Osteoimmunology of Natural and Synthetic Biomaterials Used in Dentistry for Bone Remodeling

Abstract

Bone loss in the maxillofacial region arises from multiple causes, including periodontal disease, trauma, surgical procedures, infection, congenital anomalies, and cancer. Traditional treatment relies on bone grafting, either alone or in combination with biomaterials. Advances in tissue engineering have introduced synthetic or natural scaffolds to mimic the mineralized bone matrix. Natural scaffolds offer excellent biocompatibility and similarity to native tissue but often lack sufficient mechanical strength and exhibit poor degradation rates. Synthetic scaffolds provide tunable porosity and mechanical stability; however, their biological inertness makes them poor sources of osteogenic signaling. A key factor in the success of any scaffold is its interaction with the host immune system. Upon implantation, the innate immune response is initiated, with neutrophils and macrophages being the first cells to contact the scaffold. Macrophage polarization toward proinflammatory (M1) or anti-inflammatory (M2) phenotypes determines whether the microenvironment favors inflammation or remodeling. The adaptive immune response also plays a critical role: T and B lymphocytes may promote tolerance and integration through Th2/Treg pathways and antibody-mediated regulation, or they may trigger chronic inflammation and rejection through Th1/Th17 activation. This review examines the natural and synthetic materials used for bone remodeling and their biological properties. It then outlines the sequence of immune events occurring from the moment a scaffold is implanted to its potential integration or failure. Finally, this study highlights the relevance of cellular models and in vitro assays for the early evaluation of immunogenicity and biocompatibility, which are essential for optimizing scaffold design and improving outcomes in maxillofacial bone regeneration.

1. Introduction

Bone loss in the maxillofacial region has diverse etiologies, including periodontal disease, trauma, surgical procedures (cranioplasty), infectious processes, congenital malformations, and oral cancer. The treatment for these bone defects is a surgical procedure in which bone is grafted, either alone or in combination with other materials [1]. Another alternative is the use of bone substitutes, which are often natural or synthetic biomaterials that contain or emulate a mineralized bone matrix without viable cells [2]. Some of these materials possess osteoconductive properties and can be reabsorbed by the body, releasing substances that promote the formation of new bone [3]. Recent trends in tissue engineering have focused on the use of biomaterials to either improve or restore the function of damaged tissues. However, the successful integration of a biomaterial into the host tissue remains the most significant challenge [4].

After surgical placement, the biomaterial is exposed to an inflammatory environment. The foreign body reaction, which is mediated by macrophages, is a key component of efficient and functional tissue remodeling [5]. In addition to foreign body reactions, host responses following biomaterial implantation include injury, blood–material interactions, provisional matrix formation, acute and chronic inflammation, granulation tissue development, and fibrosis [6]. During this process, macrophages play a dynamic role, as they can polarize into proinflammatory (M1) and anti-inflammatory (M2) phenotypes. Because they are the first cells of the immune system to appear in early inflammatory foci, macrophages have numerous interactions with other cells through cytokine secretion [7]. Cytokines secreted by macrophages recruit other immune cells to initiate the immune-inflammatory response and subsequent bone formation [8].

To better understand the interaction between the immune system and musculoskeletal system, the concept of osteoimmunology was originally proposed by Takayanagi [9]; osteoimmunology integrates the principles of immunology and bone biology, recognizing that immune cells and bone cells share common signaling pathways, cytokines, and transcription factors that regulate both immune responses and skeletal homeostasis [10].

The development of biomaterials requires a thorough understanding of the relationship between immune cells and bone cells, as well as the effect of the immune environment induced by implanted biomaterials on osteogenesis [11].

The aim of this review is to provide an updated analysis of the natural and synthetic biomaterials used in dentistry for bone remodeling, as well as the immunological implications that arise from their characteristics. Finally, the findings emphasize the importance of in vitro evaluation of the immune response to these materials, which should be an essential part of the evaluation protocol to aid in their design.

2. Materials and Methods

Google Scholar, PubMed, Scopus, and Web of Science databases were used for publications on the natural and synthetic biomaterials used in dentistry. Keywords included bone, bone substitutes, natural biopolymers, proteins, polysaccharides, minerals, metal alloys, synthetic polymers, ceramics, maxillofacial, 3D printing, in vitro, immune system, inflammation, graft failure, and combinations.

The research and clinical application of scaffolds for guided bone remodeling and tissue engineering began to expand in the 2000s; therefore, the following criteria were adopted: for basic descriptions of osteoimmunology, original English-language articles published in peer-reviewed journals between 2005 and 2025 were used, and for innovative applications, articles published between 2020 and 2025 were analyzed.

This review focuses on publications with specific immunological and dental implications. Facial bone structure is derived primarily from neural crest mesenchyme [12], whereas axial and appendicular bones derive from mesoderm, influencing cellular responses and regenerative potential [13]. Alveolar bone presents a higher content of trabecular bone and increased remodeling rates compared to long bones, which affects scaffold integration [14]. The oral cavity is subjected to complex, cyclic forces from mastication and occlusion, generating a heterogeneous mechanical environment distinct from long bones [15,16]. The oral cavity hosts a diverse microbiota, making it a non-sterile environment. This increases the risk of infection and influences the performance of biomaterials and the host response [17].

Variability in the origin and processing of biomaterials also complicates comparisons between studies. Many of the applications analyzed are still in early clinical phases and therefore require large-scale validation. In addition, no meta-analysis was performed to evaluate the results statistically.

3. Natural Biomaterials Used for Scaffolding

Materials of natural origin are derived from a living source without modification [2]. They possess favorable properties, such as low immunogenicity, wide availability, and affordability, and are well tolerated by organisms and biodegradable [18]. They are mainly used as mechanical supports (scaffolds), carriers, or substrates for structural modifications. These include macromolecules such as proteins (collagen, silk, elastin, gelatin, keratin, fibrin and mucin), polysaccharides (hyaluronic acid, cellulose, methylcellulose, amylose, chitin, starch, dextran, agarose and alginate) and minerals (hydroxyapatite) [19]. Within materials of a protein nature, Silk-based biomaterials have gained increasing attention in bone tissue engineering due to their biocompatibility and remarkably low immunogenicity. Silk has demonstrated anti-inflammatory and pro-angiogenic effects, which are key factors in successful tissue regeneration [20]. Figure 1 shows a schematic representation of the classification of natural-origin materials, which are grouped into protein-based, polysaccharide-based, and mineral-based categories. Information content in

Table 1. Natural-origin biomaterials for dental bone remodeling: advantages and current challenges.

