December 14, 2020
by *Zeeshan Sheikh, BDS, Dip.Dh, MSc, PhD; *Nader Hamdan, BDS, MSc, MDent (Perio); Haider Al-Waeli1, BDS, MSc, PhD; Michael Glogauer, DDS, Dip. Perio, PhD. * Both authors equally share the first authorship.
Graft materials are used to restore the physiological and anatomical function of diseased or damaged bone tissues via repair and/or regeneration.1-4 There are various graft options available for maxillofacial bone grafting which are divided into natural tissue transplants (autografts from the same individual, allografts from cadavers of same species and xenografts which are from animal sources) and synthetic materials (alloplasts). (Table 1)5 These graft materials are used because they are either osteogenic (autografts), osteoinductive (autografts and allografts) and/or osteoconductive (autografts, allografts, xenografts and alloplasts).6 Ideally, bone replacement grafts should be biocompatible, easily molded and/or carved, integrate well with the host bone and have adequate mechanical properties.7 Most hard tissue substitute graft materials elicit an immune response upon implantation that ultimately affects and dictates their biological performance in vivo. This article discusses the host immune response to bone replacement graft materials after implantation.
Table 1. Natural and synthetic bone replacement graft material options.8
What happens after implantation?
Bone graft materials implanted into the human body have the ability to trigger host responses: blood–material interactions, acute inflammation, chronic inflammation (if the acute phase is not resolved), granulation tissue development, foreign body reaction (FBR) and fibrous encapsulation.9 Blood/biomaterial interactions begin immediately post-implantation, with protein adsorption onto the biomaterial surface and the development of a blood-based transient provisional matrix (initial thrombus) that forms on and around the biomaterial. (Fig. 1)10 The types, levels, and surface conformations of the adsorbed proteins are dependent on biomaterial surface properties (physico-chemical) and are the crucial determinants of the host response to such implanted grafts.11,12 This ultimately dictates the recruitment, adhesion and survival of cells, especially monocytes, macrophages and foreign body giant cells (FBGCs), on these protein-coated graft surfaces which in turn may affect the proliferation and the differentiation of the osteoprogenitor cells.13
Schematic figure depicting post-implantation events.Immediately after implantation in vivo, a layer of proteins from the microenvironment adsorb and coat the surface of the biomaterial device. This protein adsorption results in attraction, infiltration, and attachment of various cell types such as monocytes, macrophages, and platelets. These cell types release chemokines and cytokines that recruit tissue repair cells to the site of inflammation. These cells produce collagen and result in the fibrous encapsulation of the implanted material. (Adapted from Sheikh and Brooks et al. 2015).14
Immune response to implanted hard tissue grafts
The first responders to the site of grafting (injury) are polymorphonuclear leukocytes (PMNs or neutrophils), followed closely by monocytes.14,15 These cells function to phagocytose cellular debris and foreign material, and eventually leave the site of inflammation.16 Phagocytosis is the uptake of microbial bodies, particles and debris originating from implanted biomaterials.16,17 The presence of PMNs characterizes the acute inflammatory response and are the most numerous leukocytes in the circulation, first to respond to infection, and play an important role in wound healing.18 Soon after PMN recruitment, monocytes are also recruited to the site of inflammation and become tissue macrophages. At the site of inflammation, PMNs recruit more leukocytes, phagocytose invading particles, secrete a toxic mix of enzymes and reactive oxygen species (ROS), and produce neutrophil extracellular traps (NETs).19 PMNs are typically considered to be short-lived cells that are cleared by macrophages after contributing to the acute inflammatory response;13 however, in many chronic inflammatory conditions, PMNs are present consistently at the site of inflammation.20
Monocytes are formed from the myeloid progenitor cells that give rise to monoblasts, pro-monocytes and finally monocytes. Monocytes typically circulate through the blood for 1-3 days before migrating into tissues, where they become macrophages or dendritic cells. Classically activated macrophages, also called M1 macrophages (M1s), are produced during a cell-mediated immune response21,22 and are considered to be pro-inflammatory (destructive). Wound healing (reparative) macrophages make up a group of cells that are called alternatively activated macrophages, or M2 macrophages (M2s) and associated with repair rather than defense.23,24 The timing of initiating the anti-inflammatory immune response is very important, and crucial for the healing outcome. The immune response is responsible for ending the acute phase of inflammation and triggering the repair phase through certain immunomodulatory cytokines (such as CCL2, CXCL7, and IL-10); many mesenchymal cells along with the M2 macrophage plays a significant role in this phase.25 Moreover, prolonged acute inflammation without changing into the repair phase (due to uncontrolled or chronic inflammatory response) can lead to the inhibition of proliferation and differentiation of mesenchymal cells into osteoprogenitor cells and delay regeneration.25
Multinucleated giant cells (MGCs) are a group of cells that are derived from the monocytic lineage, and are associated with bone loss, chronic inflammation, granulomatous disease and tumors.26 Monocytes undergo fusion with one another in the presence of stimuli to form cell types that are present in both health and disease.
