1.Introduction to bone regeneration

The human skeleton possesses an extraordinary capacity for self-repair, a complex biological symphony orchestrated through the intricate dance of cellular regeneration. Bone healing transcends mere physical restoration, representing a sophisticated biological response that rebuilds structural integrity after trauma or degenerative conditions like osteoporosis [1]. When bone tissue experiences damage, the body initiates a remarkable healing cascade, deploying specialized cells called osteoblasts and osteoclasts that work in precise coordination to reconstruct and remodel damaged skeletal regions [2]. Yet nature's inherent healing mechanisms occasionally encounter limitations, particularly when confronting extensive bone defects or chronic skeletal challenges. This is where biomaterials emerge as transformative technological interventions, bridging the gap between biological potential and clinical necessity. Modern biomaterials ranging from advanced ceramics and polymers to innovative composites and bioactive glasses function as intelligent scaffolds that do far more than simply fill gaps [3,4]. These sophisticated materials meticulously recreate the bone's natural extracellular environment, actively guiding cellular behaviour, stimulating tissue growth, and promoting seamless integration. The frontier of regenerative medicine now explores increasingly sophisticated biomaterials capable of dynamic interactions materials that can release targeted bioactive molecules and responsively adapt to surrounding biological conditions[5]. This chapter delves into the intricate world of these biomaterials, unravelling their complex mechanisms and examining the groundbreaking innovations that are reshaping our understanding of bone tissue regeneration.

2.Bone fracture and diseases

Bones, the intricate framework of our bodies, play a pivotal role in supporting our bodies, enabling movement, and protecting our vital organs[6,7]. However, these resilient tissues are not immune to damage and disease. Bone diseases and fractures, ranging from common conditions like osteoporosis, osteonecrosis, arthritis, tumours etc to severe bone loss due to accidental trauma etc, can significantly impact an individual's quality of life and mobility [7,8]. In certain cases, traditional treatment methods may not be sufficient, necessitating more extensive interventions such as complete bone tissue replacement or surgical procedures[9]. This discussion delves into the realm of bone diseases and fractures that demand such interventions, providing an in-depth understanding of the underlying pathology, treatment options, and associated challenges.

3.Bone ailments requiring bone tissue replacement

A spectrum of bone diseases can lead to extensive bone loss or damage, rendering them unresponsive to conservative treatment approaches. These conditions often necessitate complete bone tissue replacement or surgical intervention to restore function and prevent further deterioration[10].

3.1Osteoporosis

Osteoporosis, a systemic skeletal disorder characterized by low bone mass and increased bone fragility, is a major cause of bone fractures, particularly in the hip, spine, and wrist [7,11]. In severe cases, the weakened bone structure may fail to support even minimal loads, resulting in spontaneous fractures. For individuals with advanced osteoporosis and recurrent fractures, surgical intervention, such as hip or spinal fusion, may be necessary to restore stability and prevent further bone loss[12]. Figure 1 (a and b) below shows the comparison of bone matrix density in healthy state and in a diseased osteoporosis case respectively.

Figure 1: (a) Healthy trabecular bone (b) Trabecular bone with osteoporosis (Reproduced with permission[13])

3.2 Osteonecrosis

Osteonecrosis, also known as avascular necrosis, occurs when the blood supply to a bone is disrupted, leading to bone death. This condition can affect any bone but is most common in the hip and knee joints[14]. As the bone tissue dies, it becomes weakened and susceptible to collapse, causing pain, joint stiffness, and eventually, disability [15]. In advanced cases of osteonecrosis, joint replacement surgery may be the only viable treatment option to alleviate pain and restore function[16]. Figure 2 (a and b) below shows the comparison of healthy bone and a damaged bone tissue in the femur head causing femoral osteonecrosis.

Figure 2: (a) Healthy bone (b) Bone with osteonecrosis (Reproduced with permission [14])

3.3 Bone tumors

Bone tumours, both benign and malignant, can cause significant bone damage and require surgical intervention [17]. Benign bone tumours, while not life-threatening, can disrupt bone structure and cause pain or discomfort. Surgical removal of these tumours is often necessary to alleviate symptoms and prevent further bone damage. Malignant bone tumours, on the other hand, pose a more serious threat, as they can invade surrounding tissues and spread to other parts of the body[18]. Treatment for malignant bone tumours typically involves a combination of surgery, radiation therapy, and chemotherapy[19,20]. Figure 3 below shows a comparison between healthy bone tissue and malignant bone tumour on the periosteum surface of the femur.

Figure 3: (a) Healthy bone (b) Bone with osteosarcoma (Reproduced with permission [21])

3.4Other bone ailments

Apart from severe bone ailments conditions like Paget's bone disease, which involves dysregulated bone remodeling, and osteogenesis imperfecta, a genetic disorder affecting collagen production leading to brittle bones, could benefit from the regenerative potential of alternative bone tissue engineering approaches [22]. Osteopenia, a state of low bone mass, increases susceptibility to fractures and could be addressed by promoting new bone formation aided by tailored synthetic scaffolds [23]. Spinal stenosis and kyphosis, characterized by narrowing of the spinal canal and excessive outward spine curvature respectively, may find relief through bone regeneration and structural support provided by synthetic bone scaffolds. Even in cases of osteomyelitis, a debilitating bone infection, synthetic scaffolds could potentially facilitate bone repair and healing by providing a conducive environment for cell proliferation and tissue regeneration [24,25]. With their ability to mimic the native bone microenvironment and enhance osteogenesis, surgical interventions of synthetic bone scaffolds represent a versatile approach to address the diverse challenges posed by various bone ailments.

4. Fractures requiring bone tissue replacement

Fractures, the breaking or cracking of a bone, are a common occurrence, especially among older adults and individuals with weakened bone structures [12]. While most fractures can heal naturally with proper immobilization and support, some severe fractures may require extensive interventions, including complete bone tissue replacement or surgical procedures[2628]. Complex fractures involve multiple bone fragments, significant displacement, or damage to surrounding structures such as joints or blood vessels[1,29]. These fractures often require surgical intervention to realign the bones, stabilize the fracture site, and promote healing[26,30]. In cases where bone fragments are severely damaged or missing, bone grafting or bone substitutes may be necessary to restore bone structure and function[31,32].

Non-healing fractures, also known as non-unions, occur when a fracture fails to heal within the expected timeframe, often due to underlying medical conditions, poor blood supply, or complications from previous surgeries. These fractures can cause persistent pain, instability, and loss of function [33]. Surgical intervention, such as bone grafting or internal fixation, may be necessary to stimulate healing and restore bone integrity. Figure 4 showcases different types of fractures that can occur upon severe accidents and injuries.

Figure 4: Types of bone fractures (Reproduced with permission [34])

Bone diseases and fractures that require complete bone tissue replacement or surgical intervention pose significant challenges for both patients and healthcare providers. These interventions involve complex procedures, often with lengthy recovery times and potential complications[35]. However, for individuals with severe bone loss or damage, these interventions represent the only viable option to restore function, alleviate pain, and improve quality of life [33]. As research continues to advance in the field of bone regeneration and surgical techniques, the treatment options for these debilitating conditions are expected to evolve, offering greater hope and improved outcomes for patients.

5. Bone structure and its composition

The skeletal framework comprises two primary structural forms of bone as shown in figure 5. Cortical bone, characterized by its density and limited surface area, envelops the marrow cavity [36]. It is constitutes of haversian systems and is made up of bone tissue lamellae that are concentric and surround a central canal that is filled with blood vessels [37,38]. Trabecular alternatively referred to as cancellous bone, possesses lower density and a more extensive surface area compared to cortical bone. Located at the centre of long bones, flat bones, and vertebrae, trabecular bone is composed of an interconnected meshwork of bony trabeculae, interspersed with spaces filled with bone marrow [39].

While approximately 80% of the skeleton is composed of cortical bone, the distribution of cortical and trabecular bone varies across different skeletal regions. Trabecular bone predominates in specific skeletal regions, such as the distal ends of long bones, vertebral bodies, and the calcaneus [40]. Conversely, cortical bone is the primary component of long bone diaphysis and the femoral neck. These regional distinctions hold clinical relevance as trabecular bone, with its heightened surface area, undergoes more rapid remodeling compared to cortical bone. Consequently, sites rich in trabecular bone experience more accelerated bone loss under conditions of heightened bone turnover [41].

Figure 5: Anatomy and microanatomy of bone (Reproduced with permissions [40])

Depending on the age, species, and location, the extracellular matrix of bone is composed of a decreasing amount of mineral, collagen, water, non-collagenous proteins, and lipids [42].A normal fully grown, adult human bone generally consists of around 30% of its weight as organic phase in collagen, 65% of its weight as mineral phase of hydroxyapatite (Ca₁₀(PO4)₆(OH)₂) organized hierarchically in nano to microarchitecture of bone, and the remaining 5% of its weight as water [43,44]. Such a hierarchical structure of the bone is responsible for its range of remarkable properties and functions both biologically as well as from the mechanical strength point of view. The mineral phase of the native bone i.e. hydroxyapatite is made of combinations of carbonates, phosphates, minerals like calcium, magnesium etc along with other trace elements. The amount of these elements varies based upon the dietary habits and environment [45].

6. BONE Mechanical properties

For the essential structural integrity of the bone the mechanical properties are crucially important. Bone is a complex, hierarchical material with a diverse arrangement of structures at various length scales, which work in concert to perform diverse mechanical, biological, and chemical functions. Its mechanical properties are multifaceted and influenced by factors such as age, gender, location in the body, temperature, mineral content, water content, and disease[46,47]. At the macroscopic scale, bone can be categorized into two types: cortical bone (also known as compact bone or dense bone) and trabecular bone (also known as cancellous bone or spongy bone) [7]. Cortical bone is denser and stiffer, while trabecular bone is more porous and less stiff. The mechanical properties of bone tissue, including cortical and trabecular bone, are determined by the organic matrix, primarily composed of collagen fibres, and the inorganic matrix, primarily composed of hydroxyapatite crystals[43].