Characteristics of Materials Derived from Natural Sources
Classification		Material	Origin	Application	Advantages	Limitations	Type of Study and Outcome	Reference
Macromolecules	Proteins	Collagen	Duck, fish, porcine, bovine	Tissue scaffolds, joint disease treatments, skin regeneration, hemostatic agents	Excellent biocompatibility, degradability	High potential for adverse immune reactions, poor mechanical properties and structural integrity	Clinical trial The addition of a collagen-enriched bovine-derived xenograft significantly improved radiographic bone volume gain and probing depth reduction, and clinical attachment level gain in the treatment of isolated interdental intrabony defects	[21]
		Silk	Silkworms (Bombyx mori) and spiders	Drug delivery, films, hydrogels, scaffolds, sutures	Excellent biocompatibility, degradability, tissue integration, oxygen, and water permeability. Young’s modulus of 8.9 to 17.4 gpa	The Degradation rate of artificial bone prepared from pure silk fibroin Is regulated by sacrificing its mechanical properties. No inherent antibacterial activity	In vitro/in vivo The expression of ALP, OCN, and RUNX2 was significantly higher in silk scaffolds reinforced with silk fabric than in regenerated silk and TC4 titanium alloy scaffolds	[22]
		Elastin	Mammalian; fibroblasts and smooth muscle cells	Elastin-based materials are used to create scaffolds that promote faster wound closure, improve tissue strength and flexibility, and enhance dermal regeneration	Biocompatibility, the mechanical and biological properties of elastin-like polypeptides can be customized at the genetic level	The elastin extracted from living organisms is insoluble	In vivo A higher percentage of de novo bone formation was seen for the comprising collagen and elastin membrane (34.9%) compared to collagen (15.5%) at 21 days (p = 0.01)	[23]
		Gelatin	Partial acid or alkaline hydrolysis of collagen	Post surgical bleeding, Dressing material And drug delivery system	Biocompatibility, non-immunogenic, biodegradable, and nontoxic	Poor mechanical properties and low thermal stability. Full degradation after 42 days.	In vitro The PCL/gelatin hybrid membranes demonstrated greater calcium deposition, indicating the composite nanofibrous structure was beneficial for efficient GBR membranes	[24]
		Keratin	Mammalian; hair, nails, wool, feathers, and skin	Wound dressings, drug delivery, tissue engineering	Mechanically strong and flexible, biocompatible, low antigenicity and toxicity	Slow degradation rate	In vivo The use of the keratin hydrogel increased the % bone-to-implant contact of titanium implants by 169% in comparison to control implants	[25]
		Fibrin	Mammalian fibrinogen	Drug delivery, scaffolds, biologically active matrix, bioinks	High biocompatibility, nontoxicity, and low immunogenicity; effects in hemostasis, anti-inflammation, and promotion of wound healing. Promotes Leukocyte adhesion by binding to its surface integrin Receptor	Does not fully mimic the dynamics of the tissue environment and cannot independently change its shape and structure to conform to tissue growth	In vivo The fibrin-based bioink supported hMSCs chondrogenesis and remodeled in vivo enabling vascularization and conversion of the cartilaginous templates into bone	[26]
		Mucin	Mammalian; epithelial cells	Lubricants, cell signaling, drug delivery, wound healing	Biocompatibility, bioactivity	Poor mechanical properties	In vitro/in vivo Procyanidin/mucin coating promoted osteogenesis-related genes (col1, ON, OCN and RUNX2) in BMSCs in vitro and bone generation in vivo by activating the wnt/β-catenin pathway	[27]
	Polysaccharides	Hyaluronic acid	Mammalian	Hydration, lubrication, scaffolds, bioinks, hydrogels	Biocompatibility, biodegradability, ability to interact with cells, promote tissue regeneration	Poor mechanical properties (0.8 mpa) and rapid degradation in vivo	In vitro Biomineralized gelatin/hyaluronic acid/hydroxyapatite composite scaffolds showed increased OPG, and decreased RANKL expression compared with the unmineralized scaffolds	[28]
		Cellulose	Plants (wood, cotton); bacteria, fungi, and animals (tunicates)	Dressing material, scaffolds	Biocompatibility, biodegradability, and renewability	Integration issues, degradation variability	In vitro/in vivo Electrospun cellulose scaffolds coated with rhbmp-2 increased osteogenic differentiation of BMSCs, enhanced ALP activity and calcium content, induced in vivo collagen assembly direction, cortical bone	[29]
		Methylcellulose	Cellulose	Bioinks, scaffolds, hydrogels, drug delivery	Biocompatibility, biodegradability, thermoresponsive	Poor mechanical properties (7.8 ± 1.6 mpa)	In vitro Significantly higher levels of calcium deposition (p < 0.05) were found for methylcellulose with nanohydroxyapatite 24 formulations thus suggesting the triggering of the mineralization process	[30]
		Starch	Maize, potato, wheat, rice, sorghum	Drug delivery, tissue engineering, pharmaceutical excipient	Biocompatibility, biodegradability, low cost	Poor mechanical properties	In vitro Electrospun poly (3-hydroxybutyrate) and starch scaffolds demonstrate that the addition of starch enhances cell viability and calcium mineralization	[31]
		Chitosan	Chitin (cell walls fungi; exoskeletons of crustaceans)	Scaffolds, hydrogels, membranes, drug delivery	Biocompatibility and biodegradability	Poor mechanical properties	In vitro Chitosan/biphasic calcium phosphate scaffolds functionalized with Arg–Gly–Asp and BMP-2-loaded nanoparticles provided a favorable microenvironment for bone formation	[32]
		Dextran	Lactic acid-producing bacteria (L. mesenteroides and Streptococcus mutans)	Drug delivery, hydrogels, scaffolds, medical devices	Biocompatibility, biodegradability	Can cause allergic reactions, poor cell adhesion (need for modification)	In vivo Dextran gel containing basic fibroblast growth factor showed significantly greater bone volume and bone mineral content than sites receiving no treatment or treated with dextran gel alone	[33]
		Agarose	Red algae (Gelidium and Gracilaria)	Tissue engineering, drug delivery, wound healing, bioprinting, disease diagnosis	Biocompatibility, gelling properties, mechanical tunability, agarose Hydrogels reduce inflammatory responses by creating a hydrated microenvironment that Limits excessive immune cell infiltration	Low cell adhesion, brittleness	In vitro Cells cultured on the agarose/HA composite disks significantly increased the alkaline phosphatase activity and calcium deposition	[34]
		Alginate	Plant (brown algae), bacterial (Pseudomonas and Azotobacter)	Wound dressings, drug delivery, tissue engineering, dental impressions	Biocompatibility, biodegradability, good printability	Poor mechanical properties (storage modulus over 4 kPa), low cell adhesion	In vitro The chitosan/alginate hydrogels with nHap-PTH significantly promoted osteogenic activity via the notch signaling pathway and increased the expression levels of osteogenic proteins such as BMP-2, OCN and RunX2	[35]
Minerals		Hydroxyapatite	Animal-derived waste (eggshells, bones); red marine algae	Bone substitute, drug delivery, scaffolds	Biocompatibility, biodegradability, osteo-conductivity	Poor mechanical properties (low fracture toughness and tensile strength); critical load up to 6.90 n	In vitro Three-dimensional coating of hydroxyapatite-functionalized nanoparticles of polydopamine on implant surfaces promotes cell proliferation and upregulates the activity of alkaline phosphatase and the expression of osteogenesis-related genes in environments with high or normal ROS levels	[36]

Abbreviations: ALP, phosphatase alkaline; OCN, osteocalcin; RUNX2, runt-related transcription factor 2; PCL, polycaprolactone; hMSCs, human mesenchymal stem/stromal cells; Col1, collagen type I; ON, osteonecitn; BMSCs, bone marrow stromal cells; RANKL, receptor activator of nuclear factor kappa-B ligand; OPG, osteoprotegerin; rhBMP-2, recombinant human bone morphogenetic protein-2; BMP-2, bone morphogenetic protein-2; Arg, arginine; Gly, glycine; Asp, aspartic acid; HA, hydroxyapatite; nHAP, nanohydroxyapatite; PTH, parathyroid hormone; ROS, reactive oxygen species.

is a compilation of research where an extensive analysis of this type of material is made, describing its native characteristics, advantages, disadvantages, and applications.

Despite the qualities mentioned above, materials derived from natural sources have significant limitations that compromise their use as the sole component in the manufacture of scaffolding for bone remodeling [37]. Among the main disadvantages of these materials are their low mechanical strength; many natural polymers, such as collagen or hyaluronic acid, have tensile strengths ranging from 0.01 to 0.037 MPa (megapascals) [38], whereas the mechanical loads present in the maxillofacial region range from 1.5 to 7.5 MPa [39], which compromises their structural stability in vivo [2]. The rate of degradation varies significantly depending on factors such as the crosslink density, which is the number of crosslinks per unit volume in a polymer and determines the degree of interconnection of the polymer network [40]. Naturally, derived, uncrosslinked scaffolds degrade rapidly (7–10 days) [41], which may limit the time available for key cellular processes to occur, such as the mineralization of newly formed bone (primary mineralization), which occurs in 50% to 70% of the material during the first 2–3 months [42], or the development of vascularization between the second and fourth weeks post-implantation [43].

As biological derivatives, they present heterogeneous batches and possible risks of disease transmission or immunogenicity if not properly purified [18]. Many of these biomaterials are not easily printable or moldable via modern technologies such as 3D bioprinting or electrospinning unless they are combined with other compounds [36]. For these reasons, natural scaffolds alone rarely meet all the requirements for bone defect applications. In many cases, they are used as part of composite scaffolds or combined with synthetic materials (such as polylactic acid, polycaprolactone, or bioactive ceramics) to improve their mechanical stability, degradation rate, and osteoinductive capacity [44].

4. Synthetic Biomaterials Used for Scaffolding

Synthetic scaffolds are composed of metals, polymers, and ceramics and are manufactured artificially for the purpose of mimicking the bone extracellular matrix, offering advantages in terms of structural control, reproducibility, mechanical stability, and ease of modification [45]. Materials that fall into this category include metals such as titanium (Ti), magnesium (Mg), zinc (Zn), strontium (Sr), copper (Cu), stainless steel, chromium (Cr), silver (Ag), tantalum (Ta), zirconium (Zr), and cobalt (Co) [46]; polymers such as polylactic acid (PLA), polyglycolic acid (PLGA), polylactic-coglycolic acid (PLGA), polycaprolactone (PCL), polyetheretherketone (PEEK), polytetrafluoroethylene (ePTFE), polymethyl methacrylate (PMMA), and polyethylene glycol (PEG) [47]; among the most notable ceramics are synthetic hydroxyapatite (HA), tricalcium phosphate (β-TCP), and bioactive glasses [48]. Table 2 enlists information where an extensive analysis of this type of material is made, describing its native characteristics, advantages, disadvantages, and applications.