Osteoclasts (bone-resorbing cells), are some of the better-characterized members of the MGCs. Osteoclasts work in tandem with osteoblasts, cells responsible for bone deposition, in the process of bone remodeling.14
Immune recognition and attachment to graft materials
Host immune cells react and respond to almost all the various graft materials implanted.27 Macrophages respond to foreign bodies due to their ability to recognize self and non-self. Antigen-presenting cells, which include macrophages and dendritic cells, assist the immune system response to implanted biomaterials. Physical features such as size, substrate stiffness, and topography, can elicit and determine the body’s response to a foreign substance, in addition to surface chemistry, ligand presentation, degradation rates and release of growth factors. It has been well established that biomaterials produce micro-environmental cues that modulate the response of inflammatory cells.28 Following injury, acute inflammation occurs which is part of the natural healing process. The recruitment of other inflammatory cell types to the implant site can lead to a chronic inflammatory state. Foreign body reactions, a type of chronic tissue inflammation describes the presence of foreign body giant cells (FBGCs) at the graft interface.13,29,30 Graft based acute inflammatory response usually resolves within less than one week.29 With biocompatible implanted materials, early resolution of the acute and chronic inflammatory response occurs with the chronic inflammatory generally lasting no longer than two weeks and confined to the implantation site. Persistence of the acute inflammatory response state beyond a three week period usually indicates an infection.13 After the resolution of acute and chronic inflammatory responses has occurred, granulation tissue is observed. This is confirmed by the presence of macrophages, fibroblast infiltration, and neovascularization in the new tissue. Granulation tissue may be a precursor to fibrous capsule formation and is separated from the implanted biomaterial device by the cellular components of a foreign body reaction (consisting of macrophages, and FBGCs).31
Immune response and its effect on graft resorption
PMNs could directly participate in biomaterial resorption or indirectly promote inflammatory cell recruitment. PMNs are known to promote osteoclast-mediated resorption through secretion of interleukin-17 (IL-17). Also, PMNs can promote osteoclast-mediated bone resorption by inducing the retraction of osteoblasts.32 Macrophages respond to particulate biomaterials and engulf particles and fragments depending on their size.33 (Fig. 2) During phagocytosis, macrophage membrane reorganization leads to the complete envelopment of particles, which are contained within the cytoplasm in membrane-bound organelles known as phagosomes.34 By a complex maturation process, lysosomes, hydrolytic enzymes and other substances are fused and released into phagosomes to kill, digest, and degrade the internalized particles.34 When the particle sizes are beyond the capacity of a single macrophage to internalize (between 10 and 100 μm in diameter), FBGCs are formed. (Fig. 2) 27 These cells then attempt to engulf large particles, succeeding at times and failing at others.35
A schematic depiction of macrophage mediated phagocytosis.Macrophages respond to small fragments and particles (< 10 µm in diameter) by internalization via phagocytosis and intracellular digestion. If the particle size is larger than 10 µm and smaller than 100 µm, the macrophages fuse together forming giant cells which in turn engulf the particles and digest them. If the particles are larger, the bulk digestion is carried out via extracellular degradation by macrophages and macrophage fused giant cells through release of enzymes and/or pH lowering mechanisms. (Adapted from Sheikh et al. 2015)36
Macrophages, in addition to their function as phagocytes, are also involved in extracellular biodegradation of biomaterial matrices such as collagen by release of a variety of enzymes.37 Macrophages and FBGCs release mediators of degradation such as reactive oxygen intermediates (ROIs), enzymes, and acid between the cell membrane and biomaterial surface.38,39 It has been noted that phagolysosomes in macrophages can have acidity as low as pH 4.40 Biomaterial surfaces are susceptible to high concentrations of these degradative agents and the biomaterial surface chemistry dictates its susceptibility to biodegradation.
