts and xenografts are straightforward obtainable, but they have lazy incorporation and likely graft rejection. Furthermore, freezing or freeze drying; may modify the basic characteristics and architecture of the grafts [28,29]. Moreover, the most evident limitation of allograft is its deficiency of osteoinductive ability. For example, demineralized bone matrix (DBM) is prepared by trituration of allogenic, followed by mild acid extraction of the mineralized phase of bone. This process results in a composite of non-collagenous proteins, growth factors, and collagen [30]. Decellularized extracellular matrices (ECM) from other mammalian tissues have been used also as biological scaffolds for bone regeneration [31-33]. DBM is osteoconductive but does not extend structural support. When preparing demineralized bone matrix for implantation in the defect site, it is usually used as bone graft pasta and mixed with bone marrow, to increase osteogenic factors [34]. Bone graft substitutes consist of scaffolds made of natural or synthetic biomaterials that promote bone healing. There are broad range of synthetic bone substitutes for reconstruction of large bone defects, such as hydroxyapatite (HA), collagen, β-tricalcium phosphate (βTCP), calcium phosphate [35,36]. Moreover, an alternative to auto or allografts is titanium mesh cage as a scaffold combined with autologous bone, cancellous bone allograft, and DBM [37,38]. Many of the growth factors appear to have overlapping functions at various stages of bone healing, making it difficult to identify the specific role of a single growth factor or a combination of a few growth factors at each stage. Bone morphogenetic proteins are suggested to act locally both to recruit stem cells and to induce them to differentiate into boneforming cells such as osteoblasts [39]. Thus, scaffold for bone reconstruction should be three dimensional, accelerate osteoinductivity, increase cell migration, and release growth factors [40]. Even though new treatment techniques have been used as alternatives to traditional techniques, the obstreperous conditions, still the same, and traditional techniques must be applied.

Systemic Enhancement of Bone Healing
Recombinant human parathyroid hormone (PTH) is a new treatment for postmenopausal osteoporosis that can be systemically administered for the primary purpose of increasing bone formation. Many clinical trials showed that PTH administration induces both cancellous and cortical bone healing, enhances bone mass, and increases mechanical bone strength and bone mineral density, with a relatively safety profile [41-43]. Furthermore, systemically administered growth hormone plays an important role in bone metabolism [44,45]. Both hormones until now are under investigation to use as bone forming agents in bone defect healing. On the other hand, the use of stimulators of the prostaglandin EP2 and EP4 receptors (anabolic at cortical and cancellous sites), showed good results without adverse effects [46]. Bisphosphonates are a class of drugs that prevent the loss of bone mass, used to treat osteoporosis and similar diseases. It inhibits the digestion of bone by encouraging osteoclasts to undergo apoptosis, or cell death, thereby slowing bone resorption, so it will be useful to enhance bone healing [47].
The first report of tissues regeneration by a scaffold in humans discussed skin regeneration across a gap [49]. Scaffolds strategy for bone defect treatment focused on the mechanical properties (stiffness and compression resistance), nanoscale topography, degradability, and micro/macro porosity, and Nano scale topography. Here, we will present some of the materials used in large bone defects.

Natural Scaffolds
Natural polymers can be classified as proteins such as silk, collagen, gelatin, fibrinogen, elastin, keratin, actin, and myosin, or as polysaccharides such as cellulose, amylose, dextran, chitin, and glycosaminoglycan [50]. Poly (lactic-co-glycolic acid) (PLAGA) copolymers are among the most commonly used synthetic polymers [51]. For the healing of large bone defects, the mechanical and spacefilling features of the scaffold are at the first place. The combination of degradable polymers and inorganic bioactive particles represents the approach in terms of achievable mechanical and biological performance in hard tissue [52]. Consequently, scaffolds in large bone defects without structural support is a defiance. Therefore, decellularization of harvested tissues have been used as scaffolds, to keep the native architecture [53]. Sponge or foam porous scaffold have been used for bone regrowth, their porous network simulates the extracellular matrix allowing cells to interact effectively with their environment. The collagen consists of over 25 molecular isoforms. The most common form is type I collagen, which is organic component of bone. Sponges  . The chitosan molecule allows the material to be used for drug and gene delivery directly from the scaffold [64,65]. Biocompatible hydrogels are used in bone regeneration, and as carriers for drug delivery [66]. Hydrogel with growth factor can act directly to support the development and differentiation of cells in the newly formed tissues [67]. Hydrogels are often favorable for promoting cell migration, angiogenesis, high water content, and rapid nutrient diffusion [68]. The clinical benefit of hydrogels is that they can be applied without invasive techniques to fill the bone defects. Furthermore, they can be combined with osteoinductive factors and cells to promote healing of critical bone defects [69]. Hydrogels such as fibrin [70,71] and gelatin [72,73] showed hopeful results as transporters for therapeutic factors to promote bone regeneration. Hydrogels are insufficient for large bone healing but they can be as component of titanium cages to stimulate new bone formation.

