Inflammation, Bone Healing, and Osteonecrosis: From Clinic to Laboratory

Author:Stuart B Goodman, Stanford University Orthopedics, USA

Translator:Duan Ruimeng Reviewed by:Chen Xiantao

Abstract:The osteonecrosis of the epiphyseal and metaphyseal regions of major load-bearing bones in the limbs is a condition associated with local death of osteocytes and bone marrow in the affected chamber. Chronic inflammation is a major feature of osteonecrosis, and if persistent inflammation is not resolved, it will lead to progressive collapse and subsequent degenerative arthritis. In the pre-collapse stage, the primary treatment goal for young osteonecrosis patients is to attempt to preserve the joint rather than perform joint replacement. In this regard, core decompression with or without local injection of bone marrow aspirate concentrate (BMAC) is a recognized evidence-based approach that helps to halt disease progression and improve outcomes in early osteonecrosis. However, some patients respond poorly to this treatment. Therefore, a prudent approach is to consider alleviating chronic inflammation while addressing the defects in osteogenesis and angiogenesis. Interestingly, inflammation, osteonecrosis, and the bone healing process are highly interrelated. Thus, regulating the biological processes and interactions between cells such as the innate immune system, mesenchymal stem cells-osteoblast lineage is crucial for providing a local microenvironment for the resolution of inflammation and subsequent repair. This review summarizes the clinical and biological principles associated with osteonecrosis and provides potential strategies for modulating chronic inflammation and promoting osteogenesis and angiogenesis through local interventions. Although these studies are still in the preclinical stage, we hope to develop safe, effective, and cost-efficient interventions to rescue the patient’s own joint.

Keywords:Chronic Inflammation, Osteonecrosis, Osteogenesis, Angiogenesis, Bone Healing, Inflammation

Inflammation: General Principles

Acute inflammation is the first step in the healing of all tissues and organs in response to physical (mechanical), chemical, infectious, thermal, and other types of injurious stimuli. This trauma leads to the activation of the innate immune system, followed by the release of cytokines, chemokines, reactive oxygen species, and other pro-inflammatory factors, triggering the complement and coagulation systems. These events are initiated by pattern recognition receptors (PRRs) recognizing specific chemical motifs from damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) at the site of injury. PAMPs are derived from infectious organisms, while DAMPs are molecular byproducts of dead or dying cells, also known as endogenous danger signals. The most important PRRs include Toll-like receptors (TLRs), C-type lectin receptors, NOD-like receptors, and RIG-I-like receptors. The acute inflammatory response of the innate immune system is a widespread response aimed at eradicating or removing harmful stimuli, initiating the clearance of cellular debris, and starting the breakdown and reconstruction of normal host tissue.Interestingly, the repair and renewal phase is assisted by a pro-inflammatory environment, in which mesenchymal stem cells (MSCs) and endothelial progenitor cells migrate to the injury site along chemokine gradients.Acute inflammation can lead to restoration of the native host tissue, fibrosis, or chronic inflammation.

Chronic inflammation is a sustained state of injury characterized by intermittent or persistent acute inflammation and ongoing fibrosis, despite these attempts ultimately failing to repair. Simply put, the innate immune processes (which can be understood as a more limited antigen-specific adaptive immune system, if applicable) cannot overcome harmful adverse stimuli to reconstruct normal anatomy and physiology. Consequently, although the body continuously mobilizes all biological resources, balance is never achieved. Chronic inflammation is also a state of increased energy demand, where organelles (such as mitochondria, endoplasmic reticulum, and other essential components of the cell) become depleted, ineffective, dysfunctional, and downregulated. If chronic inflammation persists, the resilience and survival of the organism will be at risk.

The cellular distribution of the innate immune system includes cells of the monocyte or macrophage lineage, particularly local DAMP and PAMP-sensitive macrophages, polymorphonuclear leukocytes (neutrophils), dendritic cells, mast cells, and specific lymphocyte subpopulations (including NK cells) and other cell types. Chronic inflammation involves the aforementioned cells as well as other T and B cell subpopulations. Fibroblasts and vascular lineage cells appear in both acute and chronic inflammatory states. During the resolution of inflammation, pro-inflammatory M1 macrophages polarize to anti-inflammatory, pro-reparative, pro-angiogenic M2 phenotypes, interacting with local mesenchymal cells and vascular progenitor cells.

Definition and Etiology of Osteonecrosis

Osteonecrosis encompasses a range of diseases that lead to the death of bone cells and bone marrow. Osteonecrosis can be localized or widespread (multifocal). Numerous different inciting factors are associated with osteonecrosis. Generally, ONFH is caused by traumatic events (such as femoral neck displacement fractures, hip dislocations, or closed reductions of hip dislocations), but it can also be non-traumatic, meaning not caused by mechanical injury. Traumatic etiologies are believed to directly damage the blood supply to the local area. Non-traumatic causes include high-dose corticosteroid use, excessive alcohol consumption, autoimmune diseases such as systemic lupus erythematosus (SLE), radiation therapy, chemotherapy, hypercoagulable states, sickle cell disease, Gaucher’s disease, and other causes. Osteonecrosis typically occurs in the epiphyseal and metaphyseal regions and can lead to bone collapse and secondary degenerative arthritis. Osteonecrosis must be distinguished from incomplete fractures caused by overuse, abnormal pathologic fractures, and other conditions. Osteonecrosis typically occurs in weight-bearing joints such as the hip (femoral head), knee (femoral and tibial condyles), and humerus (head), but can occur in virtually any bone and location. