Collagen-hydroxyapatite based scaffolds for bone trauma and regeneration: recent trends and future perspectives

ABSTRACT

Regenerative therapy, a key area of tissue engineering, holds promise for restoring damaged organs, especially in bone regeneration. Bone healing is natural to the body but becomes complex under stress and disease. Large bone deformities pose significant challenges in tissue engineering. Among various methods, scaffolds are attractive as they provide structural support and essential nutrients for cell adhesion and growth. Collagen and hydroxyapatite (HA) are widely used due to their biocompatibility and biodegradability. Collagen and nano-scale HA enhance cell adhesion and development. Thus, nano HA/collagen scaffolds offer potential solutions for bone regeneration. This review focuses on the use and production of nano-sized HA/collagen composites in bone regeneration.

Plain language summary

Article Highlights.

Current treatment options for bone trauma

• The skeletal system's structure, consisting of cortical and trabecular bone, is vital for mobility, support, protection, blood cell distribution, nutrient regulation and endocrine function.

• High-impact incidents, such as vehicle collisions and falls, are primary causes of bone deformities and trauma, leading to significant economic, psychological and social burdens.

• Large segmental bone abnormalities are mainly treated through surgical procedures like vascularized bone transplantation and bone transfer, with treatment outcomes influenced by various patient-specific factors.

• Ideal bone transplants should be osteoinductive, osteoconductive, osteogenic, readily available and cost-effective, with autografts being the most superior option due to their high efficacy and low risk of immune reaction.

• Allografts, derived from the same species, are treated to mitigate immunogenic response and infection risks, available in forms such as intact segments, cortical-spongy segments, bone chips, powder and demineralized bone matrices.

• Xenografts, particularly from bovine sources, provide a porous hydroxyapatite-based structure that supports mechanical reinforcement, osteoconduction and angiogenesis, commonly used in maxillary sinus floor elevation treatments.

Emergence of scaffolds in bone regeneration

• Scaffolds play a crucial role in supporting tissue repair and regeneration, facilitating cellular deposition and morphogenic signals essential for bone regeneration in conditions like osteoporosis and bone malignancies.

• Exploration of biomaterials such as bioactive glass and calcium phosphate cements reveals promising outcomes, with bioactive glass demonstrating superior remodeling capabilities over autogenous bone and injectable cements providing customizable defect filling in procedures like cranioplasty.

• Surface characteristics, including chemistry, topography and roughness, profoundly influence scaffold integration and cellular behavior, impacting bone-forming cell adhesion, proliferation and protein binding essential for effective bone regeneration.

• Pore size is a critical parameter in scaffold design, with larger pores (>300 μm) favored for bone ingrowth, although optimal bone regeneration may vary with pore size, highlighting the complex connection between pore size, bone growth and scaffold strength.

• Biomaterial diversity, ranging from natural polymers like chitosan and collagen to synthetic polymers such as PCL, PLA, or PLGA, offers a variety of biodegradable options tailored for bone tissue engineering applications.

Role of collagen in bone regeneration

• Natural biopolymers, such as collagen, are gaining attention in bone regeneration due to their resemblance to native extracellular matrix (ECM) components, reducing the risk of immunological reactions and facilitating tissue regeneration.

• Collagen, a predominant structural protein in vertebrates, regulates crucial biological processes like cell attachment, migration and differentiation, making it a promising candidate for scaffolds in bone tissue engineering.

• Despite collagen's favorable biological properties, its mechanical weaknesses hinder widespread utilization, prompting research into enhancing its durability through crosslinking methods.

• Chemical and physical crosslinking agents, such as choline-based salts, have been explored to strengthen collagen scaffolds, improving their stability and mechanical properties.

• Incorporating bio-ceramics like HA into collagen matrices can further enhance scaffold strength while maintaining porosity, offering significant potential for advancing bone tissue engineering applications.

Hydroxyapatite in bone regeneration

• Hydroxyapatite (HA), a key component of bone composition, exhibits a complex crystal arrangement with uniform orientation of hydroxyl groups, contributing to its structural integrity and biocompatibility.

• HA, comprising approximately 20% of bone composition, possesses chemical elements analogous to vertebrate hard tissues, making it a biodegradable biomaterial with significant potential in orthopedics and maxillofacial surgery.

• The mechanical properties of HA can be tailored through synthesis methods like sintering, influencing its durability and in vivo biodegradability, with lower sintering temperatures enhancing biodegradability while compromising mechanical strength.

• Recent advancements in artificial bone grafts have focused on HA-based materials, either pure or in composites, due to their biocompatibility, biomineralization capabilities and porous interlinked structure, essential for osteoconduction and osteointegration.

Utilization of collagen/HA composites in bone related ailments

• Collagen/HA composites offer synergistic osteoconductive effects, making them ideal for bone regeneration applications, with potential enhancements through additional active ingredients.

• Various fabrication techniques such as gel casting, compacting and computer-assisted rapid prototyping enable precise control over the mechanical and biological properties of collagen/HA scaffolds, enhancing their suitability for tissue engineering.

• The mechanical properties of collagen scaffolds are augmented by HA incorporation, with factors like particle size influencing cellular adhesion and proliferation, demonstrating enhanced osteogenic potential compared with pure collagen scaffolds.

• Synthesis methods for HA/collagen composites, including co-precipitation and direct blending, yield hierarchical structures resembling natural bone, with potential enhancements through the incorporation of growth factors or biologically active substances.

• Porosity plays a significant role in the biological characteristics of HA/collagen composites, with increased porosity facilitating cell migration, vascularization and mechanical adaptability, despite potential reductions in mechanical strength.

• HA/collagen scaffolds can be utilized for drug delivery in bone-related disorders, enabling sustained release of therapeutic agents to target specific sites, with potential applications in tumor inhibition and antimicrobial treatment, offering a promising avenue for personalized medicine in bone therapeutics.

Future perspective

• Collagen and HA ceramics offer strong biocompatibility, tailored mechanical strength and osteoconductivity for bone regeneration.

• Ongoing research integrates bioactive compounds into HA/collagen hybrids to enhance osteoinductivity and pharmacological intervention in bone pathologies.

• Nano-HA promotes osteoblast adhesion and bone regeneration, enhancing scaffold effectiveness.

• Collagen scaffolds, though prone to degradation, are being optimized for stability and controlled degradation, ensuring suitability for bone regeneration.

• Advanced fabrication techniques like 3D printing aim to improve scaffold mechanical properties and drug delivery capabilities, advancing treatment effectiveness.

• Multifunctional composites, combining growth stimulants and antibiotics in HA/collagen scaffolds, show promise for tailored therapeutic approaches, enhancing treatment outcomes in bone defects and pathologies.