Synthetic Scaffolds
During last year's several synthetic materials have been widened for critical sized bone defects healing. Calcium phosphate or calcium carbonate-based scaffolds have shown an effect on the healing of critical bone defects, during the formation process, go through the bone-like mineral layer formed on the surface of these materials [74][75][76][77]. β-tricalcium phosphate and calcium phosphate are the earliest compounds which are used as a scaffold for bone regeneration [78]. βTCP have been used since 1920 when it has been injected into the gap of bony defect [79]. Furthermore, a composite scaffold composed of βTCP, collagen, and autologous bone fragments fixated with fibrin glue to correct cranial defects, demonstrated that the materials composed of βTCP with or without collagen would be important for cranial bone regeneration [80]. Problem with βTCP is that less new bone is placed than resorbed βTCP [81]. This reason has limited its clinical using. Calcium phosphate ceramics, such as hydroxyapatite (HA), are biocompatible materials because their composition is like the apatite in natural bone [82]. Several shapes of HA as bone substitutes are obtainable such as porous and dense blocks, powder, dense and porous granules [83]. The porous forms allow nutrient transport, cell migration, and vascularization [84,85]. Otherwise, porous spherical HA granules can be used for drug delivery systems. Previously, researches concentrated on the release of anti-inflammatory or antibacterial drug from HA, to control the infection at the implanted area [86]. Several drugs have been constructed to enhance bone formation, and the loading of HA with these drugs and agents could be a very effective method for enhancing bone formation at the defect site [87,88]. Recent studies suggest that released mineral ions such as calcium, phosphate, magnesium and strontium maybe they are responsible to some extent for the behavior of bone precursor cells [75,89,90]. Overtime, HA is ambidextrous to obtain and sustain a drug with stable drug release over time [91,92].