The mechanical properties of bone are influenced by its hierarchical structure, which includes the nanostructure, microstructure, and macrostructure. At the nanoscale, the organic matrix of bone undergoes biochemical changes, including collagen cross-linking, which affects the mechanical properties of bone [48]. Cross-links formed specifically via nonenzymatic glycation have been associated with increased brittleness of the tissue. At the microscale, bone exhibits a complex microstructure, including osteons, which are cylindrical structures composed of concentric lamellae, and cement lines, which are narrow regions around the outermost lamellae in the osteons. The presence of cement lines in the microstructure affects the fracture toughness of bone, making it more resistant to crack propagation [49]. At the macroscale, bone exhibits a complex mechanical behaviour, including elasticity, yield, fracture, fatigue, and damage. The mechanical properties of bone are influenced by bone quantity, such as density and porosity, and bone quality, such as cross-linking and protein composition [12].

In summary, the mechanical properties of bone are influenced by its hierarchical structure, from the nanoscale to the macroscale, and are affected by various factors, including age, gender, location in the body, temperature, mineral content, water content, and disease [50]. Understanding the mechanical properties of bone is crucial for developing new and more personalized treatments to mitigate the negative consequences of aging and disease on bone health. Table 1 shows a list of mechanical properties which play a crucial role for efficient functioning and mobility in day to day lives.

Table 1: Strength characteristics of the bone [43]

Property	Cortical bone	Cancellous bone
Compressive strength (MPa)	100-230	2-12
Flexural tensile strength (MPa)	50-150	10-20
Youngss (tensile) modulus (GPa)	7-30	0.05-0.5
Fracture toughness (MPa.m1/2)	2-12	-
Strain to failure	1-3	5-7

7. Bone remodelling and natural healing process

Bone remodeling is a meticulously orchestrated process that continually replaces old bone tissue with new. This intricate mechanism ensures the skeletal system maintains its essential structural and metabolic functions throughout life. Specialized cells, known as osteoclasts and osteoblasts, are the key players in this orchestrated process, working in tandem within bone multicellular units (BMUs). BMUs are activated by specific signals, initiating a precisely timed sequence of events. [6769]. First, dedicated bone-resorbing cells, the osteoclasts, meticulously dissolve old or damaged bone tissue. Following this meticulous demolition, the construction crew takes over. Bone-forming cells called osteoblasts lay down new bone matrix, replacing the resorbed tissue with freshly synthesized material. This coordinated effort ensures that the structural integrity of the skeletal system is maintained, while also allows the bone to adapt and strengthen in response to stress and biomechanical forces [51]. Through this continuous renewal process, bone remodeling ensures the skeletal system remains strong, adaptable, and capable of fulfilling its crucial structural and metabolic roles throughout life. A balance between old bone resorption and new bone formation maintains the correct bone tissue function. The figure 6 below describes the detailed process of bone remodelling into following phases i.e. resting and resorption, reversal, formation and mineralization. [52]. The bone remodeling cycle begins with resorption, driven by multinucleated osteoclasts. Adhering to bone surfaces, these specialized cells secrete acidic and enzymatic cocktails, dissolving mineralized bone matrix and releasing calcium and phosphate ions. Osteoclasts create resorption pits (lacunae) while expressing RANKL, a signalling molecule that stimulates further osteoclast differentiation and activity. Receptor activator of nuclear factor kappa B ligand (RANKL) is a protein that plays a crucial role in bone remodeling and the immune system. It's a member of the tumor necrosis factor (TNF) superfamily, and it helps control various cellular processes by binding to its receptor, RANK[33]. This targeted removal of old, damaged bone matrix prepares the surface for subsequent bone formation, ensuring efficient remodeling while maintaining overall bone architecture[53].After osteoclastic resorption, mononuclear macrophages dominate the remodeling lacunae. These cells scavenge mineral and collagen waste, facilitating their removal. They recruit and encourage osteoblast differentiation via chemotactic and paracrine signalling, establishing the groundwork for eventual bone formation with the collagen matrix. Furthermore, they downregulate osteoclast activity to ensure balanced remodeling and a seamless transition to the bone production phase[43,45]. Osteoblast production and activation occur after macrophage pit cleansing. These specialized cells are recruited by macrophage signals and fill resorption pits with collagen fibers, much like laying bricks for a new wall. Cell-to-cell interactions coordinate calcium and phosphate deposition, transforming the collagen scaffold into mineralized bone. Tiny blood vessels grow, bringing new supplies for building. Layer by layer, mineralized matrix accumulates, gradually filling and reshaping the pits into new, strong bone[53]. This regulated process, governed by paracrine and hormonal elements, completes the remodeling cycle and restores skeletal integrity. Mineralization, the final stage of bone remodeling, is critical for strengthening bone tissue. During this process, calcium and phosphate ions combine with the collagen matrix to generate hydroxyapatite crystals. This mineralization increases bone density and hardness, hence strengthening structural integrity. Osteoblasts perform an important role by secreting proteins that induce and regulate mineral deposition. The delicate balance between bone creation and resorption promotes appropriate skeletal strength, supporting critical functions including support, protection, and movement in the human body[2,40].

With this backdrop of the bone tissue regeneration, the bone tissue healing with minor fractures do not have major difficulties and require lesser healing times, however the need for external interventions becomes necessary in critical sized bone defects as highlighted in previous section figure 4. The natural healing process of bone involves osteogenic cells, extracellular matrices, and biochemical signalling working together to rapidly heal bones. However, in cases of large-scale bone injuries or defects, the body's natural healing process may not be sufficient. This is where bone grafting methods come into play.

Figure 6: Bone remodelling process (Reproduced with permission [52])

8. Bone Grafting

Bone grafting is a surgical procedure that involves the transplantation of bone tissue to repair or replace damaged or missing bone[54]. Traditional bone grafting treatments often entail the use of autografts (bone extracted from the patient's own body), allografts (bone tissue from a donor), or bone graft substitutes[55]. These methods have been the traditional approach to bone grafting and have been effective in many cases. Bone grafting methods provide an alternative solution to enhance the natural healing process by introducing additional bone tissue[56]. This can help stimulate cellular bone growth and promote the regeneration of new bone. The limitations of the natural healing process can include inadequate bone volume, impaired bone healing in certain medical conditions or elderly patients, and difficulty in achieving proper alignment and stability of the fractured or damaged bone. Furthermore, traditional bone grafting methods may require additional surgeries, have the risk of graft rejection, and cause discomfort, pain, and morbidity for the patient [57]. To overcome these limitations, advanced techniques in bone grafting have been developed. These advanced techniques include the use of 3D printing technology in creating biomimetic bone grafts. This entails shaping living tissues or cells into intricate natural shapes that resemble and promote cellular bone formation using biocompatible materials.

These advanced techniques offer significant advantages over conventional methods, such as faster manipulation of materials, the ability to create customized grafts for individual patients, and improved efficiency and effectiveness in bone regeneration[58,59]. Therefore, the development of novel bone grafting techniques, such as 3D printing of biomimetic bone grafts, has become imperative to address the limitations of natural healing processes and provide better solutions for bone injuries and defects. In conclusion, bone grafting is a method used to enhance the natural healing process of bone by introducing additional bone tissue through autografts, allografts, xenografts etc or synthetic bone grafts till the damaged bone tissue is completely healed[60,61].The two main types of bone grafts that are typically distinguished are (i) conventional natural bone grafts and (ii) artificial bone substitute grafts (synthetic bone scaffolds). The following section discusses these grafts in detail.

8.1 Natural bone grafts

The goal of the quickly evolving science of bone tissue engineering is to repair bone tissue by combining cells, biomaterials, and signalling molecules. The gold standard of bone tissue engineering approach includes use of natural bone grafts such as autografts, allografts, xenografts etc classified based on harvesting site [62,63].

8.2 Autografts

Autografts are the gold standard for bone grafting, as they have the highest rates of success and the lowest risk of complications. The primary advantages of autografts lie in the fact that they are not only osteoconductive but also osteoinductive, osteogenic and thus possess good healing potential. Autografts are obtained from healthy bone tissue within the patient's own body, commonly sourced from the fibula, ribs, iliac crest, or spinal fusion sites. For the best utilization of autograft, the harvesting site should be rich in osteocytes and osteoblasts for an enhanced osteogenic response[54,61]. For the above reason normally, trabecular porous bone rich in these cells are harvested as a choice of autograft site. The porosity of trabecular site is very conducive as it offers larger surface area because of its porous microarchitecture. Thus, also acting as a site for cell adhesion, attachment, proliferation, and vasculature growth, essential for bone remodelling at the tissue damage site. The fundamental advantage of using autograft is that they offer low risk of rejection and infection at the site of implantation sue to histocompatibility. This offers excellent bone repair and regenerative conditions. However, the amount of bone obtainable from a patient through safe harvesting restricts the use of autografts, along with problems such as donor site morbidity etc [64].

8.3 Allografts

Another popular form of bone grafting method is the allografting technique. Here the tissue to be implanted is harvested from the cadaver of donor patient [26]. Allografts as contradictory to autografts are harvested from donor patient, and thus do not require any additional surgery on the host patient for tissue, thereby avoiding issues such as donor site morbidity[26,60]. However, the primary challenges that exists for allografts are that they are found to be less bioactive and osteogenic leading to lesser preference over autografts. Handling and storage of allografts is another challenge wherein before implantation these involve preprocessing steps such as sterilization, ultrasonic cleansing, lyophilization [65]. These steps are crucial in avoiding any source of infections at the host patient site. Additionally, these processes affect the overall strength and effectiveness of the allograft which reduces the reliability [66].