Although synthetic scaffolds allow greater control over porosity, degradation rate, and composition, they do not meet all the requirements for bone remodeling [49]. Many synthetic polymers are biologically inert, do not induce osteoblastic differentiation on their own, and do not directly promote cell adhesion without being functionalized or coated with proteins or bioactive factors [50]. The degradation byproducts of some polymers, such as PLA and PLGA, can acidify the local microenvironment and trigger inflammatory responses in vitro and in vivo [51]. Although mechanical stability is an advantage, in some cases, synthetic scaffolds do not degrade at the rate necessary to allow replacement by new bone tissue, which can interfere with natural remodeling [52].

Table 2. Synthetic-origin biomaterials for dental bone remodeling: advantages and current challenges.

Characteristics of Materials Derived from Synthetic Sources
Classification	Material	Application	Advantages	Limitations	Type of Study and Outcome	Reference
Metals and alloys	Titanium	Dental and orthopedic implants, cardiovascular devices, surgical instruments, scaffolds	Biocompatibility, low density, high melting point, low thermal expansion coefficient, poor conductor of electricity, nontoxic, mechanical strength and stiffness	Difficult to machine, easily damaged by wear, expensive to produce. Although scaffolds had good osseointegration ability and osteogenic induction ability, osteolysis was still found from 20% revision surgeries	In vivo Porous titanium alloy scaffold was filled with ingrowth bone tissue at week 12, trabeculae within the range of stress shielding were not as strong as healthy control group	[53]
	Magnesium	Ligatures, cardiovascular stents, orthopedic implants	Biocompatibility, biodegradability, high strength-to-weight ratio, low density	High corrosion rate, H2 released during degradation accumulates in the surrounding soft tissue	In vitro Noninferiority of both 1.5 mm and 1.75 mm magnesium miniplates compared to 1.0 mm titanium miniplates was demonstrated in a mandible fracture model in sheep	[54]
	Zinc	Orthopedic implants, scaffolds, drug delivery, cardiovascular devices, enzymatic reactions	Biocompatibility, biodegradability, moderate corrosion	Low mechanical strength and plasticity, high concentrations of zinc ions can be toxic to cells	In vitro The Zinc scaffolds were biocompatible, as preosteoblasts were able to effectively adhere to the substrates with multiple cytoplasmic extensions, and indirect tests show the porous scaffolds retained >75% cell viability	[55]
	Strontium	Bone and tissue regeneration, drug delivery, wound dressings, bioimaging	Biocompatibility, biodegradability, can regulate bone metabolism, can promote angiogenesis, immunomodulation, matrix synthesis, mineralization, and antioxidation	Excessive strontium intake can weaken bones by displacing calcium, lower mechanical strength, limited long-term data	In vitro Strontium-doped nanorods arrays accelerate M1 to M2 transformation of the adhered macrophages, enhancing secretion of pro-osteogenic cytokines and growth factors (TGF-β1 and BMP2)	[56]
	Copper	Implant coatings, antibacterial materials, medical devices, drug delivery, bioimaging, enzyme-mimicking	Biocompatibility, biodegradability, variable porosity, good mechanical strength, and crosslinking, can promote angiogenesis, can stimulate osteoblast activity	Excessive copper can lead to oxidative stress, tissue damage, and potentially impact bone metabolism negatively	In vitro Copper in calcium phosphate scaffolds improved osteogenic, angiogenic, and antibacterial properties, facilitating better bone regeneration in vitro	[57]
	Stainless steel	Orthopedic implants, dental implants, cardiovascular devices, surgical instruments	Biocompatibility, good mechanical strength, corrosion resistance	Unmodified stainless-steel surfaces can be hydrophobic, attracting protein adsorption and promoting biofilm formation, limited bioactivity	In vitro The high corrosion resistance of the selective laser melting sample (SLM) limited the release of toxic ions into the biological environment, which resulted in better viability and proliferation of the MC3T3-E1 preosteoblast	[58]
	Cobalt-Chromium	Orthopedic implants, scaffolds, dental prosthesis, cardiovascular implants	Biocompatibility, high strength, corrosion resistance	Metal ion release, local tissue damage, inflammation	In vitro Chromium-cobalt coated scaffolds favor cell adhesion and growth of precursor cells from periodontal ligament	[59]
	Silver	Drug delivery, antibacterial coatings, scaffolds, wound healing process	Biocompatibility, broad spectrum antimicrobial activity, versatility	Long-term toxicity unknown, potential accumulation in tissues	In vitro The silver nanoparticles-loaded gelatin/β-tricalcium phosphate scaffolds demonstrated synergistic antibacterial activity, cytocompatibility, and osteogenic promotion	[60]
	Tantalum	Orthopedic implants, dental implants, scaffolds, cardiovascular devices, drug delivery	Biocompatibility, high strength, corrosion resistance, osteoconductive and osteoinductive properties	High elastic modulus can lead to stress shielding, not immune to bacterial infections, inflammatory reactions	In vitro Porous tantalum can promote the adhesion and proliferation of bone marrow mesenchymal stem cells. Osteogenic gene expression and ALP expression levels were significantly increased compared with porous Ti6Al4V	[61]
	Zirconium	Drug delivery, orthopedic implants, dental implants, scaffolds	Biocompatibility, mechanical strength, chemical stability, corrosion resistance	Zirconium nanoparticles can exhibit toxicity, potentially disrupting cells and causing oxidative stress	In vivo CAD/CAM porous zirconia scaffolds enriched with nanohydroxyapatite particles revealed significantly higher volume of new bone formation (33% ± 14) compared to the controls (21% ± 11)	[62]
Polymers	PLA	Drug delivery, medical devices, tissue engineering	Biocompatibility, biodegradability, renewable resource, versatility	Low osteoconductivity, acidic degradation products, poor cellular adhesion, low thermal stability, poor solubility in organic solvents	In vitro The porous spiral scaffold with larger surface area and better interconnections between internal porous networks could significantly improve the spatial cell compartment and promote human fetal osteoblasts growth	[63]
	PGA	Scaffolds, drug delivery, resorbable implants, sutures	Biodegradability, biocompatibility, ease of fabrication	Acidic degradation products, limited solubility, poor mechanical properties, rapid degradation	In vitro The collagen-PGA sponge was superior to the original collagen sponge in terms of the initial attachment, proliferation rate, and osteogenic differentiation of the bone marrow mesenchymal stem cells	[64]
	PLGA	Drug delivery, tissue engineering, wound dressing, medical devices, vaccines (nanoparticles)	Biocompatibility, biodegradability, controlled release, versatility	Poor drug loading and burst release, can trigger an immune response, need for surface modification, acidic degradation products	In vivo E7-BMP-2 peptides incorporated into 3D hybrid PLGA nanofiber aerogels can induce ~60–70% closure of critical-sized (8 mm) rat calvarial bone defects	[65]
	PCL	Tissue engineering, surgical sutures, drug delivery, orthopedic implants, wound dressings	Biocompatibility, biodegradability, ease of processing, tunable properties	Slow degradation rate, poor mechanical properties, low cell adhesion, low melting point	In vitro The PCL scaffolds with oxidized hyaluronic acid glycine-peptide conjugates demonstrated improved endothelial cell adhesion, proliferation and viability, suggesting the potential for vascularized tissue constructs	[66]
	PEEK	Orthopedic implants, maxillofacial implants, dental prostheses, cardiovascular devices	Biocompatibility, mechanical strength, chemical resistance	Poor osseointegration, high cost, complex manufacturing, limited cell adhesion	In vitro An APS-coated plasma-treated sulfonated bioactive PEEK scaffold facilitates M2 macrophage polarization, reduces pro-inflammatory cytokines, and enhances the secretion of anti-inflammatory factors	[67]
	ePTFE	Cardiovascular devices, tissue regeneration, dural substitute	Biocompatibility, controlled porosity, thermal stability	Susceptibility to bacterial adhesion, nonbiodegradable, needs to be removed after a certain period	In vivo ePTFE scaffolds with bone morphogenic proteins were implanted subcutaneously into SD rats to determine their in vivo ossification potential, scaffolds showed slight radiopacity 1 week after implantation and strong radiopacity 2 and 3 weeks after implantation	[68]
	PMMA	Bone substitutes, fillers, drug delivery, tissue engineering	Biocompatibility, affordability, mechanical strength, ease of processing	Non-degradable, poor bioactivity, can cause allergic reactions due to the presence of nickel in its composition	In vitro MG-63 cells were able to adhere and reside on the PMMA-CaP scaffolds up to 7 days of culture, and the large number of AR-stained cells shows that the cells are viable up to 7 days	[69]
	PEG	Drug delivery, tissue engineering, medical devices, coatings	Biocompatibility, biodegradability, versatility, solubility in water	Toxic byproducts and accumulation, PEGylation can lead to the formation of vacuoles in cells, poor mechanical properties	In vitro The hydrophilic PEG inserted into the PLA chains dissolved rapidly in the degradation environment, forming microporous channels that promoted the degradation of the PLA matrix	[70]
Ceramics	Hydroxyapatite	Bone grafts, coatings for implants, drug delivery, tissue engineering	Biocompatibility, bioactivity, tunable properties, osteoconductivity	Brittleness, low fracture toughness, slow osseointegration rates	In vivo The nanocarbonate hydroxyapatite scaffolds increase bone formation after 12 weeks implanted in a critical defect in rabbit radius compared with the control group	[71]
	β-TCP	Tissue engineering, coatings, drug delivery, guided bone regeneration	Biocompatibility, bioactivity, osteoconductive and osteoinductive properties	Poor mechanical properties, high adsorption rate, the crystallographic structure and properties of β-TCP can be influenced by impurities and the manufacturing process	In vivo β-TCP/PCL composite scaffolds demonstrated superior new bone quality and quantity in complex, mechanically demanding environments such as radial defects	[72]
	Bioactive glasses	Tissue engineering, drug delivery, wound healing, dental implants	Biocompatibility, bioactivity, versatility, tunable properties	Poor mechanical properties, brittleness, low fracture toughness, rapid dissolution	In vitro The combined use of bioactive glass and bone-conditioned medium in scaffolds synergistically promoted osteogenic differentiation and viability of MC3T3-E1 cells	[73]