1. Background

The worldwide surge in the aging population, traumatic injuries, tumor removal surgeries, bone fractures and defects, together with the economic impact of bone surgeries, have prompted researchers to investigate inexpensive materials and methods for bone repair [1]. Fractures of the bones happen frequently as a result of significant impact accidents, for instance, vehicle and motorcycling accidents or sports-related injuries. In emerging nations, the surge in economic activities and subsequent work environments have led to workplace accidents becoming a significant contributor to fractures [2]. The American Academy of Orthopedic Surgeons has reported that there are approximately 6.3 million bone fractures recorded in the US each year [3]. Additionally, there are approximately half a million surgical treatments performed yearly in the US for the treatment of fractures, and relief of acute back pain via spinal fusion treatments [4]. Furthermore, musculoskeletal issues not only have an impact on the healthcare economics aspect but also pose challenges to the broader field of economics. These issues indirectly affect the work life of individuals and consequently create a financial burden [5]. There is an anticipated rise in the prevalence of musculoskeletal diseases in coming years, primarily due to the global elderly population and the decrease in the younger population [6]. Therefore, developments in the field of bone healing has focused on discovering and creating novel materials to reduce the frequency of procedures and minimize the expenses associated with the materials utilized in regenerative procedures [7].

Implants, especially in areas that bear weight, have been constructed using metals that have the capacity to induce adverse allergic reactions and tumor [8]. Autografts, ceramic materials, polymer compounds and allografts are employed in regeneration techniques, particularly in areas subjected to minimal loads. It is crucial for implants to accurately replicate the tri-biological aspects, including wear and tear and lubrication [9]. Autografts remain the benchmark for bone regeneration [10,11]. They possess osteo-conductive and osteo-inductive properties due to their inherent ability to stimulate bone development through the presence of essential components. Furthermore, autologous transplants do not elicit an immunological response as they are derived from the individual themselves, hence reducing or eliminating the potential danger of disapproval by the body [12,13]. Still, autografts suffer from drawbacks such as extended operating hours, concurrent ailments, and a lack of available donor areas [14,15]. Other type of transplantation is allografts, when the bone graft is sourced from a different human and then transferred to the beneficiary [16]. Allografts, which constitute approximately 33% of the grafts employed by orthopedic surgeons annually in North America, are the second most utilized grafts, surpassed only by autografts. The utilization of allografts can address the drawbacks associated with autografts, such as comorbidities and scarcity of donor sites. However, the primary challenges lie in the expense, the likelihood of immunogenic reactions leading to reduced osteogenity and compromised mechanical qualities [17–19].

In order to address the limitations of existing remedies, there seems to be an increasing interest in utilizing synthetic or natural materials that possess specific biological, osteogenic and mechanical characteristics for bone tissue restoration. Tissue engineering (TE) technologies are commonly employed to facilitate tissue repair and restoration in injured organs or tissues. Large musculoskeletal defects have emerged as a prominent area of interest for biomaterial investigators, representing a significant challenge in the domain of tissue engineering [20]. Scaffolds can be the ideal candidates for bone repair that closely mirror the real bones in both architecture and chemical composition. The natural bone is characterized by a seven-hierarchical-level framework, with calcified collagen fibrils being the second level of hierarchies [21]. Composite materials comprising collagen, protein that is found in the highest quantity in the body, and hydroxyapatite (HA), the primary constituent of natural bone, are becoming important in both fundamental research and therapeutic uses for bone tissue regeneration [22]. Furthermore, it has been observed that mineralized collagen scaffolds demonstrate superior osteo-conductive capabilities and display elevated levels of osteogenic gene expression compared with unmineralized collagen scaffold [23]. Pharmaceuticals, development factors and various other small molecules were more effectively delivered over a longer period of time via these scaffolds [24]. Collagens, which are widely employed as scaffold material, possess remarkable biodegradable properties, low antigenic potential following telopeptide removal and exceptional biocompatibility [5]. Furthermore, it is widely acknowledged that raw collagen exhibits insufficient bioactivity to effectively induce bone forming capability. The incorporation of bioactive components remains a highly favored technique for enhancing the physical strength, biological activity, and the process of osteogenesis of theses scaffolds [25,26].

Numerous studies have been published on collagen-based nanocomposite structures for bone regrowth. Several reviews primarily examine the preparation, characteristics and utilization of scaffolds derived from collagen and HA. Although the collagen scaffold is often overlooked, it is crucial to consider the components employed in its construction. Given the increasing and ongoing interest in creating and using collagen-based composite scaffolds, the purpose of the article is to provide a detailed summary and classification of collagen-hydroxyapatite composite scaffolds.

This review examines the chemical compositions, methods of distribution, benefits, drawbacks and uses of both commercially available solutions and naturally occurring biopolymers for bone repair and regenerative therapies. In addition, we emphasize on the importance of biocompatibility and degradation properties in assessing the appropriateness of these biomaterials for therapeutic applications. We also aim to analyze the current state of biomaterials in regenerative medicine, with the goal of offering valuable insights into their possible applications, constraints and future prospects. Gaining a comprehensive understanding of the characteristics and effectiveness of these biomaterials is crucial for progressing the research and creating inventive approaches for tissue engineering and regenerative therapies.

2. Current treatment options for bone trauma

The musculoskeletal system consists of several types of bones, including long, short and irregular bones. These bones have multiple functions, such as facilitating mobility, providing skeletal support and protecting essential organs. Additionally, the skeletal system plays a role in distributing blood cells, nutrients and regulating the endocrine system in the human body [27,28]. The structure of every bone consists of cortical bone, which is the exterior layer that is dense and compact, and trabecular bone, which is the inner layer that is porous and spongy. These two types of bone determine the structure, firmness, and mechanical stability of the bones. The hardness of the bone is achieved by mineralization and a matrix rich in collagen [29,30]. The periosteum, an external connective tissue sheath, envelops the outer cortical bone and houses blood arteries, nerve fibers, bone marrow cells and osteoclasts, all of which play a crucial role in the development and healing of the bone. The endosteum is a membrane lining located inside the trabecular bone and bone marrow area. It contains blood vessels, osteoblasts and osteoclasts [31]. The primary responsibility for bone formation and development, which occurs continuously throughout our lifespan, lies with the osteoblasts (cells that make bone) and osteoclasts (cells that break down bone) [32].

Bone deformities or traumas primarily result from high-impact incidents, such as motor vehicle collisions, falls from significant heights, or severe blunt force injuries [33]. Postoperative infection following internal fixation might result in the development of substantial segmental sequestration, and evident bone defects will arise following debridement [34]. Managing patients with prolonged cycles and numerous comorbidities poses significant challenges in terms of treatment. Consequently, it imposes significant economical, psychological and social burdens on patients, thus, severely impacting their quality of life. Large segmental bone abnormalities are primarily addressed through surgical surgery. Currently, the primary surgical approaches for treating lengthy bone defects larger than the critical-sized bone defects (5 cm) primarily involve vascularized bone transplantation, bone transfer and similar treatments [35,36]. Several variables affect the results of therapies for bone deformities, and it is imperative to take into consideration while picking the most suitable surgical procedure and postoperative management for each individual [37]. Various treatment approaches have been outlined for addressing bone defects, considering the patient's medical history, the specific locality of the defect and the physician's personal expertise [38].

An optimal bone transplant possesses osteoinductive, osteoconductive and osteogenic qualities, while also being easily obtainable in the appropriate quantity, with minimum concerns of infectious illness transmission, and at a moderate cost [39]. Annually, the United States conducts about 500,000 bone graft operations, positioning it as the second most often transplanted tissue after blood [40]. Autografts are often regarded as the most superior option among the several materials available for bone restoration [41]. Their ability to maintain the viability of bones cellular structures at the extraction site, together with their capacity to promote the development of mesenchymal stem cells (MSC) into osteoblasts through the presence of growth factors, contributes to their osteogenic and osteoinductive capabilities. By having the same origin as the diseased tissue, the likelihood of an undesirable unwanted immune-mediated adverse reaction can be avoided, resulting in a efficacy rate above 95% [42]. The iliac crest is the most commonly used location for collecting autografts. From this area, both cortical and cancellous grafts can be obtained, depending on the specific requirements. Conversely, the acquired bone might be strategically positioned to optimize its fit with the recipient location. Other suitable sites for grafting include the distal femur, particularly for a spongy graft, the peroneal shaft for a structural graft, as well as the ribcage and distal radius [43].