Advanced Scaffold Materials and Drug Delivery
During the past few years' nanotechnology suggest that ceramics can be good stands for drug delivery and controlled extended release. Drugs have been encapsulated in hollow structures of calcium phosphate and then were triggered by ultrasound [93]. Calcium phosphate scaffolds snob the natural bone structure and provide initial structural integrity for bone cells, and their proliferation and differentiation. Thus, most ceramic Nano scaffolds serve as mechanical support, drug transporter, and promote cell growth. Researchers showed a model of using nanotubular Titania as a drug delivery platform to load antibiotics by co-precipitation of the drug and calcium phosphate crystals on the nanostructures [94]. This delivery system showed a time-delayed release of antibiotics for up to three weeks. Furthermore, another study on scaffolds included a composite of silica and calcium phosphates, showed that this material has a continuous release of gentamicin from the scaffolds for 70 days [95]. Another study has used electro spun scaffold which can be used for treating bone defects and drug delivery. This technique depends on the charging of solutions containing polymers, ceramics or metallic precursors with a high voltage. The charged solution is drawn by electric field from orifice onto collector plate to form desirable structures. The structures can be fabricated to mimic various architectures of biological systems, such as fibrous proteins in a native extracellular matrix or collagen fibrils in bone [96]. One more study, demonstrated new magnetic scaffold for bone regeneration. This study proposed new class of magnetic hydroxyapatites which can be used to develop new magnetic ceramic scaffolds with enhanced regenerative properties for bone regeneration based on hyperthermia [97]. Other common synthetic materials used to form scaffolds for bone healing are polymer materials, such as poly (alpha-hydroxy esters) [98], poly(urethanes) [99], and poly(carbonates) [100]. All of them have been applied within large bone defects, as void filler and as an osteoconductive matrix. Synthetic hydrogels can incorporate and deliver controllable dosages of robust molecules, such as growth factors and cell adhesion peptides. Furthermore, self-assembling hydrogels have been designed to nucleate mineral formation, present peptides for cell adhesion [101,102], or present peptides for growth factor binding [103]. Other studies demonstrated that mineral-coated hollow tubes composed of poly(ε-caprolactone) can stimulate bone regeneration in sheep tibia defects [104] and sheep lumbar spine fusion [105]. Moreover, it is remarkable that naturally-derived polymers can also be synthetically modified to create natural/synthetic hybrids that stimulate bone formation, such as alginate hydrogels which can be used to deliver bone-forming stem cells or osteoinductive growth factors [106]. A modern method for creating porous scaffolds composed of micro and nano scale biodegradable fibers by electrospinning is a latest development in this field [107]. Microsphere scaffolds are progressively used as drug delivery systems for antibiotic treatment of infected bone [108]. These scaffolds are a polymer matrix used for drug encapsulation for the release of drugs at a relatively slow rate over a prolonged period [109]. Polymers with low molecular weight are used in developing porous microspheres for the rapid release of the drug, while polymers with high molecular weight for developing microspheres for a slower drug release which can be achieved due to its dense nature [110]. Furthermore, particle aggregation method is proposed to make two layered scaffolds to improve bone and cartilage interface. Also, PLAGA microsphere scaffolds are used as a scaffold for load-bearing bone tissue [111]. The gel microsphere matrix and the sintered microsphere matrix were designed using PLAGA microspheres to create a 3-D porous structure for bone regeneration [112]. Polymer-ceramic microspheres are also used for bone applications [113]. Nano fibers have been used as scaffolds for bone repair and as vehicle for the controlled delivery of drugs [114]. Natural polymers and synthetic polymers inspected to produce nanofibers scaffold for biomedical application such as collagen [115], gelatin [116], chitosan [117], PLGA [118], poly (ethylene-vinyl acetate) (PEVA) [119], and PLLACL-collagen fibers [120]. Drugs, growth factors, and genes can be directly mixed into the polymer solution and electro spun to prepare drug carriers with controlled release properties [121]. It is reported that simvastatin promotes osteoblastic activity and inhibit osteoclastic activity. However, the half-life for simvastatin is 2 hours, so, it is difficult to maintain active simvastatin. To overcome this limitation, researchers proposed controlled drug delivery approaches based on microparticles which could be a promising approach for sustained-localized delivery of simvastatin [122]. Furthermore, the novel PLGA/HA/SIM Nanofibrous scaffold may be beneficial for patients who have bone deficiency soon [123].

Neovascularization
With microvascular surgical techniques, vascularized bone grafting became a good option to provide restoration of large bone defects. Vascularized bone grafts include the fibula, iliac crest and the ribs. This treatment style requires special skills; however, it is considered as procedure with limitations [124]. The limited clinical success of scaffold strategies may be explained by a lack of vascularization. Studies have demonstrated that new bone formation in porous scaffolds was significantly increased by the insertion of a vascular pedicle in the scaffold [125,126]. Therefore, promoting angiogenesis is an important aspect to enhance large bone defects healing. There are various techniques to enhance angiogenesis into the scaffold. One of these procedures is the implantation of a scaffold into a rich arterial supply area such as the abdominal mesentery [127], then waiting for the scaffold to become vascularized, removing the scaffold, and replanting the scaffold into the bone defect. The next procedure is seeding of vasculogenic cells or endothelial cells onto a scaffold. In addition, fusion of VEGF into a scaffold material has been shown to induce angiogenesis and promote bone formation [128], also a combination therapy of VEGF and BMPs seems to have a synergistic effect on bone formation during the first few weeks of treatment of critical size of bone defects [129]. Currently, Jusoh et al. have proposed 3D microvascular networks in a hydroxyapatite-incorporated extracellular matrix for designing a vascularized bone tissue model in a microfluidic device. This study concluded that hydroxyapatite enhanced angiogenic properties such as sprout length, sprouting speed, sprout number, and lumen diameter [130].