Abbreviations: MC3T3-E1, mouse preosteoblast cell line; PLA, polylactic acid; PGA, polyglycolic acid; PLGA, polylactic-co-glycolic acid; E7-BMP-2, synthetic peptide that mimics the function of bone morphogenetic protein 2; PCL, polycaprolactone; PEEK, polyetheretherketone; APS, astragalus polysaccharide; ePTFE, expanded polytetrafluoroethylene; SD, Sprague–Dawley; PMMA, polymethyl methacrylate; MG-63, human osteosarcoma cell line; CaP, calcium phosphate; AR, alizarin red; PEG, polyethylene glycol; β-TCP, β-tricalcium phosphate.

Table 2. Synthetic-origin biomaterials for dental bone remodeling: advantages and current challenges.

Characteristics of Materials Derived from Synthetic Sources
Classification	Material	Application	Advantages	Limitations	Type of Study and Outcome	Reference
Metals and alloys	Titanium	Dental and orthopedic implants, cardiovascular devices, surgical instruments, scaffolds	Biocompatibility, low density, high melting point, low thermal expansion coefficient, poor conductor of electricity, nontoxic, mechanical strength and stiffness	Difficult to machine, easily damaged by wear, expensive to produce. Although scaffolds had good osseointegration ability and osteogenic induction ability, osteolysis was still found from 20% revision surgeries	In vivo Porous titanium alloy scaffold was filled with ingrowth bone tissue at week 12, trabeculae within the range of stress shielding were not as strong as healthy control group	[53]
	Magnesium	Ligatures, cardiovascular stents, orthopedic implants	Biocompatibility, biodegradability, high strength-to-weight ratio, low density	High corrosion rate, H2 released during degradation accumulates in the surrounding soft tissue	In vitro Noninferiority of both 1.5 mm and 1.75 mm magnesium miniplates compared to 1.0 mm titanium miniplates was demonstrated in a mandible fracture model in sheep	[54]
	Zinc	Orthopedic implants, scaffolds, drug delivery, cardiovascular devices, enzymatic reactions	Biocompatibility, biodegradability, moderate corrosion	Low mechanical strength and plasticity, high concentrations of zinc ions can be toxic to cells	In vitro The Zinc scaffolds were biocompatible, as preosteoblasts were able to effectively adhere to the substrates with multiple cytoplasmic extensions, and indirect tests show the porous scaffolds retained >75% cell viability	[55]
	Strontium	Bone and tissue regeneration, drug delivery, wound dressings, bioimaging	Biocompatibility, biodegradability, can regulate bone metabolism, can promote angiogenesis, immunomodulation, matrix synthesis, mineralization, and antioxidation	Excessive strontium intake can weaken bones by displacing calcium, lower mechanical strength, limited long-term data	In vitro Strontium-doped nanorods arrays accelerate M1 to M2 transformation of the adhered macrophages, enhancing secretion of pro-osteogenic cytokines and growth factors (TGF-β1 and BMP2)	[56]
	Copper	Implant coatings, antibacterial materials, medical devices, drug delivery, bioimaging, enzyme-mimicking	Biocompatibility, biodegradability, variable porosity, good mechanical strength, and crosslinking, can promote angiogenesis, can stimulate osteoblast activity	Excessive copper can lead to oxidative stress, tissue damage, and potentially impact bone metabolism negatively	In vitro Copper in calcium phosphate scaffolds improved osteogenic, angiogenic, and antibacterial properties, facilitating better bone regeneration in vitro	[57]
	Stainless steel	Orthopedic implants, dental implants, cardiovascular devices, surgical instruments	Biocompatibility, good mechanical strength, corrosion resistance	Unmodified stainless-steel surfaces can be hydrophobic, attracting protein adsorption and promoting biofilm formation, limited bioactivity	In vitro The high corrosion resistance of the selective laser melting sample (SLM) limited the release of toxic ions into the biological environment, which resulted in better viability and proliferation of the MC3T3-E1 preosteoblast	[58]
	Cobalt-Chromium	Orthopedic implants, scaffolds, dental prosthesis, cardiovascular implants	Biocompatibility, high strength, corrosion resistance	Metal ion release, local tissue damage, inflammation	In vitro Chromium-cobalt coated scaffolds favor cell adhesion and growth of precursor cells from periodontal ligament	[59]
	Silver	Drug delivery, antibacterial coatings, scaffolds, wound healing process	Biocompatibility, broad spectrum antimicrobial activity, versatility	Long-term toxicity unknown, potential accumulation in tissues	In vitro The silver nanoparticles-loaded gelatin/β-tricalcium phosphate scaffolds demonstrated synergistic antibacterial activity, cytocompatibility, and osteogenic promotion	[60]
	Tantalum	Orthopedic implants, dental implants, scaffolds, cardiovascular devices, drug delivery	Biocompatibility, high strength, corrosion resistance, osteoconductive and osteoinductive properties	High elastic modulus can lead to stress shielding, not immune to bacterial infections, inflammatory reactions	In vitro Porous tantalum can promote the adhesion and proliferation of bone marrow mesenchymal stem cells. Osteogenic gene expression and ALP expression levels were significantly increased compared with porous Ti6Al4V	[61]
	Zirconium	Drug delivery, orthopedic implants, dental implants, scaffolds	Biocompatibility, mechanical strength, chemical stability, corrosion resistance	Zirconium nanoparticles can exhibit toxicity, potentially disrupting cells and causing oxidative stress	In vivo CAD/CAM porous zirconia scaffolds enriched with nanohydroxyapatite particles revealed significantly higher volume of new bone formation (33% ± 14) compared to the controls (21% ± 11)	[62]
Polymers	PLA	Drug delivery, medical devices, tissue engineering	Biocompatibility, biodegradability, renewable resource, versatility	Low osteoconductivity, acidic degradation products, poor cellular adhesion, low thermal stability, poor solubility in organic solvents	In vitro The porous spiral scaffold with larger surface area and better interconnections between internal porous networks could significantly improve the spatial cell compartment and promote human fetal osteoblasts growth	[63]
	PGA	Scaffolds, drug delivery, resorbable implants, sutures	Biodegradability, biocompatibility, ease of fabrication	Acidic degradation products, limited solubility, poor mechanical properties, rapid degradation	In vitro The collagen-PGA sponge was superior to the original collagen sponge in terms of the initial attachment, proliferation rate, and osteogenic differentiation of the bone marrow mesenchymal stem cells	[64]
	PLGA	Drug delivery, tissue engineering, wound dressing, medical devices, vaccines (nanoparticles)	Biocompatibility, biodegradability, controlled release, versatility	Poor drug loading and burst release, can trigger an immune response, need for surface modification, acidic degradation products	In vivo E7-BMP-2 peptides incorporated into 3D hybrid PLGA nanofiber aerogels can induce ~60–70% closure of critical-sized (8 mm) rat calvarial bone defects	[65]
	PCL	Tissue engineering, surgical sutures, drug delivery, orthopedic implants, wound dressings	Biocompatibility, biodegradability, ease of processing, tunable properties	Slow degradation rate, poor mechanical properties, low cell adhesion, low melting point	In vitro The PCL scaffolds with oxidized hyaluronic acid glycine-peptide conjugates demonstrated improved endothelial cell adhesion, proliferation and viability, suggesting the potential for vascularized tissue constructs	[66]
	PEEK	Orthopedic implants, maxillofacial implants, dental prostheses, cardiovascular devices	Biocompatibility, mechanical strength, chemical resistance	Poor osseointegration, high cost, complex manufacturing, limited cell adhesion	In vitro An APS-coated plasma-treated sulfonated bioactive PEEK scaffold facilitates M2 macrophage polarization, reduces pro-inflammatory cytokines, and enhances the secretion of anti-inflammatory factors	[67]
	ePTFE	Cardiovascular devices, tissue regeneration, dural substitute	Biocompatibility, controlled porosity, thermal stability	Susceptibility to bacterial adhesion, nonbiodegradable, needs to be removed after a certain period	In vivo ePTFE scaffolds with bone morphogenic proteins were implanted subcutaneously into SD rats to determine their in vivo ossification potential, scaffolds showed slight radiopacity 1 week after implantation and strong radiopacity 2 and 3 weeks after implantation	[68]
	PMMA	Bone substitutes, fillers, drug delivery, tissue engineering	Biocompatibility, affordability, mechanical strength, ease of processing	Non-degradable, poor bioactivity, can cause allergic reactions due to the presence of nickel in its composition	In vitro MG-63 cells were able to adhere and reside on the PMMA-CaP scaffolds up to 7 days of culture, and the large number of AR-stained cells shows that the cells are viable up to 7 days	[69]
	PEG	Drug delivery, tissue engineering, medical devices, coatings	Biocompatibility, biodegradability, versatility, solubility in water	Toxic byproducts and accumulation, PEGylation can lead to the formation of vacuoles in cells, poor mechanical properties	In vitro The hydrophilic PEG inserted into the PLA chains dissolved rapidly in the degradation environment, forming microporous channels that promoted the degradation of the PLA matrix	[70]
Ceramics	Hydroxyapatite	Bone grafts, coatings for implants, drug delivery, tissue engineering	Biocompatibility, bioactivity, tunable properties, osteoconductivity	Brittleness, low fracture toughness, slow osseointegration rates	In vivo The nanocarbonate hydroxyapatite scaffolds increase bone formation after 12 weeks implanted in a critical defect in rabbit radius compared with the control group	[71]
	β-TCP	Tissue engineering, coatings, drug delivery, guided bone regeneration	Biocompatibility, bioactivity, osteoconductive and osteoinductive properties	Poor mechanical properties, high adsorption rate, the crystallographic structure and properties of β-TCP can be influenced by impurities and the manufacturing process	In vivo β-TCP/PCL composite scaffolds demonstrated superior new bone quality and quantity in complex, mechanically demanding environments such as radial defects	[72]
	Bioactive glasses	Tissue engineering, drug delivery, wound healing, dental implants	Biocompatibility, bioactivity, versatility, tunable properties	Poor mechanical properties, brittleness, low fracture toughness, rapid dissolution	In vitro The combined use of bioactive glass and bone-conditioned medium in scaffolds synergistically promoted osteogenic differentiation and viability of MC3T3-E1 cells	[73]