Allografts are natural substances derived from a member of the identical species. They can be acquired from a suitable living donors or from deceased individuals as a source of bone material. Allograft material could be created in three major forms, namely fresh, thawed and freeze-dried. Although fresh and thawed homologous materials possess greater osteoinductive characteristics, their usage is now seldom owing to the heightened danger of host immunological response, restricted viability, and higher risk of infection transmission [44]. Allografts drawbacks are typically mitigated through lyophilization, as well as other tissue processing techniques like mechanical debridement, ultrasonic cleaning and particularly sterilizing with gamma radiations [45]. These grafts exhibit favorable histocompatibility and are offered in diverse formats, ranging from intact bone segments, cortical-spongy segments and cortical pieces, to minute fragments such as bone chips, powder and demineralized bones matrices. Customized forms can be created to fulfill the specific needs of the recipient sites [46].

Xenografts refers to the materials obtained from a species that is genetically distinct from the species it is being used on. The inorganic constituent of bovine bones tissue is isolated and processed to create a porous material based on hydroxyapatite [47]. The resultant porous structure closely mirrors the composition of humanoid bone and can effectively offer mechanical reinforcement and promote the therapeutic process through osteoconduction [48]. The presence of this permeable framework promotes the growth of fresh blood vessels via angiogenesis, which is accountable for the creation of fresh bone tissues. Bovine bone xenografts have been widely utilized in maxillary sinus floor elevation treatments and for providing implantation support, owing to their exceptional durability and little immunogenicity [49]. Table 1 entails the existing prominent commercial products for bone healing.

Table 1.

Predominant commercialized solutions available for the purpose of bone repair.

Product	Chemical composition	Mode of delivery	Advantages	Ref.
Hydroxyapatite (HA)	Ca5(PO4)3OH consists of phosphate ions/groups and calcium	In the form of beads and powder	The material is osteointegrative and osteoconductive, with a porous structure that closely resembles natural bone	[50]
Vitoss	β-TCP (tricalcium phosphate) with a high porosity of up to 90%	In the form of granules, foam pack or strips	The process of bone regeneration involves the provision of a scaffold that facilitates the remodeling of bone, enabling the penetration and adhesion of cells, rather than the gradual replacement of bone tissue	[51]
MagnetOs	βTCP and 25%–35% HA, embedded in a fast-resorbing polymer carrier	Putty formulation	Bone void filler for non-structural holes and gaps. It enhances osteogenesis, even in non-osseous tissue, resulting in a more reliable fusion process	[52]
Actifuse	Si-CaP	The product is presented as granules and is contained in a syringes loaded with a flowable substance	Promotes the growth of bone tissue via use of silicate material that promotes bone formation	[53]
Xenograft	Cancellous bovine bone that has undergone chemical and heat treatment	Typically, it is distributed in either tiny or large granules	It has shown excellent mechanical support and osteoconduction promotes angiogenesis	[54]
Accufill	Composed of amorphous CP and DCPD	Administered by syringe containing a liquid substance	Undergoes cellular remodeling to transform into real bone; possesses physical characteristics similar to cancellous bone	[55]
HydroSet	Tetra-calcium phosphate	Filled syringe containgin flowable material	It has excellent osteoconductive and osteointegrative potentials	[39]
Ceraform	Beta tricalcium phosphate and HA	Preformed granules	It is resorbable after 2 years and has osteoconductive properties	[56]
ChronOs inject	Beta tricalcium phosphate (Porous)	Preformed granules	It is resorbable and is osteoconductive	[57]
Surgibon	Beta tricalcium phosphate and HA	Preformed granules	It is resorbable and is osteoconductive	[39]
NovaBone	Calcium-phosphorus sodium–silicate particles mixed with a synthetic binder	Granules putty	It is resorbable and is osteoconductive	[52]

3. Emergence of scaffolds in bone regeneration

The word ‘scaffold’ is used to describe a constructed impermanent platform that is utilized to provide support, facilitate repairs, or improve the functionality of a structure. This can be accomplished over a range of scales and dimensions, employing diverse support mechanisms that are contingent upon the specific form and intended application. Figure 1 depicts a few important elements of scaffolds utilized in tissue engineering. The tissue engineering approach comprises several essential elements: a scaffold that replicates the specific tissue requiring regeneration, cells responsible for depositing the extracellular matrix (ECM) and morphogenic signals that facilitate cellular differentiation [58]. The primary goal of bone tissue engineering methods is to facilitate the process of bone regeneration, a necessary process in various clinical scenarios such as osteoporosis, bone illness and the removal of muscles and joints sarcoma, often leading to significant bone abnormalities. Tissue engineered materials for orthopedic treatments can be categorized into two distinct groups i.e. those that promote bone development and others that serve as a long-term replacement for bone [59]. Numerous investigations have been conducted to evaluate the utilization of several biomaterials in the context of bone repair. Bioactive glass (BG) is a fascinating biomaterial, primarily because it has the capacity to create a responsive carbonated HA layer [60]. A controlled trial was done on 21 patients who took part in a 14-year follow-up study to evaluate the safety and effectiveness of BG and autogenous bone (AB) as alternatives for bone grafts in non-cancerous bone tumors. The MRI revealed predominantly or partially adipose bone marrow, and in the cohort with big bone tumors, traces of glass grains were also detected, confirming the safety of BG. The bone substitute was highly tolerated and yielded favorable long-term outcomes without interfering with bone formation in youngsters [61].

Figure 1.

Important factors associated with scaffold development for tissue engineering.

Calcium phosphate cements have been formulated to be administered in a malleable form and subsequently solidify over a period of several hours after being implanted. Injectable cements (approved by FDA in 1998) containing minerals are commonly employed to address bone deficiencies due to their chemical makeup closely resembling the minerals of bone ECM. One benefit they have over blocks, granules and pellets is the ability to fill the defect with a specific amount [62,63]. A retrospective analysis of medical records was conducted to directly compare the efficacy and results of autografting, bone cements and demineralized bone matrix in regards to the functionality and outcomes. Six patients underwent reconstruction using demineralized bone matrix as the main reconstructive material. The reconstructed materials for cranioplasty consisted of bone cement in 17 patients, demineralized bone matrix in six patients and bone autografts in five individuals. The study found that the use of autograft or bone cement resulted in much lower incidence of residual deformities and revision [64].