8.4 Xenografts

The xenografts are the types of grafts which are obtained from an animal source. These are derived from animal sources, including porcine and bovine tissues [60,67]. Xenografts are preferred only in situations where limited supply of bone grafts along with expensive nature of autografts and allografts are the major issues. Despite extensive research aimed at improving the compatibility of animal-derived grafts (xenografts), significant challenges persist. These include a heightened risk of disease transmission, chronic rejection, and viral infection, in addition to ethical concerns. Consequently, xenografts remain a limited option for transplantation[54]. In human trials, xenografts are normally used only for orthopaedic applications involving maxillofacial regions. Based on the above-mentioned limitations, the use of xenografts is restricted to repairing substantial bone loss and fractures. [66]. The primary variables that affect the characteristics and their impact on choice of bone graft substitutes have been tabulated in table 2 below.

Table 2: Bone graft substitutes based on their characteristics.

Variable	Autografts	Allografts	Xenografts
Osteoconduction	***	***	***
Osteoinduction	**	***	*
Osteointegration	**	***	**
Osteogenesis	__	***	__

Excellent: ***; Average: **, Poor: *, None

8.5 Synthetic bone grafts (scaffolds)

Synthetic bone grafts or bone substitutes also commonly referred to as bone scaffolds are artificial materials used with the aim of scaffolding the bone tissue damage or fracture site [68]. Synthetic bone scaffolds offer a promising alternative to traditional bone grafting techniques, addressing their limitations, and providing a more versatile approach to bone regeneration. Synthetic scaffolds can be engineered to mimic the natural bone structure and composition, creating a biomimetic environment conducive for new bone formation. Their customizable design allows for optimized porosity, surface topography, and mechanical properties to promote cell attachment, proliferation, and differentiation. Functionalization with bioactive molecules further enhances their osteoinductive and osteoconductive properties [69]. Additionally, synthetic scaffolds can be produced consistently and in large quantities, overcoming the scarcity of autologous and allogeneic grafts. This scalability and reproducibility potentially lead to more cost-effective and accessible treatments across various bone-related conditions. The rationale for synthetic bone scaffolds lies in their ability to provide a tailored, biomimetic platform for bone regeneration while circumventing the drawbacks of traditional grafting techniques, making them a promising therapeutic avenue in orthopaedics and regenerative medicine. In this line, several engineered biomaterial candidates have been explored till date with variety of applications. At the same time, they also demonstrate some or the other limitations either with respect to physical, chemical, or mechanical properties [7073]. All such engineered bone scaffold biomaterial variants have been elaborately discussed ahead. Figure 7 schematically highlights the strategies to be considered while designing and developing bone scaffolds for tissue repair and regeneration.

Figure 7: Bone tissue healing: strategic design of scaffolds

(Reproduced with permission[74])

9. Biomaterials for bone regeneration

A bone scaffold is a three-dimensional artificial material designed to support new bone formation [16,18]. The biomaterials development for bone scaffold is carried out based on some preset characteristics, that can help the material to qualify as a viable option for bone tissue engineering. In the above context, several bone scaffold biomaterials, with their structural and biological properties along with their techniques of fabrication have been reported. Biocompatible bone scaffolds are engineered to replicate the intricate architecture of natural bone, providing a game-changing approach for healing bone lesions. These three-dimensional marvels should be permeable to promote blood vessel growth and cell infiltration and strong enough to sustain daily stressors. Ideally the bone scaffolds should demonstrate a capability where they can be injected with growth hormones for speedier healing, outfitted with sensors for real-time monitoring, and even pre-seeded with blood vessels for improved integration. These ground-breaking "building blocks" indicate a future in which bone repair transcends traditional boundaries, offering not only tissue restoration but also a higher quality of life for countless people. Overall, the bone scaffolds idealistically are expected to have a biocomposite, osteoconductive and biomimicking architecture which closely replicates the native bone tissue healing characteristics along with appropriate mechanical support throughout its hold in the tissue damage site.[45,75]Based on these characteristics the biomaterials for bone tissue regeneration are broadly categorized from the materials perspective as metallic, polymeric, ceramic, and composite biomaterials. The scaffolds for bone regeneration however must have some characteristic features which meet the biological requirements, must have compatible structural features and composition with respect to native bone and lastly should have an ease of fabrication with the available state of the art [76,77]. The biomaterial reported till date based on these ideal characteristics have been elaborated ahead.

In the process of development of biomaterials, it is very important to choose a starting material with regards to the properties that we expect to have from the bone scaffold material. With the current state of the art, several material options have been explored and reported earlier. These are categorised fundamentally into metallic, polymeric, bioceramic and biocomposite bone scaffold-based biomaterials. Each of these categories have been elaborately discussed ahead.

9.1 Metallic biomaterials

The concept of artificial bone scaffolds and implants till date has been widely explored with metallic materials for their fundamental advantage of having better mechanical strength. Range of metallic biomaterials such as stainless steel, cobalt nickel chromium, titanium, tantalum and magnesium-based alloys have been explored as choice of materials for bone tissue engineering [7881]. For an implant it is important to establish an interfacial bonding with the host bone during period of healing. The presence of trace elements like Mg, Zn, Sr, Fe, Cu, Mn, Co, Li, Ag etc in the native bone have a significant impact on the bone growth and metabolism. A study reporting fabrication of metal reinforced polymer composite bone scaffolds with bioactive doping of above elements established the role of these metallic ions on the hydrophilicity and thus improving interfacial bonding, imparting antibacterial activity, and improving mechanical properties along with the promotion of vascularization of the scaffolds. However, doped metals should not only exist on the surface of the composite scaffold but should be evenly dispersed throughout the whole composite scaffold, so as to ensure the smooth completion of the entire bone repair process[82]. Nevertheless, metallic materials consist of components that may be harmful or trigger allergies. Additionally, they are prone to releasing undesirable metallic ions in extremely harsh conditions caused by corrosion, wear, and friction within the physiological system[83]. This occurrence can potentially provoke inflammatory responses, resulting in gradual osteolysis of surrounding tissues and causing additional harm to local tissues. Revision surgeries required in such scenarios cause further complications and severe pain to patients. Therefore, a need for potentially less harmful alternative development seems apparent with metallic implants.

9.2 Polymeric biomaterials

The vast application of polymeric materials in implantable devices stems from their advantageous combination of fabrication ease, inherent flexibility, and biocompatibility [84]. Additionally, composites formed by combining polymers with other materials offer a remarkable spectrum of mechanical, electrical, chemical, and thermal properties, granting significant design flexibility[85,86]. Furthermore, these polymers exhibit excellent tensile strength, ensuring they can securely enclose the implant throughout its intended lifespan. In essence, the selection of polymeric materials for implantable devices revolves around their ability to create a safe, durable, and functionally optimal environment for the device while simultaneously achieving harmonious interaction with the human body [87]. Table 3 below shows a list of different polymeric biomaterials used in the cases of bone scaffolding and implantation. Based on the data from US food and drug administration (FDA), these polymeric biomaterials shown below are divided into categories.

Table 3: Categorial medical implant materials and their applications[86]

Category	Applications	Materials Used
Anaesthesiology	Catheters	Polyethylene Polytetrafluorethylene Polyamide
Cardiovascular	Pacemaker, Implantable cardioverter/defibrillator, Left Ventricular Assist device.	Polypropylene Polyethylene Polytetrafluoroethylene Polyamide
Dental	Dentures Dental Implants	Polymethylmethacrylate
Orthopaedic	Implants for bone fracture and injuries	Polyethylene Polyether ether ketone Polyhydroxyalkanoates
Ear, nose, and throat	Strapes implant Nasal implants for nose reconstruction	Polydimethylsiloxane Silicone Parylene Polyethylene
General and plastic surgery	Synthetic blood vessels Breast implants Cheek, jaw and chin implants Lip implant. Titanium surgical implants Hip implant	Polypropylene Polyethylene terephthalate Polytetrafluoroethylene Silicone Polydimethylsiloxane

From the table 3 and the respective data, as obvious as it looks, polymeric implant as biomaterials have major applications in the non-load bearing tissues of the body with a limited range of materials for load bearing[88]. This being an inherent limitation of the polymeric implants, only composite bone scaffolds consisting of polymeric matrix have been found to influence the load bearing bone tissue scaffolds.

9.3 ceramic biomaterials

Bioglass and bioceramics as bone scaffolds and implants have showcased salient features and performance in terms of their biocompatibility, bioactivity, bioresorbability etc which makes them a good choice for bone tissue regenerative applications in the long run [64,69,8992]. Range of bioceramics such as tricalcium phosphate cements, hydroxyapatite, wollastonite-apatite, apatite-mullite, zirconia, alumina, bioglass etc have been developed for variety of bone tissue growth and rehabilitation scenarios[9397]. Among these, bioactive glasses have gained significant traction due to their unique ability to form a hydroxyapatite layer on their surface, facilitating bonding to bone. Bioglasses exhibit excellent biocompatibility, osteoconductivity, and even osteoinductivity, making them promising candidates for bone regeneration applications.

The application of bioactive glasses is restricted, particularly in load-bearing bone defects, because of their intrinsic brittleness and low mechanical strength. To overcome these limitations, bioceramics have been explored, combining the bioactivity of glasses with the superior mechanical properties of ceramics. Bioceramics like hydroxyapatite, tricalcium phosphate, and their composites have demonstrated excellent biocompatibility, osteoconductivity, and in some cases, even osteoinductivity.

In the quest for development of advanced bioceramics as biomaterials, specifically, glass-ceramics have always remained as a better choice for their inherent characteristics tuning. The glass-ceramics with their precursor glass compositions can be tuned preferably to achieve the desired performance from the final material [98]. One of the significant challenges with glass-ceramics and bioceramics in general is their poor mechanical properties especially in terms of their fracture toughness. Glass-ceramics typically demonstrate very high modulus and very high hardness making them brittle[99]. This characteristic restricts their application to limits of only compressive loading applications and development of whole implant with bioceramic material becomes difficult. However, the mechanical properties of the glass-ceramic too can be tuned to some extent with the selective tuning of their precursor glass compositions thereby holding their role and position as the most preferred choice of bioceramic for bone tissue regenerative applications[74]. With this background, chain silicate glass-ceramics have been known to demonstrate better properties compared to other bioceramic materials available till date[100,101]. Chain silicates play a crucial role in the regeneration of bone tissue due to their bioactive and biocompatible properties. Chain silicates are a structural family of silicate minerals where the silicate tetrahedra (SiO₄) are linked together to form single or multiple chains. Interesting characteristics like regulated bioactivity and anisotropic mechanical behaviour are imparted by this structural arrangement. A well-known quadruple chain silicate, wollastonite (CaSiO₃), has been thoroughly investigated as a bioactive ceramic phase in glass-ceramics for bone regeneration [102,103]. Strong attachment to living bone is made possible by its capacity to produce a surface layer that resembles hydroxyapatite when exposed to physiological conditions. Moreover, glass-ceramics containing wollastonite have mechanical characteristics that can be tailored, which qualifies them for load-bearing applications[104]. Similar to this, diopside (CaMgSi₂O₆), a bioactive phase in glass-ceramics that is a member of the single chain pyroxene group, has shown great promise. Its structure contains magnesium ions, which have been shown to increase osteoblast activity and improve bone formation[94]. For chain silicate glass-ceramics used in bone tissue engineering, a great deal of study has been done to optimize glass compositions, crystalline phase assemblages, and processing techniques to obtain desired mechanical, structural, and biological qualities [163165]. With such consideration, one interesting chain silicate known for its excellent fracture toughness and bioactivity is fluorcanasite glass-ceramic. Fluorcanasite glass belongs to the quadruple chain silicate category of chain silicate glass-ceramics[99]. Acting as biomimetic agents, fluorcanasite glass-ceramic closely imitates the natural minerals present in bone tissue, creating an environment that is favourable for regeneration[101]. Additionally, fluorcanasite glass-ceramics expedite bone mineralization by encouraging the development of fluorapatite along with hydroxyapatite crystals, a key component of bone, thus aiding the integration of implants with surrounding tissues[90]. In addition to their structural contributions, fluorcanasite glass-ceramics enhance mechanical strength, which is essential for applications that involve bearing heavy loads[99]. With high crystallinity in its microstructure, made up of interpenetrating sharp crystals, fluorcanasite (FC) glass-ceramic exhibits high flexural strength (?300 MPa), fracture toughness (?5 MPa.m^1/2), and a reasonably excellent crack deflection [27,179]. It has been discovered that FC GCs have volume nucleation qualities that make them appropriate for casting into intricate shapes [28,180].

Furthermore, their capacity to promote angiogenesis ensures sufficient blood supply, crucial for delivering oxygen and nutrients to newly formed bone tissue. By influencing cellular activities such as adhesion, proliferation, and migration, fluorcanasite glass-ceramics manage a wide range of essential events for successful bone regeneration[101]. Essentially, these diverse characteristics of fluorcanasite glass-ceramics render them indispensable in the development of biomaterials for bone tissue engineering, offering promising advancements in the field of regenerative medicine by promoting a holistic approach to bone healing and reconstruction.

9.4 composite biomaterials

It is desirable to create a biomaterial that is comparable to native bone in terms of composition and structure when it comes to bone tissue engineering. Bone is a naturally occurring biocomposite structure composed of a well-organized composite material of apatite-based mineral phase (5070%) as a bioactive reinforcement and collagenous protein (2040%) as a matrix [105]. Therefore, to match the physico-chemical and mechano-biological performance of the native bone, biomimicry serves as the most reliable option where in biocomposite bone scaffolds can be developed for bone related ailments. The biocomposite approach also allows for tuning the mechanobiological performance of the bone scaffolds. A wide range of biocompatible and biodegradable materials have been extensively explored for bone scaffold development, including metals such as magnesium, titanium and its alloys, ceramics like hydroxyapatite and zirconia, and polymers including polycaprolactone and gelatin etc [106109].

9.4.1Metal matrix composites

Conventional metallic bone scaffolds, encompassing stainless steel, chromium-cobalt, titanium, and tantalum alloys, present a challenge for regenerative medicine. While appreciated for their bioinertia, these materials suffer from inherent limitations. Their high density translates to substantial weight, potentially impacting biomechanics and osseointegration. Furthermore, their low corrosion resistance exposes them to potential degradation in hostile biological environments, compromising implant longevity. Additionally, their high elastic modulus creates a mechanical mismatch with native bone, leading to stress shielding and potential bone resorption. Finally, their insolubility renders them permanent implants, necessitating revision surgeries in case of complications or growth requirements. To counteract these challenges at the same time maintaining the reliability factor of metallic implants, metal matrix biocomposite bone scaffolds are preferred. In the metallic biocomposite bone scaffolds the choice of metals is based upon the targeted properties ranging from strength factor to osteogenetic response. One such choice of metallic biocomposite bone scaffolds is Magnesium (Mg) based alloys. Magnesium and its alloys emerge as promising candidates for next-generation bone scaffold materials due to their inherent biodegradability and favourable weight profile compared to established metals like stainless steel, chromium-cobalt, titanium, and tantalum. Capitalizing on these attractive properties, the strategic incorporation of bioactive molecules into Mg-based composites represents a compelling avenue for developing advanced bone scaffolds capable of sustaining mechanical loads and promoting biological integration[71,79,110112].

For bone tissue engineering, magnesium-based composite bone scaffolds have a number of potential benefits, such as improved biocompatibility, regulated biodegradation, necessary bioactivity, and appropriate mechanical performance [113]. These improvements can minimize scaffold failure due to stress shielding and potentially eliminate the need for additional surgery [74]. Several studies explored the use of different materials within Mg-based scaffolds. For instance, Xiaong et al. incorporated hydroxyapatite (HAp) at various weight percentages. While showing improved mechanical properties, higher HAp content worsened corrosion resistance and cell viability [114]. Khanra et al. found improved mechanical properties but a shift from ductile to brittle behaviour with HAp addition [115]. Huang et al. observed that ?-tricalcium phosphate (?-TCP) in Mg-alloy (Mg-2Zn-0.5Ca) matrix enhanced micro-hardness and grain refinement but significant corrosion and pitting after immersion in simulated body fluid [116]. Dutta et al reported silicate-based bioactive glass (BG) in Mg-matrix. This improved corrosion resistance up to 10% BG loading, with significant deterioration beyond that [117]. These studies highlight the potential of Mg-based composite bone scaffolds but also the need for careful material selection and optimization to balance various properties for successful bone tissue engineering applications.

9.4.2 Polymer matrix composites

Polymer biocomposite bone scaffolds offer a promising solution for regenerating damaged or lost bone tissue. This approach holds a significant importance as it is also in line with the composition of the native bone, where the matrix phase of the bone consists of collagenous protein with biomineralized hydroxyapatite as bioceramic natural reinforcement[74]. The polymer matrix biocomposite bone scaffolds are typically made from a combination of synthetic or natural polymers and other biocompatible materials like ceramics or bioglass. They mimic the natural bone structure, providing crucial features for successful bone regeneration. Several polymeric matrix materials have been reported with use of compatible bioceramics as bioactive reinforcements[118,119]. Some of these are hydroxyapatite, bioglass, tricalcium phosphate cements etc. Hydroxyapatite (HAp) as bioreinforcement has a primary limitation of inherent brittleness, which can be compensated to some extent by use of polymeric matrix. Chitosan has been reportedly used as a biopolymer along with HAp. Pioneering research into CS/HA biocomposite materials explored the creation of bone cement. This involved mixing powdered hydroxyapatite (HA), zinc oxide (ZnO), and calcium oxide (CaO) with a chitosan solution. The resulting paste exhibited several desirable properties, including a rapid setting time, a pH level compatible with the body, and significant compressive strength.[118]. Chitosan (CS) and hydroxyapatite (HA) link together through a specific interaction. This bond forms when calcium ions from HA bind to the amino groups present on chitosan[120]. This interaction acts as a starting point, or "nucleation site," for the growth of HA crystals, ultimately resulting in a material with enhanced mechanical flexibility. Additionally, as a biocomposite, the integration of bioceramics into a polymer matrix also aids in the gradual transfer of load to the healing bone as the bioceramic is resorbed and replaced by natural tissue. On the matrix side, materials like poly-L-lactide are widely utilized due to their biodegradability and bioresorbability, which ensures that the scaffold is gradually replaced by natural tissue over time [121,122].

Composite bone scaffolds are designed to revolutionize bone repair by combining enhanced mechanical strength, mimicking natural bone, with an improved cellular environment. This means they offer a suitable space for crucial nutrients like calcium and phosphate ions, or other beneficial components, to be delivered to cells, ultimately accelerating the healing process. On the polymeric matrices front, flexible and easily decomposable biopolymers, like polyglycolic acid, polyvinyl alcohol, polylactic acid, and polycaprolactone, are emerging as promising materials for scaffold matrices. These biocompatible and lightweight materials offer key advantages over traditional options, such as brittle bioceramics and rapidly degrading magnesium alloys. However, these biopolymeric materials although biodegradable they lack the appropriate bioactivity and structural stability to form new bone tissue at defect site[87,123126]. To tackle these inherent limitations and improve the scope for these materials in bone tissue repair and regeneration, bioactive glass, glass-ceramics etc have been found to be as suitable alternatives as osteoconductive tailored high strength reinforcements[97,127]. The potential of these reinforcements lies in their tuneable precursor compositions which can tuned to achieve near human bone like compositions and strength. These fillers possess additional characteristics such as bioactivity, hydrophilic osteoconductive surfaces, chemical stability and potential biocompatibility for enhanced bone tissue healing [119,128130]. With regards to the strength enhancement, apatite forming glass ceramics such as wollastonite, mullite and fluorcanasite have demonstrated encouraging possibilities for mechanical functionality. Especially, the structural stability of fluorcanasite glass-ceramic and thereby its inherent fracture toughness and flexural strength has demonstrated a good potential[99,131]. No prior work has ever demonstrated the use of fluorcanasite glass-ceramic reinforcement for its enhanced fracture toughness and flexural strength in a biocomposite bone scaffold.