Abbreviations: MC3T3-E1, mouse preosteoblast cell line; PLA, polylactic acid; PGA, polyglycolic acid; PLGA, polylactic-co-glycolic acid; E7-BMP-2, synthetic peptide that mimics the function of bone morphogenetic protein 2; PCL, polycaprolactone; PEEK, polyetheretherketone; APS, astragalus polysaccharide; ePTFE, expanded polytetrafluoroethylene; SD, Sprague–Dawley; PMMA, polymethyl methacrylate; MG-63, human osteosarcoma cell line; CaP, calcium phosphate; AR, alizarin red; PEG, polyethylene glycol; β-TCP, β-tricalcium phosphate.

5. Immunological Interaction

Although natural and synthetic scaffolds are designed according to the principles of biocompatibility, biofunctionality, and osteoconductivity [74]. The immune system response remains a determining factor in immune system integration and clinical success [75]. Excessive activation of the immune system can induce chronic inflammation, fibrosis, bone resorption, or graft failure, whereas a controlled and regulated response promotes osteogenesis and bone remodeling [76].

The study of immunogenicity through in vitro models is a key strategy for predicting the biological behavior of a biomaterial before its evaluation in animal models [77]. This approach eliminates systemic variables, such as hormones that participate in physiological bone remodeling (calcitonin, parathyroid hormone, vitamin D3, and estrogen) [78]; reduces the use of experimental animals in early stages (following the 3Rs: replacement, reduction, refinement) [79]; and facilitates direct comparisons between materials under identical conditions [80]. In this context, in vitro tests not only assess the safety of a material but also provide information on its immunomodulatory properties, which is particularly relevant in bone remodeling, where the balance between inflammation and tissue restructuring is essential [81].

5.1. Innate Immune Mechanisms Triggered by Biomaterials

The innate immune response constitutes the first line of defense against foreign materials and is activated immediately upon contact between the scaffold and the tissue or cellular microenvironment [82].

During the immediate phase of the implantation process, upon contact with biological fluids, the scaffold undergoes the formation of a protein layer composed of albumin, fibrinogen, fibronectin, immunoglobulins, and complement proteins (C3, C3b, C4, and C5) [83]. This layer conditions the way in which immune cells recognize the material (through receptors such as integrins or complement receptors) [84]. The interaction between proteins at the scaffold surface and adhesion receptors on inflammatory cell populations is the main cell recognition system for implantable materials and medical devices [85].

In the early stage, within hours of implantation, the protein layer generated in the immediate phase triggers the activation of the complement system, releasing anaphylatoxins C3a and C5a, which attract neutrophils and monocytes [86]. Once neutrophils arrive at the site, they release reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) to degrade or isolate the material [87].

Throughout the intermediate phase, during the first 3 days after implantation, monocytes are attracted by CCL2/MCP-1 and C5a [6]; once on the scaffold surface, they differentiate into macrophages, initiating polarization toward the M1 (proinflammatory) phenotype with the production of TNF-α, IL-1β and IL-6 [88]. M1 macrophages attempt to phagocytose fragments of the material; if they cannot remove them, they secrete degradative enzymes (metalloproteinases, collagenase, elastase, and hyaluronidase) [89].

In the late phase, if the scaffold is properly designed (biocompatible, degradable, with osteoinductive and immunomodulatory signals), the initial inflammation subsides [41]. Macrophages switch to an M2 (anti-inflammatory) phenotype, releasing IL-10, TGF-β, VEGF and BMP-2, promoting angiogenesis, osteoblast recruitment and bone remodeling [90]. This timely shift from M1 to M2 polarization (day 3–7) is essential for creating a conducive microenvironment for bone remodeling and successful scaffold integration.

If the material is not well tolerated, M1 macrophage activation is perpetuated, resulting in the formation of granulomas, multinucleated giant cells, and fibrous encapsulation, indicating scaffold failure [82]. Figure 2 illustrates the coordinated sequence of innate and adaptive immune events involved in the foreign body in response to implanted biomaterials, from initial inflammation to chronic immune activation or resolution. Table 3 lists the cytokines and growth factors implicated in the innate immune response.

5.2. Adaptive Immune Mechanisms Triggered by Biomaterials

The adaptive immune response plays a determining role in the long-term integration of biomaterials, in the resolution of inflammation or, in unfavorable cases, in the rejection of the implant [91]. This response develops in a later phase (day 3–week), modulated by signals from innate immune cells such as macrophages and dendritic cells [11]. Macrophages and dendritic cells phagocytose fragments of the scaffold or adsorbed proteins and act as antigen-presenting cells (APCs) [92]. They present those antigenic peptides associated with MHC class II molecules to CD4+ T cells [93]. Th1 cells promote chronic inflammation by releasing IFN-γ and TNF-α, maintaining the M1 phenotype of macrophages [94]; Th2 cells secrete IL-4, IL-13 (favoring M2 polarization and a pro-regenerative environment) [95], IL-4, IL-5 and IL-6 (stimulating B lymphocyte and antibody production) [96]; Th17 cells produce IL-17, which stimulates neutrophil recruitment and contributes to the development of fibrosis [97]; and finally, regulatory T cells (Tregs) secrete IL-10 (suppressing macrophage activation and proinflammatory cytokine production) and TGF-β (regulating lymphocyte proliferation), which are fundamental in the resolution of inflammation and biomaterial tolerance [98]. In the case of scaffolds of natural origin (such as collagen, chitin, and decellularized extracellular matrix), there is a risk of generating antibodies against conserved epitopes such as α-Ga, a carbohydrate epitope abundantly expressed on cell membrane glycolipids and glycoproteins in nonprimate animals (bovine and porcine bone grafts) [99]. The production of IgG and IgM antibodies can contribute to opsonization and favor phagocytosis by macrophages, although excess IgG and IgM can induce chronic inflammation [100,101]. Knowledge of these combined immune responses has motivated the development of scaffolds with immunomodulatory capacity [102]. Figure 3 illustrates the activation and differentiation of T and B lymphocyte subpopulations in response to biomaterial implantation. The main cytokines and growth factors involved in the adaptive response are presented in Table 3.