Various calcium phosphates graft materials are commonly used for bone regeneration and repair applications,5,6,41 and are resorbed in vivo via passive dissolution and cellular processes.42,45 Cell mediated calcium phosphate resorption occurs due to the particle formation and fragmentation due to implant disintegration. The cells that take part in cell-mediated resorption are osteoclasts, which create an acidic environment on mineral surfaces by release of protons, macrophages and FBGCs.13 PMNs and monocytes/macrophages are among the first cells to colonize the surface of these calcium phosphate particles and play a crucial role in biodegradation.46 PMNs and macrophages that encounter these particles are activated to phagocytose these grafts (Fig. 3) dependent on particle size as discussed earlier. (Fig. 2)47
A: A scanning electron microscopic micrograph showing human neutrophils (red arrows) trying to phagocytose calcium phosphate (monetite) based graft material crystals (blue arrow). B: A scanning electron microscopic micrograph showing a phagocytosed monetite graft crystal being broken down intracellularly into three fragments (blue arrow) by a human neutrophil (red arrow).
The effect of graft material chemistry and physical features on the host immune response
The surface chemistry,48 form and topography29,49 of the graft surface determine the composition and severity of the foreign body reaction and immune response by affecting recruitment and activation of phagocytes and the adherence and activation of leukocytes.50 Surface geometry has also been researched and explored with regards to understanding its influence on macrophage behavior and host responses. Substrates with micro-pattern grooves and ridges have been used to study and better understand the behavior of macrophages and foreign body reactions towards micro-sized topography.49,51,52 Also, various studies have investigated macrophage and/or tissue responses toward nano-sized surface geometry.53-56 Experimental studies have also confirmed the role of particle shape in drug delivery via implantable graft materials.57 It is known that local geometry of the particle at the point of cell attachment, not the overall particle shape, dictates whether macrophages initiate internalization.57 It has been seen that an elliptical shaped disc (sufficiently small to be phagocytized at its pointed end), is fully internalized by neutrophils and macrophages in a few minutes, (Fig. 3)19 whereas macrophages attached to the flat region of an elliptical disc do not succeed for over 12 hours.57 The phagocytosis or internalization of cylindrical shaped particles depends strongly on their aspect ratio (ratio between the width and height). It has been seen that particles possessing an aspect ratio of three get phagocytosed about four times as fast as their spherical counterpart materials of the same volume.58 This suggests that the local geometry might be very important in this context, rather than the material’s chemistry alone.
Development of new biomaterials for biomedical devices and tissue-engineered constructs requires an in-depth understanding of the biological responses to implanted materials. It is clear that the chemistry, size, shape and surface texture profoundly impact the function of the implanted graft materials. Further work remains to map the dependence of immune response to biological properties and to categorize the relative effect and role of different physical and chemical factors towards foreign body reactions. For each application, comprehensive mechanisms of how physical properties affect biological performance and the interplay between various physico-chemical properties are required to be elucidated. Having a clearer understanding of the exact role of macrophages in tissue healing processes, especially in events that follow biomaterial implantation, will aid in the design of novel biomaterials and tissue-engineered constructs that elicit a favorable immune response upon implantation and perform suitably in their intended applications.
Oral Health welcomes this original article.
About the Authors
Dr. Zeeshan Sheikh is a dental clinician and a biomaterial scientist with degrees of BDS, MSc. PhD and two Post-Doctoral Fellowships. He is currently a Periodontics & Implant Surgery Resident in the year two of training at Dalhousie University.
Dr. Nader Hamdan is an Assistant Professor and Director of the Graduate Periodontics Program, Faculty of Dentistry at Dalhousie University.Dr. Hamdan treats patients in multiple private practices limited to Periodontics and Dental Implant Surgery in Canada.
Dr. Haider Al-Waeli has a BDS, M.Sc., and Jordanian board in Periodontics. He finished his PhD from McGill University and his interest is chronotherapy and bone healing after surgery. He is in the second year of Periodontics Residency at Dalhousie University.
Dr. Michael Glogauer is a clinical periodontist and is an expert in inflammation, immunology, and oral diseases. He is a Professor at the Faculty of Dentistry. He is the Head of Dentistry for University Health Network and Chief of Dental Oncology at Princess Margaret Cancer Centre.
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