The surface characteristics of materials are of paramount importance in their efficient design and implementation in healthcare, as the surface serves as the primary point of contact with the living system. To promote bone development in TE scaffolds, it is critical to optimize factors like surface features and their chemistry, surface energies, wettability, pores size, form and mechanical characteristics [65]. Surface characteristics, including chemical composition and topography, can impact the adherence and growth of cells. These features play a crucial role in various biological processes that occur upon implantation, such as protein binding and bone regeneration [66]. A study revealed that cells displayed projecting filopodia within honeycomb-like structures made of Poly-capro lactone (PCL)/nano-hydroxy apatite (nHA), indicating a favorable cell spread. In order to achieve optimal scaffold integration, it is advantageous to have a material surface with the degree of roughness as this can promote the connection, expansion and differentiation of bone-forming cells that rely on anchoring [67]. Another study discovered that factors such as the wettability and interface free energies can also have an impact on cell proliferation. The study found that surface free energy, rather than roughness, had a crucial role in cellular adhesion and multiplication [68].

A study demonstrated the significance of utilizing a permeable scaffold in bone restoration. This research evaluated the usefulness of a HA porous scaffolds for delivering bone morphogenetic protein-2 (BMP-2) [69]. Pore size is a crucial characteristic as excessively small pores can lead to blockage by cells, while also influencing protein adsorption, cellular motility and osteoconduction. Pore diameters larger than 300 μm are preferable for bone ingrowth compared with lower pore sizes [70]. Fukuda et al. conducted a comparative analysis of osteoinduction in comparable conditions using varied pore sizes (500, 600, 900 and 1200 respectively) of Ti implants. The study found that a pore size of 500–600 μm demonstrated exceptional osteoinductive properties [71]. In a separate investigation conducted by Prananingrum et al., showed that after a period of three weeks of being inserted into the rabbit calvaria, the scaffolds with a pore size of 600 μm exhibited a more significant growth of bone tissue. However, after a duration of 20 weeks, the pore size measuring 100 μm exhibited an advanced level of bone ingrowth related to the others. The results of this study suggest that the regeneration of bone into porous scaffolds is influenced by the size of the pores, with the most significant bone growth observed in the 100 μm. The pores size is a crucial parameters that enhance biological processes by facilitating bone ingrowth and the infiltration of cells and nutrients. However, these properties can conflict with others as the pore size increases, the loss in the durability of the scaffold is enhanced. This drop in strength can potentially contribute to failures in vivo [72].

Currently, scaffolds can be made from either natural or manmade biomaterials, which can be either biodegradable or non-degradable. For instance, natural polymers such as chitin, chitosan and collagen are currently being utilized in tissue regeneration applications [73]. Biodegradable synthetic polymers, such as PCL, poly-lactic acid (PLA), or poly(lactic-co-glycolic) acid (PLGA), can be tailored with various characteristics for use in bone tissue engineering [74].

4. Role of collagen in bone regeneration

While polymers made from synthetic materials that are engineered to possess specific elasticity and molecular weights exhibit encouraging outcomes, they are susceptible to acidic degradation, which subsequently leads to the release of detrimental byproducts that impede the process of tissue healing. Synthetic polymers exhibit some drawbacks, including reduced durability, inadequate form retention, restricted cell adherence and the possible production of hazardous by-products, among others [75]. In recent times, there has been a notable increase in the investigation of natural biopolymers for their potential application in medicine, particularly as biomaterials for the regeneration of bones. The majority of biopolymers found in nature are composed of polysaccharides, proteins, matrix proteins and ECM components, among others. These materials exhibit similarities to the cellular elements of the human body's tissues, therefore enabling them to imitate the native ECM of tissues. Therefore, the likelihood of allergy or immunological reactions to these biopolymers is significantly reduced when they are utilized as scaffolds in implantation procedures [76,77]. Table 2 presents a concise summary of the various kinds of naturally derived biopolymers that are frequently employed in the domain of regenerative therapies.

Table 2.

Diverse range of naturally occurring biopolymers and their usage within the field of regenerative therapies.

Biopolymer	Source	Application	Limitations	Ref.
Fibrin	The polymer of glucose derived from botanical sources, such as natural fibers like cotton	The application of this material encompasses various medical purposes, such as dressing wounds, skin regeneration and healing of injured peripheral nerves	Demonstrate inadequate morphological coherence	[78]
Starch	Derived from botanical origins such as grain, tapioca and potatoes	Application in cartilage and bone tissue regeneration	The structural properties of the material are substandard	[79]
Agarose	Agarose is a biopolymer with a high molecular weight that is derived from sea red algae	Assists in the process of regenerating diverse organs and promoting the regenerative process of skeletal tissues	It does not promote cellular adhesion	[80]
Xylan	A polysaccharide that is present in the cell walls of dicots and grass	Utilized in the context of bone regrowth, healing wounds and other related applications	The functionality is compromised when it gets immobilized on surface	[81]
Collagen	Collagen is derived from several sources such as bovine, porcine, ovine and human placenta	The application of collagen is prevalent in various fields such as wound treatment, delivering drugs, tissue restoration	One significant limitation pertains to its inadequate mechanical characteristics	[82]
Chitosan	The exoskeletons of several organisms, such as insects, nematode, yeast fungus and mushrooms, are sources of derivation	This polymer finds utility in various domains like as orthopedics, cornea and heart repair, wound recovery and delivery of drugs	The primary constraint associated with this polymer is in its suboptimal mechanical strength	[83]
Hyaluronic Acid	Naturally present in the human body, particularly in connective tissues	Used in tissue engineering, wound healing and as a Visco supplement in joints	May cause inflammatory response in some individuals	[84]
Silk	Produced by silkworms or spiders	Used for bone and cartilage; neural tissue engineering; tendon and ligament; sutures in wound treatment	The significant brittleness of the material poses challenges in its handling as a scaffold biomaterial	[85]
Cellulose	Obtained from several sources (i.e., bacteria, wood, plants, pulp and cotton)	Used in bone, cartilage, adipose, cardiac, skin and vascular tissue engineering	Cellulose's insolubility in water and other common solvents is a significant drawback for its use in biological applications	[86]

In vertebrate organisms the biological processes involved in support, including attachment of cells, migration, differentiation and multiplication, are predominantly regulated by the structure protein known as collagen. The collagen composition exhibits the ability to express ligands necessary for cell attachment and plays a vital part in the adsorption of osteoblasts and the accumulation of minerals [87]. Hence, collagen possesses the capacity to facilitate the regeneration of compromised bone through the process of mineralization. Moreover, collagen is highly prevalent in tissues, which accounts for its capacity to interact with a diverse range of cells inside those tissues. Despite the numerous advantages, the diminished mechanical qualities impede its extensive utilization. Nevertheless, recent reports have indicated that the application of an appropriate crosslinking agent can enhance the durability and strength of the material [88]. Various types of cross-linking agents can be classified into two main types: chemical cross- linking agents and physical cross-linkers. Glutaraldehyde is a commonly employed chemical a cross-link for collagen. Nevertheless, due to its increased toxicity, it has been substituted by choline-based salts for the purpose of scaffold synthesis [89]. Tarannum and colleagues provided support for the claim that the durability of the collagen structures is enhanced by the electrostatic bonds that occur between collagen and choline salts. The occurrence of this event was enabled through the interaction that took place between the OH groups present in collagen and choline. The electrostatic attraction, in its turn, induces conformational alterations that contribute to the stabilization of the protein structure [90].