Table 3. Osteoimmunological cytokines and growth factors regulating bone remodeling.

Cytokines and Growth Factors Involved in Bone Remodeling
Immunity	Cytokine/ Growth Factor	Origin	Subexpression	Overexpression	Reference
Innate	IL-1β	Macrophages M1	Decreased initial cell recruitment and inflammatory signaling. Delay in the early phase of the foreign body reaction	Chronic inflammation, increased RANKL production, osteoclastogenesis, bone resorption and possible fibrous encapsulation	[103]
Innate	IL-4	Th2	Decreased polarization toward the M2 phenotype, persistent inflammation, reduced support for osteogenesis	Increased polarization toward the M2 phenotype, reduces inflammation; however, excessive levels can inhibit necessary initial responses	[94]
Adaptative	IL-5	Th2	Limited direct impact on bone; possible alteration in humoral immune regulation	It may indirectly contribute to fibrosis	[95]
Innate/Adaptative	IL-6	Macrophages M1, Th2	It decreases the early activation of remodeling and initial osteo-immune signaling	Increased osteoclastogenesis, sustained inflammation, bone loss and failure of biomaterial integration	[96,104]
Innate/ Adaptative	IL-10	Macrophages M2, Treg	Lack of inflammatory resolution, prolonging the M1 polarization causing tissue damage	Decreases inflammation and osteoclastogenesis; very high levels can suppress early osteogenic signals	[90]
Adaptative	IL-13	Th2	It decreases polarization toward the M2 phenotype and angiogenic support	May promote fibrosis over functional bone formation	[105]
Adaptative	IL-17	Th17	Decreased initial neutrophil recruitment may affect implant site clearance	Chronic inflammation, increased RANKL production, osteoclastogenesis	[106]
Innate/ Adaptative	TNF-α	Macrophages M1	Deficient early inflammatory activation (delayed repair)	Inhibition of osteoblasts, increased osteoclasts, bone loss, chronic inflammation	[107]
Innate/ Adaptative	TGF-β	Macrophages M2, Treg	Decreased MSC recruitment and osteoblastic differentiation	Can induce fibrosis and encapsulation if not finely regulated	[108]
Innate	VEGF	Macrophages M2	Decreased angiogenesis, immature bone, poor scaffold integration	Disorganized vasculature, deficient synergy with osteogenesis if not temporarily controlled	[91]
Innate	BMP-2	Macrophages M2	Decreased osteoblastic differentiation and mineralization	Ectopic bone formation, local inflammation, and adverse clinical effects	[109]
Adaptative	IFN-γ	Th1	Reduced control of osteoclastogenesis in specific phases	Increased inflammation, osteoblast inhibition, Th1 activation (possible scaffold rejection)	[95]

Abbreviations: IL-1β, Interleukin-1 beta; IL-4, Interleukin-4; IL-5, Interleukin-5; IL-6, Interleukin-6; IL-10, Interleukin-10; IL-13, Interleukin-13; IL-17, Interleukin-17; TNF-α, Tumor Necrosis Factor Alpha; TGF-β, Transforming Growth Factor Beta; VEGF, Vascular Endothelial Growth Factor; BMP-2, Bone Morphogenic Protein-2; IFN-γ, Gamma Interferon; Th2, T helper 2; Th1, T helper 1; Th17, T helper 17; Treg, T regulatory.

Table 3. Osteoimmunological cytokines and growth factors regulating bone remodeling.

Cytokines and Growth Factors Involved in Bone Remodeling
Immunity	Cytokine/ Growth Factor	Origin	Subexpression	Overexpression	Reference
Innate	IL-1β	Macrophages M1	Decreased initial cell recruitment and inflammatory signaling. Delay in the early phase of the foreign body reaction	Chronic inflammation, increased RANKL production, osteoclastogenesis, bone resorption and possible fibrous encapsulation	[103]
Innate	IL-4	Th2	Decreased polarization toward the M2 phenotype, persistent inflammation, reduced support for osteogenesis	Increased polarization toward the M2 phenotype, reduces inflammation; however, excessive levels can inhibit necessary initial responses	[94]
Adaptative	IL-5	Th2	Limited direct impact on bone; possible alteration in humoral immune regulation	It may indirectly contribute to fibrosis	[95]
Innate/Adaptative	IL-6	Macrophages M1, Th2	It decreases the early activation of remodeling and initial osteo-immune signaling	Increased osteoclastogenesis, sustained inflammation, bone loss and failure of biomaterial integration	[96,104]
Innate/ Adaptative	IL-10	Macrophages M2, Treg	Lack of inflammatory resolution, prolonging the M1 polarization causing tissue damage	Decreases inflammation and osteoclastogenesis; very high levels can suppress early osteogenic signals	[90]
Adaptative	IL-13	Th2	It decreases polarization toward the M2 phenotype and angiogenic support	May promote fibrosis over functional bone formation	[105]
Adaptative	IL-17	Th17	Decreased initial neutrophil recruitment may affect implant site clearance	Chronic inflammation, increased RANKL production, osteoclastogenesis	[106]
Innate/ Adaptative	TNF-α	Macrophages M1	Deficient early inflammatory activation (delayed repair)	Inhibition of osteoblasts, increased osteoclasts, bone loss, chronic inflammation	[107]
Innate/ Adaptative	TGF-β	Macrophages M2, Treg	Decreased MSC recruitment and osteoblastic differentiation	Can induce fibrosis and encapsulation if not finely regulated	[108]
Innate	VEGF	Macrophages M2	Decreased angiogenesis, immature bone, poor scaffold integration	Disorganized vasculature, deficient synergy with osteogenesis if not temporarily controlled	[91]
Innate	BMP-2	Macrophages M2	Decreased osteoblastic differentiation and mineralization	Ectopic bone formation, local inflammation, and adverse clinical effects	[109]
Adaptative	IFN-γ	Th1	Reduced control of osteoclastogenesis in specific phases	Increased inflammation, osteoblast inhibition, Th1 activation (possible scaffold rejection)	[95]

Abbreviations: IL-1β, Interleukin-1 beta; IL-4, Interleukin-4; IL-5, Interleukin-5; IL-6, Interleukin-6; IL-10, Interleukin-10; IL-13, Interleukin-13; IL-17, Interleukin-17; TNF-α, Tumor Necrosis Factor Alpha; TGF-β, Transforming Growth Factor Beta; VEGF, Vascular Endothelial Growth Factor; BMP-2, Bone Morphogenic Protein-2; IFN-γ, Gamma Interferon; Th2, T helper 2; Th1, T helper 1; Th17, T helper 17; Treg, T regulatory.

6. In Vitro Tests to Evaluate Scaffold Immunogenicity

The study of the immunogenicity of scaffolds in vitro allows for controlled analysis of how biomaterials influence the activation of the immune system prior to in vivo studies [110]. The selection of a cell model depends on the immune phase to be analyzed [77]. These cell models allow analysis of everything from early phagocytic cell-mediated inflammation to lymphocyte activation and interaction with resident bone cells.

6.1. Monocytes and Macrophages

The lining tissues of bone (periosteum and endosteum) contain a discrete population of resident macrophages that perform immune surveillance in the bone microenvironment, phagocytosis, detection of bacterial products, and antigen presentation [111]. Upon bone injury, macrophages derived from monocytes secrete various cytokines (IL-1β, IL-6, IL-12, IL-23, IFN-γ, and TNF-α), chemokines (C-X-C), and growth factors (TGF-β) to initiate the recruitment of fibroblasts, mesenchymal stem cells (MSCs), and osteoprogenitor cells from their local niches [103]. Inflammatory mediators produced by macrophages, including IFN-γ, TNF-α, and IL-6, promote either the proliferation of preosteoclast cells or osteoclast differentiation [104]. Some studies suggest that macrophages can produce BMP-2 (bone morphogenic protein 2), which promotes the osteogenic differentiation and proliferation of hBMSCs (human bone marrow mesenchymal stem cells) [109]. They constitute the first line of response to biomaterials and are the most widely used model [112]. They respond to a wide range of biomaterials in vivo and in vitro, including polymers, metals, collagen, and ceramics [113]. To determine the polarization profile, M1 (proinflammatory) or M2 (anti-inflammatory) cytokine secretion can be assessed [114]. Refai et al. [115] demonstrated that the surface topography of titanium, particularly the SLA surface (sandblasted and acid-etched), modulated the expression of proinflammatory cytokines and chemokines by macrophages in a time-dependent manner. In a study conducted by Kazimierczak et al. [116], a biomaterial composed of chitosan, agarose, and nanohydroxyapatite was developed. Activated macrophages were cultured on the surface of this scaffold, and an increase in the release of anti-inflammatory cytokines (IL-4, IL-10, IL-13, and TGF-β), characteristics of the M2 phenotype, was observed.