When it comes to the choice of suitable biopolymer, several explored options are available till date ranging from polypropylene fumarate to polycaprolactone as biodegradable grade photopolymer in bone tissue regeneration[132134]. The specific limitation for these biodegradable grade photopolymers lies in their load bearing capabilities[135]. In bone tissue engineering, fundamentally, the scaffold structure should be able to bear the load of the damaged bone at the site of implantation, with the polymeric matrix having load bearing capabilities of its own. In this line leaving aside biodegradability, several photopolymeric resins which are typically biocompatible but not biodegradable are employed in dental applications[136]. These biocompatible dental grade resins have sufficient strength from the load bearing perspective and have been least explored for the bone scaffolds especially with critical sized bone defect healing[74,137,138].

9.4.3Ceramic matrix composites

Bioactive ceramic matrices are combined with reinforcing phases to form mechanically strong structures with customized bioactivity for bone regeneration in ceramic matrix biocomposite scaffolds. Alumina, zirconia, or silicon carbide are high-strength ceramic fibers or whiskers that are used to reinforce bioactive glass matrices and hydroxyapatite (HAp) for enhanced fracture resistance[139]. To combine mechanical strength and bioactivity, inert ceramic matrices (zirconia, alumina) have been mixed with bioactive glass/ceramic particles. Apatite-forming capabilities and customized mechanics are demonstrated by zirconia and akermanite bioceramics composites[139,140]. To improve qualities and offer nanotopographical cues, ceramic nanotubes and fibers have been combined with bioactive glasses, glass-ceramics, and reinforced calcium phosphates. The fracture resistance of HA and bioglass scaffolds has been enhanced using carbon nanotubes[141,142]. The bioceramics used in these composites, such as bioactive glass and hydroxyapatite, play a crucial role due to their inherently high bioactivity, which allows them to form strong bonds with bone tissue and support new bone formation [143].

The combination with bioglass has shown high bioactivity, indicating strong potential for osteoconductive and osteoinductive reinforcement. Conventionally, biocomposite bone scaffold fabrication approaches incorporating hydroxyapatite (HAp), beta tricalcium phosphate (-TCP) cement based bioactive fillers have already been tried out. [144,145]. However, these materials have demonstrated weak mechanical properties and are primarily used as filler materials at the fracture site[29]. In such scenarios, glass and glass-ceramics come to the rescue. The prime advantage of glass and glass-ceramics being its tuneable mechanical and biological properties, finds its applications where multitude of performance characteristics are required. For a bone scaffold not only just the biological properties such as osteoconductivity, biocompatibility, cell viability and proliferation are required but along with its appropriate mechanical properties for the load bearing of the bone are required as well[122]. Glass-ceramic, bioceramics and bioglass find their suitability for such applications with their reinforcement capabilities. In case of glass-ceramics, the selectively tuned glass precursor compositions in glass-ceramics synthesis can be regulated to achieve desired crystallite presences and achieve favourable mechano-biological performance[131,146].

For bone tissue and its repair, the necessary crystalline phases contributing for tissue regeneration are the apatite-based crystallites occurring naturally in the native bone in the form of hydroxyapatite (HAp). Similarly, the tailoring of glass-ceramics for tissue regenerative applications requires inducing apatite based crystalline phases specifically such as hydroxyapatite or fluorapatite, both of which have significant contribution in regulating the bioactivity such as osteoconductivity as well as osteoinductivity[101]. This can be obtained from oxides of silicate-based glass-ceramics. Also, for bone scaffolds with load bearing applications, requires strength enduring reinforcement as well. Chain silicate glass-ceramics are one such class of materials which can be conducively tailored to achieved polycrystalline phases contributing to each individual performance characteristics such as strength in terms of toughness, chemical stability and essential biological characteristics for bone scaffolds. They notably promote osteogenesis by influencing the differentiation of mesenchymal stem cells into osteoblasts, which is vital for the formation of new bone tissue. There are various methods to improvein-vitrocell viability in silicate ceramic-polymer composite scaffolds. These include enhancing surface roughness, generating ionic dissolution products, maintaining a stable pH and forming an apatite layer, increasing three dimensionality and adsorbing phosphorous from the environment, decreasing ion release and the surface area exposed to cells, improving hydrophilicity, providing more active sites for protein adsorption, and achieving a high surface-area-to-volume ratio. Figure 8 shows an ionic release mechanism in silicate-based glass-ceramic used as composite bone scaffold reinforcements.

Figure 8: Bioactive interaction factors and mechanism in silicate-based glass-ceramic-polymer composite scaffold with native bone (Reproduced with permission [147])

10Critical properties of biomaterials

The properties associated with bone tissue engineering crucial to bone scaffold development primarily are the materials biocompatibility and cytotoxicity. For enhanced osteointegration further, the bioresorbability, osteoconductivity, osteoinductivity have a vital function in the process of tissue development. Additionally, in the context of bone tissue repair and regeneration, the biodegradation of the bone scaffold materials is also very important consideration. All the above-mentioned characteristics of the bone scaffolds are mentioned in figure 9 and elaborated further in this section.

Figure 9 : Bone scaffold characteristics for bone tissue repair and healing (Reproduced with permissions [148])

10.1Biological properties

The effectiveness and clinical performance of bone scaffolds are significantly influenced by their biological characteristics. A crucial requirement is biocompatibility, or the capacity to blend in with living tissues without causing negative reactions [149]. For tissue integration to be effective, scaffolds need to be non-cytotoxic and promote cell adhesion, proliferation, and differentiation. Additionally preferred are bioresorbability and biodegradability, which enable scaffolds to eventually be replaced by regenerated tissue by degrading at a controlled rate in line with the creation of new bone [150,151]. In addition, scaffolds ought to demonstrate bioactivity, which is the capacity to generate an apatite layer that is physiologically active and resembles bone, hence promoting osseointegration and bonding with native bone [26]. Developing safe and efficient bone scaffolds that can stimulate and direct the complex process of osteogenesis requires optimizing these biological features through material composition, selection, and structural design.

10.2Biocompatibility

Ensuring biocompatibility is a top priority in bone tissue engineering, confirming the safety of a bone scaffold for human use without causing harm to the body. Biocompatibility, a crucial characteristic of a bone scaffold, indicates its ability to support cellular activities during the bone regeneration process, while avoiding toxic, inflammatory, or immune-related reactions when interacting with the body's physiological fluids[152]. Evaluation of a bone scaffold's biocompatibility can be carried out through three methods:in-vitrotesting,in-vivocytotoxicity tests, and human clinical trials. In vitrocytocompatibility assays use living cell culture lines in conjunction with cell-free systems such phosphate-buffered saline (PBS) or simulated bodily fluid (SBF), where the ion concentrations are like those in human blood plasma[153,154]. When it comes to evaluating bone scaffold biocompatibility,in-vitrocell cultures have proven to be more sensitive thanin-vivotechniques. Although human clinical trials are the best test medium available, there are frequently ethical issues with this type of research.

10.3Cytocompatibility

The cytocompatibility test is a key component of biological evaluation and screening protocols, employing tissue cellsin-vitroto assess cell growth, reproduction, and morphological impacts induced by bone scaffolds or implant materials. It is highly favoured as a preliminary assessment, serving as a vital toxicity indicator for bone scaffolds due to its simplicity, speed, high sensitivity, and potential to reduce animal testing requirements[155].In-vitrocytocompatibility assessments utilize living cell cultures alongside cell-free systems such as phosphate-buffered saline (PBS) or simulated bodily fluid (SBF) with ion concentrations similar to those in human blood plasma[152]. In evaluating bone scaffold biocompatibility,in-vitrocell cultures have shown greater sensitivity thanin-vivotechniques. While human clinical trials are the most effective testing medium, ethical concerns often surround this type of research.[155]

10.4 Bioresorbability and biodegradability

As new research keeps evolving towards development of biomaterials for bone tissue regeneration, each new class of material builds up upon limitations of previously researched materials. One such important characteristically important criteria for synthetic bone scaffold materials is their biodegradability and bioresorbability. In its true form the genuine advantage of biodegradability and bioresorbability is that it eliminates the need for a second revision surgery post implantation and in tandem matches up with the pace of bone tissue healing. A biodegradable bone scaffold material should be non-cytotoxic and the byproduct of biodegradation should be eliminated from the body within the clinically acceptable tenure[156]. As far as biodegradation is concerned, the mechanism of biodegradation crucially determines the rate of biodegradation. Typically, this biodegradation mechanism is categorized into enzymatic and hydrolytic biodegradation. Enzymatic degradation is carried out by specific enzymes which are secreted by the bodys cells, target the bonds within implant material, and break them. It is possible that the process may be highly selective, depending on the types of enzymes that are present around the implant[157]. Hydrolytic degradation, on the other hand, takes place when water molecules come into direct contact with the implant material, breaking it down over time. Both mechanisms contribute to implant resorption or disintegration, which has an impact on long-term functionality and the potential for removal. Understanding these degradation processes is critical for developing implants with optimal performance, biocompatibility, and long-term safety in the complex biological environment of the human body [158].

10.5 Bioactivity

In terms of bone tissue engineering, bioactivity refers to the biological characteristics of bone scaffold biomaterials which enhances the osteoconductive and osteoinductive performances at the bone and scaffold interface. This is essentially important for bone scaffolds to establish their potential as suitable candidates for bone tissue regeneration. Biomaterials with superior bioactivity for bone repair are the subject of recent research in bone tissue engineering. For instance, the existing bioactive glasses are being upgraded through the development of novel compositions, e.g. from silicate glasses, borate glasses, and the addition of trace elements [159]. These upgrades may enhance bone and vascular tissue regeneration, including differentiation of osteoblasts from stem cells. Tuneable mechanical strength and biodegradation rate values make bioactive glasses even more attractive. Elastomeric composites are being investigated to match the elasticity of collagen an important bone protein with an eye toward materials that mimic both the strength and flexibility of bone proteins like collagen, paving the way for next-generation bone scaffolds that can promote bone regeneration effectively[157]. Table 4 below showcases a list of bioactive materials with their characteristics highlighting advantages and disadvantages.