The structural characteristics of scaffolds can be significantly enhanced through the process of crosslinking collagen, which can be achieved through either chemical or physical means. These crosslinking processes has been shown to greatly facilitate cellular adhesion and promote cellular proliferation within the scaffold. The prepared scaffolds, despite their biological activities, are crucial for their robustness due to their reduced strength. However, the scaffold's interconnected porosity is essential for efficient cell seeding, as it improves the rate of cell proliferation when seeded with cells [91]. In this context, the incorporation of bio-ceramics into the matrix of collagen is considered an optimal approach to enhance resilience while maintaining porosity. Mineralized collagen, comprising HA, is a primary constituent of the inherent bone structure [92]. The inclusion of HA within collagen structures has the potential to augment the mechanical strength of porous collagen, hence offering significant assistance in the progression of bone regeneration. According to the reported data, the integration of HA into the collagen matrix has been observed to enhance the compression modulus of the scaffolds [93].

5. Hydroxyapatite in bone regeneration

The composition of bone consists of a complex bio composite including collagen type I fibrils and HA in the form of Ca10(PO4)6(OH)2. Figure 2 depicts the schematic illustration of the crystal arrangement of HA crystal. In monoclinic HAP, the hydroxyl groups exhibit uniform orientation inside a given column and exhibit opposing orientation across columns. Conversely, in the hexagon structure of the crystal, adjacent hydroxyl groups have opposing orientations [94]. The relative proportions of collagen, HA and water inside bone are approximately 70%, 20% and 10% each, of the total amount of bone composition [95]. Among the several varieties of calcium phosphate cements, it is noteworthy that HA bioceramic possesses chemical elements that are analogous to those found in the bones and hard tissue of vertebrates. HA is classified as a biodegradable bio ceramic, making it a notable material within the area. Its application as an implant in orthopedics, dentistry and maxillofacial surgery has been found to facilitate both growth and osteointegration [96]. The surface of HA implant that is in contact with its surroundings exhibits an affinity for natural apatite found within the human body, hence facilitating the process of osteogenesis in the context of orthopedic implants. The mechanical properties of HA can be manipulated by modifying many factors throughout the synthesis process, including sintering temperatures, pH levels, concentrations of substances and duration. In order to enhance their mechanical properties, the HA transplants are typically subjected to a sintering process. The durability of the scaffold is reduced while the in vivo biodegradability is improved when the temperature at which it is sintering is below 800°C. The presence of several calcium phosphate forms in the ceramic graft may account for this phenomenon. Among these forms, HA exhibits the lowest level of biodegradability [97].

Figure 2.

Schematic illustration of the crystal arrangement of HA crystal. Adapted with permission from [98].

In recent decades, substantial progress and exploration have been undertaken in the realm of artificial bone grafts, specifically focusing on those composed solely of HA or including surface coatings of HA or polymer/HA based composites. In addition to its biologic compatibility and biomineralization capabilities, its mechanical properties and porous interlinked structure are also significant factors to consider. Furthermore, it is imperative that HA-based materials possess adequate osteoconductive and osteointegrative characteristics [99]. The characteristics of HA can be manipulated and customized through the implementation of various synthesis methods, as extensively documented in existing scholarly works. One innovative synthesis method that has gained attention is the creation of macroporous HA scaffolds through the pyrolysis of wood. This particular approach shows great promise in achieving three-dimensional hierarchy with linked porosity, making it very suitable for applications in orthopedic procedures [100,101]. HA demonstrates a comparatively lower degradation rate in relation to other CaP. HA has greater stability in aqueous environments in comparison to other CaP within the pH ranging from 4.2 to 8.0 [102].

HA demonstrate minimal cytotoxicity and immunological reactivity. Moreover, they have the ability to stimulate bone regeneration and offer substantial mechanical integrity, making them promising candidates for usage in bone substitute applications. The adjustability of the porosity of the HA material allows for the promotion of bone formation during the entire development process. This property enables the binding of antibodies, bone growth-promoting agents and bone regeneration regulators to the HA material, hence possibly facilitating its utilization in diverse applications for tissue engineering [103]. In contemporary medical practice, there is a growing utilization of HA and collagen-based formulations that have been enhanced through the incorporation of pharmaceutical agents, bioactive substances, metallic elements and carefully chosen nanoparticles. These modified compositions have found extensive application in the therapeutic management of conditions affecting the skeletal system. Compositions including of HA and collagen that have been modified with diverse modifications are employed in a wide range of biomedical applications, including tissue engineering of bones, vascular transplantation, cartilage regeneration and other implanted medical implants.

6. Utilization of collagen/HA composites in bone related ailments

The fields of tissue engineering and regenerative therapy are emerging disciplines within the scientific community that facilitate the creation of artificial replacements. HA and collagen have been identified as key components in enhancing bone regeneration within the field of materials science. The ideal characteristics of such compositions would include biodegradability, non-toxicity, non-immunogenicity and mechanical strength comparable to the native tissues they are intended to substitute. The concurrent utilization of these two substances has a synergistic osteoconductive impact. Optimal outcomes are achieved by the utilization of collagen-HA compositions that have been enhanced with additional active ingredients [104,105]. Multiple approaches have been employed to generate collagen/HA combinations suitable for tissue engineering applications. Currently, the prevailing techniques comprise gel casting process, compacting, computer-assisted rapid prototyping and three-dimensional printing. The collagen/HA composite offers the potential to create materials with precisely regulated mechanical characteristics and targeted biological function [106].

The utilization of collagen resorbable matrix enables the creation of multipurpose implants, wherein the HA phase serves as a platform for osteogenic cell proliferation subsequent to completing its biomechanical role of tissue fixation through the sorption process. When formulating a tissues-engineered scaffolds, it is imperative to consider that its morphology should align with the injured tissue that necessitates replacement. The scaffold must possess suitable structural and functional characteristics. Presently, there is a significant amount of ongoing research focused on bioactive scaffolds that have been modified with growth stimulants. These growth factors have the capability to speed up cellular proliferation and facilitate the mechanism of tissue repair [107]. The collagen-HA compositions with finely fibrous structure demonstrate favorable characteristics as a suitable substrate for the in vitro culture of osteoblasts. However, it is important to acknowledge that porous collagen/HA composites provide a favorable surface for the formation and development of osteogenic cells found in the bone marrow [108].