6.2. Dendritic Cells

It acts as a bridge between innate and adaptive immunity, presenting antigens to T lymphocytes via MHC-II [117]. Dendritic cells (DCs) are present in oral gingival tissue. In chronic periodontitis, DCs cluster in periodontal lesion tissue to destroy periodontal bone tissue by forming aggregates with T cells [118]. Dendritic cells (DCs) can differentiate into functional, multinucleated osteoclasts, particularly in the presence of inflammatory cytokines such as macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor kappa-b ligand (RANKL), and IL-17 [119]. Under inflammatory conditions, osteoclasts and DCs cooperate to mediate bone destruction and bone remodeling [120]. The function of DCs depends mainly on their degree of maturation. Mature DCs can effectively initiate T and B-cell activation, thereby triggering adaptive immunity and immune memory.

In contrast, immature DCs can promote tolerance induction. Human monocyte-derived DCs can be cultured in contact with scaffolds, and their maturation can be assessed by measuring CD86 expression [121]. Chan et al. [122] demonstrated that Ti(IV) ions alter the properties of dendritic cells, leading to increased T-cell reactivity and a shift toward a Th1-type immune response. This effect may account for the inflammatory side effects observed in patients with titanium implants. Park et al. [123] evaluated the immunological outcome of DC treatment with different biomaterials (PGLA, chitosan, alginate, hyaluronic acid and agarose) to demonstrate the variety of DC phenotypes induced by biomaterials commonly used in combination products such as vaccine delivery vehicles or tissue-engineered scaffolds. The results showed that the PLGA or chitosan films promoted DC maturation, whereas HA inhibited DC maturation. Agarose was also useful for maintaining an immature DC (iDC) phenotype.

6.3. Neutrophils

When tissue damage occurs, neutrophils are the first to be recruited and migrated to the injury site. They are responsible for eliminating invading pathogenic microorganisms, initiating an acute inflammatory response, and improving the host’s defenses [124]. Under conditions of chronic inflammation, such as periodontitis, neutrophils can produce signals such as RANKL, stimulating osteoclastogenesis and promoting bone resorption [125]. Although they are rarely used in prolonged assays because of their short half-life [126], they are used in studies of NETosis, reactive oxygen species (ROS) release, and IL-8 secretion [127]. Li et al. [128] developed a zinc-doped ferric oxyhydroxide nanolayer-modified plasma electrolytic oxidation (PEO)-coated Mg alloy (PEO-FeZn). They evaluated its antibacterial, immune-anti-infective, and osteogenic abilities. The nanolayer inhibited immune evasion-related gene expression and contributed to the formation of neutrophil extracellular traps (NETs) by activating the extracellular release of DNA fibers and granule proteins, thereby suppressing bacterial invasion and promoting osseointegration in vivo in Staphylococcus aureus-infected rat femurs. Herath et al. [129] evaluated a triple-cell coculture model (including osteoblasts, endothelial cells, and neutrophils) and demonstrated that neutrophils significantly increased angiogenesis by inducing the expression of proangiogenic markers such as VEGF-A, CD34, EGF, and FGF-2. However, their excessive activation may be associated with chronic inflammation and fibrous encapsulation [91].

6.4. T Lymphocytes

Under inflammatory conditions, T cells produce a series of cytokines and growth factors that play key roles in bone remodeling, such as RANKL, which can promote the differentiation of monocytes into osteoclasts [130]. The participation of these cells depends on the subtype into which they differentiate: polarization toward Th1 (promotes inflammation), Th2 (promotes remodeling), Th17 (persistence of inflammation), and Treg (suppresses the inflammatory response) [131].

Li et al. [132] used biphasic calcium phosphate (BCP) granules in a model of ectopic bone formation in female mice. They demonstrated that BCP activates the calcium signaling pathway in macrophages, leading to the release of the chemokines Ccl3 and Ccl17. These chemokines recruit Tregs, which are crucial in BCP-mediated bone regeneration. Song et al. [133] used a rat alveolar bone defect model to evaluate a deoxyribonucleic acid-crosslinked collagen scaffold (DNA-Col). These authors reported that differentially expressed genes (DEGs) were positively correlated with genes involved in T-cell development and that the number of Tregs increased.

6.5. B Lymphocytes

Osteoblast lineage cells promote the differentiation and growth of hematopoietic stem cells (HSCs) and B cells in endosteal bone niches [134]. The interaction between B cells and bone cells is reciprocal, and defects in the RANKL-RANK-OPG signaling axis result in altered bone phenotypes [135]. Although the role of B cells in normal bone remodeling is minimal, activated B cells play an important role in many inflammatory diseases associated with bone changes, as well as in periodontal disease [136].

In recent years, regulatory B lymphocytes (Bregs) have been shown to be crucial modulators of various inflammatory diseases [137]. Hetta et al. [138] analyzed changes in circulating Bregs and proinflammatory (IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10, IL-35, and TGF-β) cytokines in patients with periodontitis. These results suggest that the increase in peripheral Breg cells and serum cytokine levels among periodontitis patients is closely associated with disease progression.

In the context of bone tissue engineering, to date, no studies have been conducted using B lymphocytes to evaluate antibody secretion (IgG/IgM) in the presence of scaffolds. This lack of information presents an opportunity to evaluate how these antibodies promote opsonization and macrophage phagocytosis [139].

6.6. Cocultures of Mesenchymal Stem Cells and Osteoblasts

MSCs are derived mainly from bone marrow; however, they can also be found in other tissues of the body [140]. Adult MSCs are found in the oral cavity. In the context of bone tissue engineering, the most relevant MSC niches are periodontal ligament stem cells (PDLSCs), gingival tissue stem cells (GMSCs), bone marrow stem cells (BMSCs), and oral mucosal stem cells (OMSCs) [141]. MSCs can differentiate into osteoblasts and migrate to the surface of bone defects to modulate the microenvironment [142].

Osteoblasts, the cells responsible for bone formation, arise from the commitment of mesenchymal precursors to osteoprogenitor lineages through the sequential action of transcription factors and ultimately differentiate into osteocytes [143]. Osteoblasts produce extracellular proteins (osteocalcin and alkaline phosphatase) and type I collagen, which constitute more than 90% of the protein in the bone matrix [144]. Jain et al. [145] prepared biocomposite scaffolds comprising chitosan (CHT), polycaprolactone (PCL) and hydroxyapatite (HAP) via the freeze–drying method to assess the role of the scaffolds in the spatial organization, proliferation, and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in vitro. The scaffold compositions supported mesenchymal stem cell attachment, proliferation, and osteoblast differentiation. Li et al. [146] created a three-dimensional composite hydrogel scaffold composed of sodium alginate microspheres encapsulated in type I collagen. A 3D indirect coculture system was established in which osteoblasts and endothelial cells were used to evaluate the osteogenic differentiation potential. The results demonstrate that endothelial cells significantly promote osteogenic differentiation of osteoblasts.

It is important to design experiments to understand how MSCs regulate osteogenic differentiation and bone regeneration in the presence of scaffolds and how these cells interact with each other to promote bone formation [147].

6.7. Cocultures of Osteoblasts and Macrophages

This model is essential for testing immunological biocompatibility [107]. A successful scaffold should favor the transition from M1 to M2 macrophages and allow osteoblasts to maintain their ability to mineralize [148]. Jacho et al. [149] created a cell-laden 3D tissue analog to study indirect crosstalk between osteoblasts and macrophages, as well as their direct synergistic effect when cultured on a 3D scaffold. Compared with osteoblasts exposed to only media, osteoblasts exposed to secretions from proinflammatory macrophages presented elevated RANKL expression and decreased alkaline phosphate activity. In contrast, anti-inflammatory markers (CCL18) were downregulated, and osteoclastogenic markers (TRAF6 and ACP5) were unchanged. These data suggested that osteoblasts inhibited the osteoclastogenic differentiation of macrophages while preserving their proinflammatory lineages. Shi et al. [150] investigated the role of macrophage behavior on deproteinized bovine bone matrix (DBBM, BioOss) and its impact on the microenvironment for bone tissue remodeling. These results demonstrated that RAW 264.7 cells could polarize into M2 macrophages in response to DBBM. Conditioned media from macrophages cultured with DBBM seeded with MC3T3-E1 cells (osteoblast-like cells derived from mouse calvaria) markedly increased osteoblast differentiation.