Table 4 Advantages and limitations of bioactive bone scaffold biomaterials [160]

Bioactive materials	Advantages	Disadvantages
Calcium phosphates (e.g HAp, ?-TCP and biphase CaP)	Excellent biocompatibility Supports cell viability. High osteoconduction	Brittle Slow biodegradability in crystalline state Low strength in amorphous state
Silicate bioactive glasses	Excellent biocompatibility Supports cell viability. High osteoconduction Tailorable mechanical properties and degradation Vasculature Rapid gene expression	Low strength in amorphous state
Bioactive glass-ceramics and bioglass	Tunable mechano-biological performance Excellent biocompatibility Supports cell viability. High osteoconduction	Brittle Toxicity risk due to release of borate ions in case of borate bioactive glasses Slow biodegradability in crystalline state
Bulk biodegradable polymers (Poly (lactic acid)) Poly (glycolic acid) Poly (lactic-co-glycolic acid) Poly (propylene fumarate) Poly (polyol sebacate)	Good biocompatibility Biodegradable (with a wide range of degradation rates) Bioresorbable Good processability Good ductility Elasticity	May cause inflammatory response because of acidic byproducts of biodegradation. Scaffold structural breakdowns due to accelerated biodegradation profiles
Composites (containing bioactive phases)	Good biocompatibility Supporting cell activity Good osteoconductivity Tailorable degradation rate Improved mechanical reliability.	Not as good as native bone matrix There is a need to improve fabrication techniques.

10.6Mechanical properties

A bone scaffold requires structural integrity which keeps it intact enough to take care of the load bearing and non-load bearing regions where the scaffold implantation surgery has been performed. The inherent heterogeneity of bone mechanics, characterized by variations in fracture toughness, compressive strength, and Young's modulus across anatomical locations, necessitates the development of bone scaffolds that exhibit site-specific mechanical compatibility[160]. Additionally, adequate mechanical strength is crucial to withstand surgical manipulation during implantation.

Furthermore, the scaffold must retain sufficient structural integrity throughout the period from implantation to osseous remodeling to sustain physiological loading. Notably, the rate of bone remodeling exhibits dependence on both age and the specific defect location. This likely reflects the established disparities in bone quality between younger and older populations, as manifested by differences in porosity and bone mineral density. While advancements have been made in the development of biomaterials boasting superior mechanical properties, this often comes at the expense of porosity, a critical factor for cellular infiltration and differentiation [161]. Unfortunately, numerous bone scaffolds demonstrating promisingin-vitroresults have ultimately failed in vivo due to limitations in mechanical strength along with vascularization, insufficient porosity for cellular activity, and inadequate nutrient delivery[162]. Therefore, it is evident that the efficacy of a bone scaffold hinges on achieving an optimal balance between its mechanical characteristics and a porous architecture that facilitates cellular infiltration and vascularization.

10.7Structural properties

10.8 Porosity and pore size distribution

One of the key attributes of the native bone which plays a crucial role in maintaining its biological activities as well as mechanical strength is the porosity. Porosity is defined as the presence of holes and a network of these interconnected holes acting as channels essential for bone health and function. Structurally, most porous structures of the bone are referred as the trabecular bone or cancellous bone. The other denser part of the bone is referred to as compact or cortical bone. While cancellous bone porosity varies from 30% to 90%, compact bone porosity ranges from 5% to 30%. Changes in disease, aging, and changed stress can all affect bone porosity, which is not fixed. Figure 10 shows the structural entities of the native bone and pore sizes they accommodate to perform their respective biological functions in the process of bone tissue regeneration. Table 5 below shows the pore size range and their respective biological relevance.

Table 5: Variations in pore sizes for an ideal bone scaffold [105]

Pore Size (m)	Biological significance
<1>	Protein interaction and bioactive response
1-20	Cell adhesion and directed cell growth
100-1000	Cell proliferation and bone growth
>1000	Tuning the structure and functionality of bone scaffold

Figure 10: Hierarchical porosity scale of the native trabecular and cortical bone (Reproduced with permission [48])

Pores serve as blood vessel highways, transporting nutrients and oxygen to living bone cells while also eliminating waste. This porosity also contributes to bone's remarkable balance of lightness and strength, which distributes stress and prevents fracture. Furthermore, these pores provide the required room for bone remodeling, a constant process in which old bone is replaced with new bone, ensuring that bones remain strong and adaptive throughout life. Native bone is a hierarchically distributed physical structure in terms of range of porosities occurring from cortical bone to trabecular bone[163]. Bone quantity and quality which are heavily dictated by bone porosity, though crucial, are often neglected in diagnosing bone diseases and assessing fracture risk. Henceforth, while formulating bone scaffold solution for native bone ailments, porosity, pore size distribution, pore architecture, type of porous lattice, pore surface area etc, these characteristics hold a significant importance in determining the structural integrity and quality of biological functions required for bone tissue regeneration[7,164]. The type of porosity is also a very essential feature. Crucially for rapid osseointegration, open, interconnected pores are the key factor, allowing blood vessel growth and enhanced bone ingrowth within the biomaterial[165]. The porosity levels at different hierarchies have specific function that dictate the overall performance of the native bone and its mechano-biological functions[105,166]. Thus, it is necessary to take into consideration such pore hierarchies in the design of bone scaffolds for bone tissue healing.

It is important to note that the porosity of the bone specifically the randomness of the porosity shape and size at a particular site provides necessary strength and vasculature scaffolding [7]. Also, the interconnected random porosities offer sites for the cells to anchor, adhere, attach, and proliferate at a faster rate, thereby facilitating the bone reconstruction process [167]. Consequently, it is crucial to design scaffolds which have replicative lattice structure similar to the porous (trabecular) bone architecture. Of the several scaffold architectural variations, most commonly preferred lattices for bone tissue regenerative phenomena are simple cubic porous lattices, diamond lattices, schwarz lattice, gyroid lattice and voronoi lattice architectures. However, each of these categories possesses a set of advantages as well as limitations. Therefore, the better choice of lattice is always the one that offers simplicity of fabrication, has high strength, surface area and pore interconnectivity[168].

While simple cubic lattices offer basic designs, their mechanical properties are not ideal and they do not exhibit biomimicry [169]. Although diamond lattices are more robust, their restricted interconnectedness may prevent cell migration [170]. Schwarz lattices although with balance strength and porosity, may have a less even distribution of stress [171]. For gyroid, the continuous surfaces of gyroid lattices improve fluid flow, however, their uniform nature restricts their adaptation to particular bone areas [172]. Voronoi lattices are the best option for bone scaffolds since they are modeled after naturally occurring cellular structures [173]. They have outstanding mechanical qualities, pore diameters that can be customized, and a biomimetic architecture. Better cell adhesion, proliferation, and differentiation are also encouraged by voronoi lattices[174]. Although there are advantages to each type of lattice, Voronoi lattices are the most similar to the hierarchical structure of natural bone and offer the finest conditions for bone tissue creation and regeneration. A voronoi lattice design works on an algorithm which divides up a space into cells based on the distances to certain points. Each cell contains all the points that are closest to one point. This creates a lattice-like pattern of interconnected cells and pores. The porous network of Voronoi lattice makes it useful for designing bone scaffolds[175].

Voronoi lattice architecture potentially has demonstrated highest surface area and has been found to closely mimic the trabecular bone microarchitecture [165,176]. Figure 11 showcases the voronoi lattice design along with other interconnected porous lattices which are commonly used in bone scaffolds fabrication to achieve osteointegrative features.

Figure 11: Types of interconnected porous lattices for bone tissue regeneration

10.9 Biomimicry

The biomimicry stands as an important approach in the development of any biomaterial candidate. In terms of bone tissue engineering, biomimicry aims to replicate the structural and mechano-biological properties of the bone[165]. Biomimicry as a fabrication criterion provides an ideal benchmark in the design process of bone scaffold biomaterials where structural features like microporosities, interconnected porous architectures, mechanical strength, material composition and overall biological properties such as bioactivity, bioresorbability are crucial considerations. The need for biomimicry arises from the compatibility point of view where not just the biocompatibility but also biological mechanisms, mechanical strength and structural features matching with the native bone leads to a most reliable bone scaffold biomaterials with least amount of limitations demonstrating its suitability for bone [177]. Strategic biomimicry seeks to prevent unnecessary complexity in scaffold design. It achieves this by understanding what principles can be learned from nature and then focusing on replicating only the critical characteristics that are crucial for the scaffold's function. For bone tissue repair, the primary attributes of biomimicry are the bone scaffold material and the macro as well as microarchitecture. These attributes should be replicated to the maximum extent possible to achieve synergy between the host bone tissue and the bone scaffold biomaterial. Consequently, it is essential to choose materials that are first biocompatible, non-cytotoxic and are osteointegrative enough to achieve bone and bone scaffold interfacial integrity and proliferation[177,178]. Bone as a material is a biocomposite consisting of bioceramic hydroxyapatite mineral reinforcement and natural biopolymeric collagenous matrix. Henceforth, novel attempts to imitate this native combination of suitable bioceramic reinforcement along with suitable biopolymer matrix essentially needs to be explored with several suitable material combinations.