The mechanical properties of collagen scaffolding in their pure form exhibit a remarkably low strength, hence significantly constraining their potential for broader utilization in tissue regeneration. The incorporation of HA has been observed to enhance the compressive modulus of collagen scaffolds. The extent of this improvement is primarily influenced by factors such as the proportion of collagen, the quantity of HA, the procedures used for composite formation and the crosslinking techniques employed [109]. Furthermore, the enhancement of cellular adhesion can be achieved by HA with collagen scaffolds, hence increasing their surface area. Compared with micro-sized HA components to nano-sized HA particles, it can be observed that the latter exhibit enhanced effectiveness due to their increased surface area. Moreover, it is crucial to recognize that apatite possesses the capacity to form direct chemical bonds with the adjacent bone tissue. The presence of this particular property is expected to facilitate enhanced and expedited osseointegration between the scaffolds and the adjacent host bone tissue, particularly due to the inclusion of the HA. Cells exhibit greater proliferation and improved bioactivity when cultured on rough surfaces. Consequently, scaffolds including HA demonstrate increased cellular proliferation compared with scaffolds lacking the HA component [26]. It is also believed that utilization of micron-sized HA particles may result in inadequate resorbability, uneven distribution and fragile structures inside the resulting composite scaffold [110]. Cunniffe et al. proposed the incorporation of nano-sized HA inside collagen scaffolds. The collagen/nano-HA scaffolds that were produced had a significant porosity and interlinked structure. Additionally, the compressive modulus of the scaffold noticed a notable rise of 18-times [111]. Liu et al. created a three-dimensional, porous, human-like collagen (HLC)/n-HA scaffold cross-linked by 1,2,7,8-diepoxyoctane (DEO) for bone tissue regeneration with outstanding mechanical and biological properties (Figure 3). The scaffold with 6% (v/v) DEO (called nHD-2), had better mechanical properties and enzymatic hydrolysis resistance than those with 4 and 8%. The nHD-2 scaffolds had low cytotoxicity and good cytocompatibility compared with the other two scaffolds in cell viability, live cell staining and cell adhesion tests. After 1, 2 and 4 weeks, rabbits subcutaneously injected with nHD-2 scaffolds had better anti-biodegradation and histocompatibility than scaffolds containing 4 and 8% (v/v) DEO. The repair of rabbit bone lesions showed that nHD-2 scaffolds were better at directed bone growth and restoration than commercially available composite hydroxyapatite/collagen (HC). The results indicate that nHD-2 scaffolds' superior mechanical qualities, anti-biodegradation, biocompatibility and bone repair effects make them promising for bone tissue engineering [89]. Table 3 below summarizes recent studies on the use of collagen-hydroxyapatite based scaffolds for the treatment of bone trauma and regeneration.

Figure 3.

Schematic illustration of the preparation and evaluation of three-dimensional, porous HLC/ n-HA hybrid scaffold crosslinked via 1,2,7,8-diepoxyoctane. The scaffold having 6% v/v content of DEO had excellent mechanical properties, less cytotoxicity, good cytocompatability and bone repair effects. Adapted with permission from [89] under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Table 3.

Prominent research studies focused on investigating the use of collagen-hydroxyapatite based scaffolds for the treatment of bone trauma and regeneration.

Title of the study	Major finding	Ref.
Antimicrobial effect and cytotoxic evaluation of Mg-doped hydroxyapatite functionalized with Au-nano rods	Mg-doped hydroxyapatite functionalized with Au-nano rods exhibits antimicrobial and cytotoxic properties	[112]
Au, Pd and maghemite nanofunctionalized hydroxyapatite scaffolds for bone regeneration	Nanofunctionalized hydroxyapatite scaffolds enhance bone regeneration	[113]
Dense collagen-based scaffolds for soft tissue engineering applications	Dense collagen-based scaffolds are suitable for soft tissue engineering applications	[114]
Recent advances in hydroxyapatite-based biocomposites for bone tissue regeneration in orthopedics	Hydroxyapatite-based biocomposites enhance bone tissue regeneration in orthopedics	[115]
Medicated hydroxyapatite/collagen hybrid scaffolds for bone tissue engineering	Medicated hydroxyapatite/collagen hybrid scaffolds enhance bone tissue engineering	[116]
Recent advances in bioengineering bone revascularization based on composite materials comprising hydroxyapatite	HA composites promote vascularization in bone regeneration through angiogenesis	[117]
Three-dimensional bioactive hydrogel-based scaffolds for bone regeneration in implant dentistry	Bioactive hydrogels enhance bone regeneration	[118]
Generation of bone grafts using cryopreserved mesenchymal stromal cells and macroporous collagen-nanohydroxyapatite cryogels	Cryopreserved mesenchymal stromal cells and collagen-nanohydroxyapatite cryogels generate bone grafts	[119]
VEGF-loaded heparinized gelatine-hydroxyapatite-tricalcium phosphate scaffold accelerates bone regeneration via enhancing osteogenesis-angiogenesis coupling	VEGF-loaded scaffolds enhance bone regeneration	[120]

6.1. Synthesis of HA/collagen composites

So far, three techniques have been employed in the fabrication of collagen/nHA scaffolds, co-precipitation, including direct blending and SBF immersion. The utilization of spontaneous co-precipitation of collagen fibrils and nHA is regarded as a promising approach to attain the equivalent hierarchical structure of bone, as compared with the non-uniform spread of HA in direct blending and the slow and unpredictable process of HA synthesis in SBF [121]. The nucleation process of HA nanostructures onto fibrous collagen is accomplished in an aqueous suspension that has a significant amount of Ca2+ and PO43- in a specific ratio. This process is initiated by increasing the pH of the collagen solution from 9 to 10 at the ambient temperature. The chemical reaction that occurs between HA and collagen leads to the alignment of the c-axes of blade-shaped HA crystals along collagen strands, resembling the structural arrangement observed in bone tissue [122]. In their study, Calabrese et al. successfully extracted human MSCs from adipose tissues and subsequently incubated them on scaffolds composed of collagen and magnesium-HA composites. The findings from the Alizarin red staining and the analysis of osteogenic markers indicate that collagen/Mg-HA hybrid scaffolds can promote the differentiation of loaded MSCs into osteocytes, despite the absence of osteoinductive stimuli [123].

Hybrid scaffold materials have been a viable strategy in regenerative medicine in recent years. These scaffolds utilize a combination of various materials, typically synthetic and natural polymers or inorganic components, to produce structures that possess improved mechanical characteristics, biocompatibility and bioactivity. A prominent study conducted by [124] showcases the promising capabilities of hybrid scaffolds in the fields of tissue engineering and regenerative therapies. The study examines the methods of creating hybrid scaffolds, their physical characteristics and their effectiveness in biological applications, such as repairing bones or regenerating cartilage

Hybrid scaffolds, which include synthetic and natural components, have the ability to imitate the intricate structure and composition of real tissues. This enables them to promote cell adhesion, proliferation and differentiation. Furthermore, the adjustable characteristics of these support structures enable accurate manipulation of their mechanical durability, rate of breakdown and release patterns of bioactive substances, thereby maximizing their therapeutic effectiveness.

The conversation on hybrid scaffold materials highlights the significance of multidisciplinary methodologies in biomaterials research, which involves the integration of knowledge and skills from materials science, engineering, biology and medicine. Moreover, it emphasizes the capacity of hybrid scaffolds to overcome the constraints of traditional biomaterials and expedite the implementation of regenerative treatments from laboratory to clinical practice [124].

Cuniffe et al. offered a compelling comparison between two techniques used to prepare HA/Collagen composites. The first approach involved the introduction of HA particles to the collagen suspension for an extended period followed by lyophilization. In the other approach, the frozen collagen, in the condition of a porous scaffold, was immersed in a nHA suspension and eventually lyophilized. Both scenarios resulted in the production of composite scaffolds featuring extensively porous and linked components [125]. The study conducted by Cholas et al., utilized HA microspheres produced through the process of spray drying to fabricate the HA/Col hybridization. The paper proposes the potential utilization of this composite material as a vehicle for a pharmaceutical compound, which would be inserted into the microspheric HA [126]. Tampieri et al. acquired composites of HA/Collagen by two different methodologies. Initially, a collagen solution was combined with HA that had been previously acquired through precipitation from a Ca(OH)2 solution using an H3PO4 solution. In the second approach, the formation of HA from the chemicals employed in method 1 was conducted in the presence of collagen, resulting in precipitation. The results indicate that the method relying on the immediate precipitation of apatites in collagen solution is the most optimal [127]. The shape of the scaffold is primarily influenced by the method used for fabrication. Thus, it is crucial to discuss the techniques utilized in preparing the scaffolds, which are illustrated in Figure 4.