Cell Based
This strategy depends on seeded scaffolds with cells before implantation, or acellular scaffolds that require in vivo recruitment of autologous cells [131]. Studies have been shown that fresh mesenchymal stem cells from the bone marrow can lead to improved bone healing if more than 1,500 colonies forming units of mesenchymal cells are applied per 1 cm 3 of defect [132]. Therefore, for large bone defects, exogenous cells may be necessary. Some studies suggested that allogeneic MSCs are hypoimmunogenic relative to other cell types [133]. Adipose-derived mesenchymal stem cells are multipotent cells that can differentiate into numerous cell types including osteogenic cells [134]. Studies demonstrated that osteoinduced ADMSCs successfully repaired the defect when seeded on coral scaffolds, also when seeded on polylactic acid scaffolds [135,136]. Finally, chondrocytes may help in healing of critical size defects of the calvarias. Doan et al. demonstrated that chondrocytes, when implanted directly into a critical size cranial defect in mice, heal the defect by 6 weeks' post implantation [137]. Although application of MSCs as cellular material facilitates the construct innovation, there is still some issue with MSC preparation. Furthermore, natural bone is a composite of Nano hydroxyapatite particles with collagen nanofibers which impart the tissue's unique properties [138].

Gene Therapy
This procedure depends on delivery of protein of interest by a vector to the bone defect. The encoding gene can be obtained from the recipient and implanted at the defect site [139,140]. Vectors are important to optimize the transduction of encoding genes. Vectors can be viral or non-viral (polyplexes, DNA plasmids, lipoplexes etc.), but generally vectors still did not achieve the intrinsic efficiency [139][140][141]. Although, a great number of articles which demonstrated successes in healing of large bone defects, but all have been applied on animal's models, without attentions to toxicology and other matters [142][143][144]. An update review [140] listed experimental studies conducted to evaluate the in vivo and ex vivo gene transfer, using viral and non-viral and thus establishing parameters of efficiency and safety, major difficulties, advantages and disadvantages of each method. The authors referred to expected future improvements in gene therapy, emphasizing that although promising results have been achieved in animal models, human trials have not yet been reported.

Conclusion
Large bone defects do not heal by itself. Despite therapeutic strategies discussed in this review, there is insufficiency of confirmed product which give effective results clinically. It is difficult to maintain active drug delivery even proposed controlled drug delivery based on microparticles [123] Vascularized bone grafting Requires special skills; however, it is considered as procedure with limitations [124] Cell based Although application of MSCs as cellular material facilitates the construct innovation, there is still some issue with MSC preparation. Furthermore, natural bone is a composite of Nano hydroxyapatite particles with collagen nanofibers which impart the tissue's unique properties [138] Gene therapy Although, demonstrated successes in healing of large bone defects, but all have been applied on animal's models, without attentions to toxicology and other matters [139][140][141]  Possibly those strategies have based on inappropriate understanding of bone defects healing. However, autografts still the best therapeutic strategy for large bone defects. The limitations of allografts and xenografts, imposed to look for an ideal composite graft and optimal delivery system for osteoconductive materials, osteogenic cells, and osteoinductive growth factors. Unfortunately, the synthetic and organic biomaterials available to stimulate osteogenesis does not meet the expectations required for bone graft substitute. Local stimulation with growth factors or drugs still needs a proper carrier and a definition of dose and time sequence appropriate for the kinetics of bone healing. Moreover, a small number of clinical trials with inconclusive results do not guarantee growth factors effectiveness in the medical settings. There is still little information available about the cellular basis for MSC mediated bone healing in humans. Finally, knowledge concerning the interaction of nanoparticles within the body are still nominal (Table 1). 28. Bigham