10.9.1 Osteoconduction and osteoinduction

Osteoconduction and osteoinduction represent complementary yet distinct mechanisms essential for effective bone tissue regeneration. Osteoconduction refers to a biomaterial's capacity to function as a passive structural scaffold that facilitates bone growth along its surface by providing an appropriate three-dimensional architecture with optimal porosity (typically 100-500 ?m), interconnected pores, and surface properties conducive to cell attachment[126,164]. This structural framework enables the migration and proliferation of osteoblasts and supports neovascularization, ultimately guiding the spatial organization of newly formed bone. In contrast, osteoinduction involves the active recruitment and stimulation of undifferentiated mesenchymal stem cells to differentiate into the osteoblastic lineage, initiating de novo bone formation through biochemical signalling[30]. This process is primarily mediated by growth factors such as bone morphogenetic proteins (BMPs), transforming growth factor-beta (TGF-?), and insulin-like growth factor (IGF), or through the controlled release of bioactive ions like silicon, strontium, or zinc from the biomaterial[179,180]. The most clinically effective bone regeneration strategies incorporate biomaterials that seamlessly integrate both osteoconductive and osteoinductive properties, creating a synergistic environment where the structural template guides the spatial organization of new bone while biochemical cues accelerate cellular differentiation and matrix production[127,181]. This dual functionality allows these advanced biomaterials to potentially outperform traditional autografts by eliminating donor site morbidity while providing customizable properties that can be tailored to specific defect sites, patient populations, and clinical scenarios, representing a significant advancement in the treatment of challenging bone defects[182,183].

10.9.2 Cell attachment, migration and differentiation

In tissue engineering, cell attachment, migration, and differentiation are fundamental processes that underpin the success of regenerative strategies. Cell attachment refers to the initial adherence of cells to biomaterial surfaces, a crucial first step for tissue regeneration. The surface properties of biomaterials such as surface chemistry, topography, and hydrophilicity can significantly influence this process by promoting the binding of cells through molecular interactions with adhesion proteins like fibronectin and collagen [184186]. Once attached, cells must migrate to the injury site or throughout the scaffold to promote tissue formation. Migration is largely governed by chemical signals, such as growth factors, that direct cell movement toward areas of injury (chemotaxis), as well as the physical structure of the material, which can guide the direction of movement through aligned microstructures or channels[187]. Additionally, the mechanical properties of the biomaterial, such as its stiffness or flexibility, affect the efficiency of migration, with cells generally responding better to substrates that resemble the mechanical properties of the native tissue they are meant to regenerate[178].

Once cells are positioned at the injury site, they undergo differentiation, a critical process in which stem or progenitor cells transform into specialized cell types like osteoblasts or chondrocytes, depending on the tissue being regenerated[188,189]. This process is regulated by biochemical cues from the biomaterial, such as growth factors embedded within the scaffold, and by the physical properties of the material, which can mimic the natural extracellular matrix. The stiffness or elasticity of the material can further influence which lineage the cells differentiate into; for example, stiffer materials may promote bone formation, while softer ones may favour neural or adipogenic differentiation[33]. Together, these interconnected processes attachment, migration, and differentiation are essential for creating functional, integrated tissues. By understanding and optimizing how biomaterials influence these cellular behaviors, we can improve the regenerative capacity of implants and scaffolds, facilitating the successful repair of complex tissue defects and disorders[190].

11. Fabrication techniques for bone scaffolds development

When developing biomaterials for bone tissue regeneration applications, the best fabrication technique depends on a number of factors, including the intended use, the materials selected, the method's efficiency of fabrication, and how well the fabricated part performs in achieving the intended functionality. In the light of using a biomimetic bone scaffold, this section throws light on various fabrication techniques which can help in achieving biocomposite bone scaffolds with bone mimicking attributes. These fabrication techniques are elaborated further in this section.

11.1 Conventional techniques

Conventional fabrication techniques for bone tissue engineering scaffolds have historically provided the foundation for clinical approaches to bone regeneration. Solvent casting/particulate leaching represents one of the most widely employed methods, where polymer solutions are cast over porogen particles (typically salt or sugar) that are subsequently leached out to create porous structures[191]. This relatively simple approach allows for control of porosity through porogen size and concentration, though it often results in limited pore interconnectivity and residual solvent concerns. In parallel, freeze-drying techniques exploit the formation of ice crystals within polymer solutions that, upon sublimation, leave behind a porous architecture with pore sizes dictated by freezing parameters[11,192]. While offering good control over overall porosity, freeze-drying struggles to produce consistent pore morphologies or mechanical properties suitable for load-bearing applications [191].

Gas foaming presents another conventional approach, where pressurized gas (typically carbon dioxide) is used to create bubbles within polymer melts or solutions that form pores upon depressurization or solvent evaporation [193]. This technique advantageously eliminates the need for organic solvents but often yields structures with limited interconnectivity and inconsistent pore distribution. Thermally induced phase separation similarly provides solvent-free processing by leveraging temperature changes to cause polymer-rich and polymer-poor phase separation, ultimately creating microporous structures after solvent removal [194,195]. While these techniques have enabled the creation of numerous clinically used bone graft substitutes, their application for complex defects remains constrained by processing limitations that typically result in random, poorly controlled internal architectures.

The limitations of conventional fabrication techniques ultimately stem from their inability to precisely control scaffold microarchitecture and incorporate spatial heterogeneity. Most conventional methods produce scaffolds with randomly distributed pores that inadequately mimic the hierarchical structure of native bone tissue. These techniques generally cannot create precisely controlled patient-specific external geometries necessary for complex defects [169]. The inability to spatially control the distribution of bioactive factors or mechanical properties further limits their biological performance. Additionally, conventional methods often struggle with reproducibility between batches, making quality control challenging. Perhaps most significantly, these approaches typically cannot produce scaffolds with vascular channels or defined nutrient transport pathways, which severely limits construct size due to diffusion constraints and results in poor core viability in larger implants [196]. These collective limitations have driven the development of advanced manufacturing techniques that offer greater precision and architectural control for next-generation bone tissue engineering.

11.2 Additive manufacturing techniques

11.2.1 Selective laser sintering (SLS)

Selective laser sintering (SLS) has emerged as a promising additive manufacturing technique for fabricating three-dimensional porous bone scaffolds with precise microstructural control. In SLS, a high-power laser selectively fuses or "sinters" thin layers of powdered material one upon another to build up the final three-dimensional part according to a computer model. A key advantage of SLS for bone tissue engineering is the ability to produce highly porous scaffolds from biocompatible and bioresorbable materials like polymers, ceramics, and metals. The ability to process both metallic and polymeric biomaterials via SLS allows tailoring the scaffold properties to meet the specific mechanical strength, biocompatibility, and degradation requirements for different bone regeneration applications. The laser energy of SLS can be modulated to achieve customized pore sizes, porosities, and interconnected pore networks critical for promoting cell migration, vascularization, and bone ingrowth [197]. Polymers like polycaprolactone (PCL) are commonly used for SLS bone scaffolds due to their processibility and controlled degradation rates. PCL scaffolds fabricated with porosities up to 70% and fully interconnected pores ranging from 600-800 ?m utilizing SLS have been reported earlier. These constructs supported adhesion and proliferation of human bone marrow stromal cells [77].

On the metallic side, titanium and its alloys like Ti-6Al-4V have been extensively investigated for SLS bone scaffolds due to their excellent biocompatibility and mechanical characteristics. Porous Ti-6Al-4V scaffolds via SLS with compression strengths up to 454 MPa demonstrated porosities ranging from 41-63%, making them suitable for load-bearing bone defects.[198]. Other biocompatible metals like stainless steels, cobalt-chrome alloys, and shape memory alloys like nitinol have also been processed by SLS for bone scaffolds. Porous NiTi scaffolds with super elastic behaviour, making them promising candidates for implant applications where flexibility and shape recovery are desired have been reported so far as showcased in figure 12 below [199].

While challenges like surface roughness, residual stresses, and process optimization remain, SLS shows great promise as a reproducible technique to manufacture biocompatible metallic and polymeric scaffolds with tailored architectures and properties to match the intended bone regeneration application.

Figure 12: Process workflow of SLS 3D printing to achieve porous bone scaffolds (Reproduced with permission [199])

11.2.2 Fused deposition modelling (FDM)

In the realm of bone tissue engineering, fused deposition modelling (FDM) has garnered a lot of attention as an additive manufacturing technology for creating bone scaffolds[200,201]. Bone scaffolds are three-dimensional constructions that offer a chemical and physical environment that supports cell adhesion, proliferation, and differentiation all of which ultimately aid in bone regeneration. The process of FDM involves heating a thermoplastic material, such as polycaprolactone (PCL), poly (lactic acid) (PLA), or poly (lactic-co-glycolic acid) (PLGA), to a semi-molten state before it is extruded, layer by layer, through a nozzle onto a build platform [202]. The schematic representation of FDM approach is shown in figure 13 below. A computer-aided design (CAD) model sets the nozzle's predefined course, giving exact control over the scaffold's geometry and architecture. FDM offers several advantages in bone tissue engineering, including its capacity to produce scaffolds characterized by extensive porosity and interconnected pore structure [203]. The movement of cells, the diffusion of nutrients, and the elimination of waste products are all made possible by these porous structures and are necessary for the effective regeneration of bone. With FDM it is possible to replicate the complex architecture of actual bone tissue and manipulate the scaffolds porosity, pore size, and overall shape by varying process parameters like layer height, nozzle diameter, and printing speed. The possibility to include bioactive substances into the scaffold during the creation process, such as proteins, growth hormones, or medications, is another benefit of FDM. These bioactive compounds can provide localized and sustained administration to improve bone regeneration and encourage tissue integration. They can also be coated onto the scaffold's surface or encapsulated within the polymer matrix [204].

Figure 13: Schematic representation showcasing the working principle of fused deposition modelling (FDM) 3D printing (Reproduced with permission[205])

11.2.3 Digital light processing (DLP) and Stereolithography (SLA)

Several fabrication techniques although, offer range of efficacies, digital light processing (DLP) and stereolithography (SLA) offer some unique advantages because of their ability to form into complex shapes, fabrication of pore sizes of different ranges[92,194]and the possibility of incorporation of particulate reinforcement[206]. Besides, the detailed and accurate nature of these methods allows for the production of unique porous microarchitectures that closely resemble the natural composition of bone tissue[176]. Furthermore, the layer-by-layer nature of these AM techniques enables the incorporation of functional components, such as growth factors, drugs, or bioactive molecules, within the scaffold during the fabrication process[207]. This can enhance the biological performance of the scaffold by promoting tissue regeneration, angiogenesis, or targeted drug delivery.