Figure 4.

Various fabrication techniques that are commonly used to create nHA/collagen scaffolds.

6.2. Osteoconductivity & vascularization of HA/Collagen composite

The term ‘osteoconductivity’ pertains to the capacity of a scaffold to promote the proliferation of fresh osseous tissue by attracting and supporting the movement of biological components such as MSCs, osteoblasts and osteoclasts, as well as promoting the development of blood vessels [128]. Multiple investigations have verified the capacity of HA/Col composite materials to promote the generation of fresh osseous tissues. For most of the biological investigations conducted to produce HA/Col composites, type I collagen derived from pig dermis or bovine tendon has been utilized. In a study, HA/collagen scaffolds were fabricated by the freeze-drying process and investigated their osteoconductivity. The investigation of cell proliferation shown that the combination of HA and collagen resulted in a higher percentage of cell viability compared with using HA or collagen alone. This finding supports the notion that the HA/collagen combination synergistically enhances the scaffold's ability to promote bone formation. Additionally, it was established that the presence of BMP7 in collagen and BMP2 in HA contributed to the scaffold's osteoconductive properties [129]. Kikuchi et al. conducted a study with the aim of synthesizing a composite material, consisting of HA/collagen, that closely resembles bone tissue. The intention was to utilize this material for evaluation of dog tibia bones. The production of composite involved selecting the most favorable pH and temperature conditions to enhance the process of organizing collagen fibers. The composite material was utilized as a substitute for a 20 mm imperfection. The bone condition was assessed utilizing x-ray techniques over a period of 12 weeks [130].

Vascularization is the primary process by which a functional blood network forms within or on the scaffold, ultimately determining its success. The utilization of the nHA/collagen scaffolding has demonstrated successful development of a vascular network, as stated. Chen et al. confirmed the development of the circulatory network by including a nHA/collagen composite during the synthesis of a chitosan-based scaffold. In this study, the usage of chitosan as a scaffold material did not result in any blood flow under in vivo settings. Nevertheless, the introduction of nHA/collagen into the chitosan scaffold resulted in an atypical distribution of blood flow surrounding the scaffold on the 14th and 28th day post-implantation [131]. Calabrese et al. introduced HA/Col composites into mice via implantation. Magnesium ions were used to further enhance HA. The results demonstrate the material's capacity to attract host cells and stimulate the formation of bone at abnormal locations within a living organism. The accuracy of angiogenesis was also verified using FMT analysis. The scientists highlight that the materials possess a notable level of safety, as they do not contain growth stimulants or cells that have undergone in vitro alteration. Consequently, these materials have the potential to serve as a secure and encouraging therapeutic option for bone illnesses [132].

6.3. Porosity of HA/collagen composite

The biological characteristics of HA/Col composites are greatly influenced by their porosity. An animal model was used to test the effectiveness of porous scaffolds, which were implanted into the tibias of rabbits. Implants with increased porosity exhibited accelerated development of fresh bone tissues and its infiltration into composite material structures. The porous structure enhances the ability to support bone growth. Enhances cell migration and promotes the development of blood vessels to support the nourishment of newly generated bone tissue [133]. When porous composites come into touch with water, they exhibit elastic behavior. This characteristic enables their convenient utilization in surgical procedures and their ability to conform to the intricate contours of cavities. Nevertheless, it is widely accepted that these materials possess reduced mechanical strength as a result of their porous architectures. The study involved the implantation of HA/Col and TCP materials into the tibia bones of rabbits in order to evaluate their biomechanical capabilities. It was discovered that while the HA/Col composite is less mechanically resilient than TCP, it exhibited superior mechanical strength at the site of the defect after implantation compared with the TCP material [134]. Although excessive porosity diminishes its mechanical robustness, the sponge-like flexibility facilitates convenient manipulation during surgical procedures. Once wet, HA/Collagen porous composites acquire elasticity similar to that of a sponge, facilitating their insertion into bone lesions [135]. From a surgical perspective, the incorporation of collagen into HA offers several advantageous characteristics during surgery. These include the convenience of tailoring the material to fit the defect site, the ability to easily adapt to the shape of the defect, the capacity to adhere the material to the transplantation site and the capability to enhance blood clot formation and stabilization due to collagen's hemostatic attributes [136].

6.4. Drug delivery using nHA/collagen scaffolds for the treatment of bone disease

The composites can be utilized not only for bone regrowth but also for the treatment of bone-related disorders. In this application, the scaffold structure will serve as a carrier for encapsulating medicinal molecules. Consequently, as the scaffolds undergo biodegradation, the drug compounds can be deliberately delivered to the intended location. Rong et al. utilized PLGA combined with adriamycin in the nHA/collagen scaffold. They investigated the scaffold's ability to release the drug over an extended period and its effectiveness in inhibiting osteosarcoma MG63 cells. The scaffold exhibited a prolonged drug release profile for a duration of 28 days, during which 65% of the medication was delivered from the scaffold within the specified timeframe [137]. Having antimicrobial activity is a beneficial attribute of scaffolds because it allows for the development of scaffolds that can avoid bacterial diseases at the site where they are implanted. This can be achieved by including appropriate antibacterial medications throughout the process of scaffold fabrication. Lian et al. incorporated vancomycin onto a framework made of nHA/collagen and PLA. Long-term antibacterial activity was detected in this scaffold. Following a 72-hour incubation period, the presence of a zone of inhibition measuring 21.7 ± 0.6 mm was found [138]. Recently, Mulazzi et al. developed a medicated (Vancomycin and gentamicin-loaded) osteoinductive and bioresorbable hybrid bone-mimetic scaffold of collagen fibers biomineralized with magnesium doped-hydroxyapatite to treat infection and repair bone defects. Microbiological and in vitro tests showed that the the medicated scaffolds were safe and effective for extended duration therapy, preventing bone defect-related infections in orthopedic surgeries [116].

Conventional treatments for bone tumors have progressed gradually, encompassing surgical procedures, chemotherapy, radiation, hormone treatment and immunotherapy. These therapeutic interventions are accompanied by several limitations, such as the development of resistance to multiple drugs, the recurrence of tumors, the occurrence of severe adverse effects and the emergence of notable bone abnormalities [139]. In order to overcome the constraints associated with existing treatment modalities, the implementation of bone-specific drug focused treatment emerges as a notably efficacious therapeutic strategy. This strategy enables the pharmaceuticals to be focused specifically on the tumor site, extending the duration of drug release and minimizing systemic adverse responses. nHA is considered a favorable carrier for anti-tumor medications because of its strong surface attraction and exceptional osteogenic characteristics [140]. Rong et al. fabricated adriamycin (ADM) loaded in PLGA nanoparticles (NPs) and applied them onto porous nHA/collagen scaffolds. The fabricated ADM-PLGA-nHA composite scaffold exhibits a pore diameter ranging from 100 to 200 μm, with a porosity of 82 percent. It demonstrates excellent pore connectivity and achieves continuous and extended release of ADM over a period of 28 days. In contrast to the direct peritoneal administration of ADM, the implantation of this drug-loaded scaffold in a nude mice model demonstrates a substantial inhibition of osteosarcoma cell proliferation, enhanced anti-tumor efficacy and reduced occurrence of side effects [137]. The drug can be loaded in scaffolds through several methods including mixing the pharmaceutical with the scaffold material before manufacturing, encasing the drug carrier and incorporating it into the scaffold materials, coating the scaffold with polymers, or impregnating the scaffold with the drug (Figure 5).