7. Discussion

Bone remodeling in the oral cavity, driven by natural and synthetic biomaterials, is a coordinated process regulated by a complex network of cytokines that mediate communication between immune cells and bone-forming cells [9]. The literature indicates that the interplay between the scaffold and the immune system is a crucial factor in determining whether a graft will be successful [81]. Innate immune responses are activated within the initial hours following implantation with the foreign body reaction [83], which results in early infiltration of neutrophils and monocytes, followed by macrophage polarization to either a proinflammatory (M1) or anti-inflammatory (M2) phenotype [113]. This sequence is critical because prolonged predominance of M1 macrophages can generate a hostile microenvironment, leading to the release of TNF-α, IL-1β, and IL-6, which inhibits osteogenesis and promotes osteoclastogenesis [107]. In contrast, an efficient transition to the M2 phenotype, which is mediated by IL-10 and TGF-β, promotes matrix formation [90]. One example of how the transition from the M1 to M2 phenotype can be modulated is bioactive scaffolds that incorporate osteoinductive factors like calcium silicate, HA, tricalcium phosphate or surfaces with specific nanotopography, achieving improved integration in vivo [151]. Therefore, the success of a biomaterial depends not only on its structural or mechanical capacity but also on its ability to regulate the immune system. Most recent advances in tissue engineering are focused on understanding the interplay between chemical and textural characteristics and the immune system; one example is the effect of substrate porosity on macrophages, since the infiltration of oxygen and nutrients could determine their polarization [152].

Although sometimes underestimated, adaptive immunity plays an important regulatory role. Dendritic cells and macrophages present antigen-associated scaffold proteins to CD4+ T lymphocytes via MHC II, which can polarize toward Th1, Th2, Th17, or Treg subpopulations [152]. A shift toward Th1/Th17 subpopulations tends to favor chronic inflammation, whereas Th2/Treg expansion negatively modulates inflammatory activity and protects the viability of osteoblasts and mesenchymal stem cells [106]. B lymphocytes and their antibodies contribute to the opsonization of scaffold fragments and to complement activation, processes that, if unregulated, trigger excessive inflammation. However, they can also play a role in bone repair by secreting osteoprotegerin (OPG), a negative regulator of osteoclastogenesis [134].

Within the osteoimmunological framework, cytokines act as molecular switches that determine whether the immune microenvironment favors inflammation, bone resorption, tissue remodeling, or successful biomaterial integration [10]. Cytokine profiling provides critical insight into the immunomodulatory properties of biomaterials. In vitro assessment of cytokine secretion enables early prediction of scaffold performance by revealing its capacity to steer the immune response toward an anti-inflammatory phenotype [153]. Consequently, the ability of biomaterials to modulate cytokine signaling should be considered a key criterion in biomaterial design and preclinical evaluation.

In this context, in vitro assays and cell-based models represent essential tools for the preliminary assessment of scaffold suitability for bone remodeling [77]. These systems allow the direct impact of the scaffold on essential cells, including osteoblasts, osteoclasts, mesenchymal stem cells, macrophages, and lymphocytes, to be studied under conditions that are controllable and reproducible [154]. They are early predictors of biocompatibility, osteoinductivity, and immunogenicity, accelerating scaffold design and optimization. However, in vitro systems provide a limited view of the bone microenvironment, omitting factors such as vascularization and dynamic mechanical loads [50]. Therefore, their use should be understood as an initial and complementary filter.

As our understanding of the mechanisms regulating bone remodeling expands, new therapeutic targets and signaling pathways that could optimize biomaterial design have emerged [11]. In dentistry, bone remodeling is addressed clinically through various therapeutic strategies, ranging from autologous grafts (the gold standard) to synthetic or natural scaffolds (hydrogels, collagen matrices, chitosan, and hydroxyapatite) [3], which are designed to fill alveolar defects, repair peri-implant fenestrations, or facilitate alveoloplasty after tooth extraction [8]. These options have clear strengths (biocompatibility, osteoconductivity, bioactivity) but also significant limitations, such as biological variability, low mechanical resistance underload, poor vascularization in critical defects, imprecise control of factor release, and the risk of a chronic inflammatory response or premature resorption [18]. Faced with these limitations, efforts have focused on two main areas: the design of immunomodulatory biomaterials that control macrophage polarization [110] and the development of physicochemical platforms that improve mechanical properties, porosity, and vascularization [75]. For example, scaffolds loaded with nanoparticles that release anti-inflammatory drugs or pro-M2 peptides have been shown to guide the local immune response toward reparative phenotypes, reducing persistent inflammation and promoting osteogenesis [155]. In parallel, surface modification techniques for implants (ion doping, HAp coatings, nanotexturization) increase cell adhesion and bone integration while also incorporating immunomodulatory pharmaceuticals [156]. However, several methodological and regulatory challenges must be addressed before widespread clinical implementation, replicability in the production of natural biomaterials (batch-to-batch), long-term safety testing (biodegradation and byproducts) [45], industrial scaling of advanced techniques (3D printing with hybrid materials), and controlled clinical trials demonstrating clear benefits over standard approaches (autografts, conventional membranes) [157].

Despite growing interest in the development of biomaterials for tissue engineering, the evaluation of immune responses continues to receive insufficient attention in many studies. A considerable number of studies continue to focus primarily on physicochemical and mechanical properties, overlooking the complex interactions between biomaterials and the host’s immune system. This lack of information results in a limited understanding of immunogenicity and immunomodulatory effects, which ultimately hinders the translation of these materials into clinical applications.

This manuscript does not provide a guideline for selecting a specific biomaterial or scaffold design. Its purpose is to present a comprehensive overview of the properties and biological interactions of different materials. The selection of a biomaterial in clinical practice must be based on multiple factors, including the patient’s specific clinical condition, the characteristics of the defect and mechanical requirements. In addition, practical considerations such as resource availability and the clinical setting may also influence the final decision.

8. Conclusions and Future Perspectives

Bone remodeling in dentistry is entering a phase of rational biomaterial design guided by osteoimmunology, and the most promising interventions not only support or drive new bone formation but also actively shape the local immune response to promote inflammation resolution and repair. Innate and adaptive immunity play crucial roles in the integration or rejection of biomaterials; understanding and guiding this interaction is an essential requirement for the design of the next generation of biomaterials. The evidence discussed in this review highlights that the balance between proinflammatory cytokines (e.g., TNF-α, IL-1β, IL-6, and IL-17) and anti-inflammatory or pro-remodeling cytokine mediators (e.g., IL-4, IL-10, and TGF-β) critically influences macrophage polarization, lymphocyte activation, and osteoblast–osteoclast coupling. A better understanding of the initial interaction between the biomaterial surface and the host will help in designing biomaterials that promote balanced immune system activation.

Here we propose an integrated figure (Figure 2 and Figure 3) that includes immune cells and cytokine interaction with the biomaterials in the early and late phases. As we describe in Figure 2, this process begins with complement system activation, resulting in the recruitment of monocytes, which then differentiate into macrophages. In Figure 3, we describe that macrophages and monocytes act as antigen-presenting cells with proteins absorbed onto the scaffold surface. These antigens are presented on MHC-II, resulting in the activation of CD4+ T lymphocytes, which, upon differentiation into Th2 cells, begin to secrete IL-4, promoting the polarization of M2 macrophages, and into Treg cells, which begin to secrete IL-10 and TGF-β, regulating the proliferation of immune cells and promoting tolerance to the scaffold (late-phase scaffolding success). In contrast, in the late phase, excessive activation of the immune system could lead to the differentiation of CD4+ T lymphocytes into Th1 and Th17 cells, promoting a state of chronic inflammation, ultimately resulting in rejection of the material with fibrous encapsulation.

Therefore, cytokine profiling should be regarded not merely as a descriptive readout but also as a functional indicator of biomaterial performance and immunological compatibility. In this context, in vitro tests and cell models are strategic options for early assessment of the biocompatibility and immunogenicity of scaffolds before they are moved on to animal or clinical trials. However, owing to the limitations of in vitro models, more complex strategies, such as three-dimensional (3D) cultures, bone organoids, bone-on-a-chip systems, and multicellular cocultures that more faithfully reproduce the human bone microenvironment, need to be developed. These models represent an ethically and scientifically superior alternative to animal use, enabling the evaluation of cellular and inflammatory responses that more closely resemble human physiology. Its implementation could accelerate the development of safer, more effective, and customized scaffolds, significantly reducing the time and cost of the preclinical phase.

The main challenges include engineering scaffolds to incorporate biomolecules, nanotopographies, or coatings that direct the immune response toward profiles favorable for remodeling, as well as advanced evaluation models that combine immune cells, osteogenic cells, and biomechanical conditions in platforms that are more representative of the bone microenvironment.

This field is advancing toward multifunctional systems, including composites that combine natural and/or synthetic materials with bioactive molecules to modulate cellular responses. At the same time, 3D printing and biofabrication technologies are enabling the development of patient-specific scaffold architectures with controlled porosity and mechanical properties that mimic native bone. Complementing these innovations with artificial intelligence (AI) would allow us to predict immune responses and optimize material design.

This approach represents a promising route toward the next generation of therapies for complex bone defects tailored to each patient’s specific biological needs, guiding bone remodeling through the coordinated regulation of immune, vascular and skeletal systems, enhancing long-term integration and clinical outcomes.