Both DLP and SLA are light based 3D printing technique which utilize a photo-polymeric liquid resin as a raw material which upon irradiation with UV light (DLP) or laser (SLA) results into a completely cross-linked material with rigid structures[208]. The major difference between both the technologies, apart from the irradiation source, is the method of light projection. The DLP 3D printing operates on a mask projection technique wherein an entire part cross sections based on CAD slices is printed at once layer by layer, whereas in stereolithography a 2-photon polymerisation (2PP) approach is used where spot crosslinking mechanism takes places resulting in tracing the CAD slice point by point [132,209]. Other advantages of DLP and SLA 3D printing techniques include the possibility of selection from a range of biocompatible and biodegradable photopolymers available. Selective photopolymers have been shown below in table 6. These materials can be tailored to exhibit desired mechanical properties, degradation rates, and biological responses, ensuring optimal integration with the surrounding tissue and facilitating the gradual replacement of the scaffold with newly formed bone [210].

Despite their advantages, DLP and SLA techniques face some challenges too. Issues such as material shrinkage, and the need for post-processing steps should be addressed to enhance their practical applications in bone scaffold development. Additionally, concerns related to the potential cytotoxicity of unreacted monomers and photoinitiators must be carefully evaluated. As a result, appropriate choice of photopolymeric resin, additives and reinforcements are crucial for their potential use in bone scaffold development [137].

Table 6: Biopolymeric photocurable resins with their features

Photo polymer	Tissue application	Advantages	Disadvantages	References
Poly (lactic-co-glycolic acid) (PLGA)	Bone, Cartilage, Muscle, Nerve, Skin	Biodegradable, tunable degradation rate, FDA approved	Can be brittle, potential inflammatory response	[211]
Poly (?-caprolactone) (PCL)	Bone, Cartilage, Skin, Vascular Grafts	Biodegradable, good printability, tunable properties	Low mechanical strength, slow degradation rate	[125]
Poly (ethylene glycol) (PEG) Hydrogels	Cartilage, Neural tissue, Skin	Highly tunable properties, good biocompatibility	Lower mechanical strength compared to some other options	[126]
Poly(?-glutamic acid) (PGA) Hydrogels	Bone, Cartilage, Neural tissue	Biodegradable, excellent cell adhesion	Can be brittle, pH sensitivity depending on formulation	[109]
Polydopamine (PDA)	Neural tissue, Skin, Vascular Grafts	Biocompatible, promotes cell adhesion	Limited printability depending on formulation	[212]
Polypropylene Fumarate (PPF)	Bone, Cartilage, Neural tissue, Skeletal muscle	Biodegradable, tunable mechanical properties, injectable	Lower printability compared to some other options	[213]
PEGDA (Poly (ethylene glycol) diacrylate)	Cartilage, muscle, and neural tissue engineering	Highly tunable properties, good biocompatibility	Potentially limited default mechanical strength	[214]
Thiol-ene based polymers	Cartilage neural tissue, skin, blood vessels	Highly tunable properties, good biocompatibility	Limited commercially available options, requires specific curing mechanisms.	[207]

Overall, DLP and SLA have emerged as promising fabrication techniques for bone scaffold development, offering versatility, precision, and the ability to create complex structures tailored to specific biological and mechanical requirements. The above techniques, however, have been explored not just with polymeric resin material but also in fabricating porous ceramic bone scaffold, in which case, polymeric resin is used as a binder. The process involves preparation of polymer ceramic suspensions in suitable proportions followed by 3D printing with DLP/SLA and later debinding and sintering the green bodies to achieve porous ceramic scaffolds [215]. However, no reported studies have significantly explored the possibility of using either DLP or SLA technique towards fabrication of composite bone scaffolds through a biomimetic approach with native bone like materials and microarchitecture.

DLP's mask projection technique allows for the quick fabrication of entire layers at once, leading to quicker build times compared to SLA. This makes DLP ideal for situations where speed is crucial, such as in making customized implants [216]. On the other hand, SLA's point-by-point polymerization method offers superior resolution and surface finish, making it an attractive choice for applications that require intricate details or smooth surface finishes[34,217]. The high accuracy of SLA also allows for the fabrication of complex internal architectures, which can be tailored to specific mechanical or biological requirements. Figure 14 shows schematically the working principle of both the DLP and SLA 3D printing technique.

Figure 14: Schematic representation showing working principle of a) stereolithography (SLA) and b)Digital light processing (DLP) 3D printing (Reproduced with permission [218])

11.2.4 Binder Jetting

Within the realm of 3D printing methodologies, the generation of intricate porous architectures that replicate native bone's structural organization holds immense potential for bone tissue regeneration applications. Binder jetting as a 3D printing technique stands out as a prominent approach in this context due to its inherent capabilities. A schematic representation of binder jetting process has been shown below in figure 15. It is an additive manufacturing method in which a liquid binder is selectively deposited on successive layers of powder bed to form 3D objects [219]. In the field of bone tissue engineering, binder jetting is used to prepare scaffolds which have uniform porosity, pore size and interconnectivity on the microscale, highly critical for cell infiltration, nutrient diffusion and vascularization [220]. By adjusting these characteristics, biomimetic structures that approximate the hierarchical structure of natural bone can be created, which may improve osteoconductivity and osteoinductivity [3]. Binder jetting's compatibility with a broad variety of materials is one of its main benefits for bone tissue regeneration. Because of their resemblance to the inorganic component of natural bone, calcium phosphate-based ceramics, like hydroxyapatite (HA) and ?-tricalcium phosphate (?-TCP), are frequently utilized [59]. The production of biocompatible and osteoconductive scaffolds is possible by processing these materials into fine powders appropriate for binder jetting [221]. Additionally, the printing of structures with growth factors and bioactive substances is made possible by binder jetting. As an illustration, scientists have effectively added bone morphogenetic proteins (BMPs) to calcium phosphate scaffolds, exhibiting increased osteogenic potential bothin-vivoandin-vitro[221]. One major benefit for encouraging bone regeneration is the scaffold's three-dimensional structure, which allows for the local delivery of bioactive chemicals.

Binder jetting's adaptability even extends to the design of implants tailored to individual patients. Custom-fit scaffolds that precisely match the geometry of bone abnormalities can be designed and manufactured with the use of medical imaging data, such as CT scans[222]. This customized strategy has demonstrated potential in treating difficult maxillofacial and craniofacial abnormalities, where accurate anatomic reconstruction is essential. Binder jetting for bone tissue regeneration has potential, however there are a few drawbacks. Post-processing procedures like sintering are frequently necessary for printed scaffolds mechanical qualities in order to attain sufficient strength for load-bearing applications [223]. The inclusion of biologics that are sensitive to temperature may be limited by this heat treatment. Furthermore, it is still very difficult to match the pace of scaffold breakdown to the rate of new bone production [224].

Figure 15: Schematic representation of binder jetting process (Reproduced with permission[3])

11.2.5 Bioprinting

Bioprinting is an additive manufacturing process, which has the capacity to produce patient-specific structures that closely resemble the intricate structure and makeup of the native tissues ranging from bones to skin. By utilizing a layer-by-layer deposition technique, this technology blends biomaterials, cells, and growth factors to produce three-dimensional (3D) structures with exceptional accuracy and repeatability[225]. One of the key advantages of bioprinting for bone tissue regeneration is the ability to create scaffolds with tailored porosity and interconnected pore networks, both necessary for cell migration, nutrition transport, and vascularization [220]. These scaffolds can be made to resemble the trabecular structure of bone, offering both mechanical support and a setting that is favourable for the attachment, growth, and differentiation of cells [226]. A range of biomaterials, including synthetic polymers like polycaprolactone (PCL) and poly (lactic-co-glycolic acid) (PLGA) and natural polymers like collagen, gelatin, and alginate, have been investigated for the bioprinting of soft tissue and hard tissue structures. Figure 16 shows a schematic representation of process of tissue engineered scaffolds via bioprinting approach. For the purpose of improving osteoconductivity and osteoinductivity, bioink can be mixed with cells and bioactive substances such as tricalcium phosphate, hydroxyapatite, or bioactive glasses[227,228]. Bioprinting techniques enable the integration of living cells, such as osteoblasts or mesenchymal stem cells (MSCs), into the printed constructions, in addition to biomaterials [229]. These cells aid in regeneration and have the ability to develop into bone-forming osteoblast cells. Moreover, the addition of growth factors can encourage angiogenesis and osteogenesis, respectively, such as vascular endothelial growth factors (VEGFs) and bone morphogenetic proteins (BMPs) [230].

Bioprinting has been used to generate bone tissue models for long bone deformities, maxillofacial defects, and craniofacial defects. Drug screening,in-vitroresearch, and biomaterial testing prior to in vivo applications can all be conducted using these models. Furthermore, the ability of bioprinted constructions to regenerate bone tissue has been demonstrated by their effective implantation in animal models[231]. Many obstacles still need to be overcome in spite of the encouraging developments in bioprinting for bone tissue regeneration. Developing biomaterials with the best mechanical qualities is one of these, as is preserving cell viability and function when printing, and including vascularization techniques to guarantee that the printed constructions receive enough oxygen and nutrients [232]. The creation of patient-specific constructions with regulated design, composition, and biological cues is made possible by bioprinting, which provides a potent method for bone tissue regeneration. To translate this technology into clinical applications and enhance patient outcomes, it will be imperative to tackle the outstanding hurdles as the technology progresses.

Figure 16: Schematic representation of tissue engineering using bioprinting approach (Reproduced with permission [227])

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