Figure 5.

Various drug loading techniques on a nano-hydroxyapatite (nHA) scaffold. Adapted with permission from [141] under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

7. Conclusion

HA exhibits considerable potential as a material for uses in theranostics, encompassing both therapeutic and diagnostic functions. However, the potential for producing composites that incorporate HA and collagen presents an opportunity to modify their physio-chemical properties in a manner that is advantageous for biological uses. In the field of biomedical engineering, the successful restoration of tissue-engineered bone defects necessitates the utilization of a 3D biocompatible scaffold. This scaffold must possess the ability to facilitate cellular attachment and development, while also offering adequate mechanical support to promote the healing of bone tissue. In recent years, the field of orthopedic tissue engineering has experienced significant advancements. However, the successful repair of diseased bone lesions remains a formidable objective. Moreover, this paper extensively examines the diverse range of data that substantiates the superior performance of the nHA/collagen scaffold in in-vitro studies and in-vivo investigations. Due to advancements in research technologies and the availability of in-vitro tools, the collagen-hydroxyapatite based scaffolds will offer more realistic and safer tools for Bone trauma and regeneration in coming years.

8. Future perspective

The utilization of traditional approaches for bone regeneration is associated with several limitations, including accelerated degradation, suboptimal clinical outcomes and little resemblance to natural bone properties. In recent times, there have been reports suggesting that the aforementioned limitations can be effectively addressed by the utilization of scaffolds derived from biopolymers. The field of medical tissue engineering and the science of materials is experiencing significant progress, leading to the development of diverse formulations that hold great significance in medical fields. Collagen and HA ceramics are widely recognized for their significant contributions to bone regeneration, owing to a multitude of factors. Numerous ongoing studies are investigating the integration of active compounds into HA/collagen hybrids. It is widely adopted owing to its favorable characteristics, including its strong biocompatibility, lack of immunogenicity and absence of toxicity. The integration of HA with other materials can yield a 3D scaffold for bone grafting that possesses adequate mechanical strength, appropriate pore size and porosity and osteoconductivity. This combination effectively addresses the limitations in mechanical qualities exhibited by HA alone. The utilization of HA composite scaffolds in conjunction with bioactive factors is a promising approach to address the limited osteoinductivity shown in certain scaffold composites. This is attributed to the favorable drug loading capabilities exhibited by HA. In the interim, the achievement of guided release of pharmacological factors has been observed, with the concurrent preservation of their activity. In addition, bone pathologies such as arthritis, bone tumors, bone loss and bone tuberculosis give rise to bone abnormalities that necessitate bone restoration and concomitant pharmacological intervention. Hence, the integration of HA synthetic scaffolds with various pharmaceutical agents not only serves as a means of reinforcing bone deficiencies but also hinders the proliferation of germs, cancer cells and osteoclasts via controlled drug release, thereby facilitating favorable outcomes in bone healing. The utilization of a sustained-release system enhances the potential of HA-based scaffolds as a promising approach for applications in bone tissue creation.

Moreover, the nHA target sites exhibit a preference for the adherence of osteogenic cells via the mechanism of cell signaling. Nano-hydroxyapatite serves as a pro-adhesive agent, facilitating osteoblast adhesion, while the presence of calcium in HA contributes to enhanced bone regeneration. The utilization of collagen derived from mammalian sources has the potential to facilitate the spread of diseases to human beings [142]. The strength of collagen scaffolds is insufficient for bone regeneration, necessitating the use of chemical cross-linkers to enhance scaffold stability. However, this process may potentially damage the biological capabilities of the scaffold. Physical crosslinkers, like as ultraviolet rays, possess the capacity to induce the breakdown and denaturation of collagen, hence potentially compromising the mechanical properties of the material. The degradation property is a fundamental element that significantly influences the process of bone repair [143]. The rapid degradation of collagen within the body poses challenges in monitoring its degradation behavior in vivo, mostly due to the existence of collagenase. Additionally, the task of forecasting the rate of degradation for scaffolds at various in vivo target regions is arduous and necessitates extensive experimental protocols. Comprehensive optimization methods are necessary to provide commentary regarding how degradation behavior might be adjusted according to specific requirements. Despite the existence of certain limitations associated with collagen-based scaffolds, researchers are actively pursuing various research methodologies in order to address these limitations and enhance their suitability as a leading candidate for scaffolds used in bone regeneration.

Significant advancements have been achieved in the examination of n-HA composites utilized as bone scaffolds. Nevertheless, there remain some apprehensions. For instance, a notable disparity exists between the strength of the material or elastic modulus of materials and bone. Additionally, the degradation rate of these composites may not align with their rate of formation of fresh bone. Furthermore, the loading dose and sustained release duration of bioactive substances and drugs may not satisfy the necessary criteria. The ongoing advancement in the field of composite artificial bone materials has paved the way for further investigation into the healing of bone defects using HA composites. Future study in this area may prioritize the exploration of novel preparation techniques aimed at enhancing the mechanical properties and breakdown rate of HA composite scaffolds. One such instance involves the utilization of 3D printing technologies to construct composite scaffolds with varying pore sizes and porosity in order to manipulate their physicochemical characteristics. The achievement of increased loading rates of bioactive components and slow releasing pharmaceuticals can be attained by altering the surface of the synthetic scaffold or by including drug-loaded nano- or micro-spheres that can be adsorbed onto the scaffold. Multiple bioactive factors and pharmaceuticals are included onto the HA composite scaffold in order to enhance the therapeutic outcome. An example of this is the simultaneous administration of growth stimulants and antibiotics, which promotes the regeneration of new bone at locations where bone defects have occurred due to infection.

In the future, the use of HA and collagen-based composites in bone regeneration shows great potential for treating bone defects and improving therapeutic approaches. As research advances, we expect more investigation into innovative preparation methods with the goal of improving the mechanical properties and degradation rates of HA composite scaffolds. Advanced manufacturing techniques such as 3D printing have the potential to produce scaffolds that can be customized with different pore sizes and porosities. This allows for the optimization of their physicochemical properties to suit specific applications. In addition, there will likely be ongoing efforts to enhance the loading rates of bioactive components and prolong the period of drug release. This may involve modifying the surface of synthetic scaffolds or incorporating drug-loaded nano- or microspheres. Multifunctional composites, incorporating various bioactive factors and pharmaceuticals, may become more common, enabling tailored therapeutic approaches for bone defects, including those resulting from infection or trauma. To summarise, the continuous progress in HA/collagen composite scaffolds has promising possibilities for the future of bone tissue engineering. This could lead to significant gains in both the effectiveness of treatments and the results seen in clinical settings.

Author contributions

D Han: data gathering and organization; W Wang: initial draft preparation; J Gong: drafting the manuscript and critical revision; Y Ma: conception and design of the study; Y Li: finalization and critical reviewing of the manuscript; All the authors read the final manuscript and agree to publish this work.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.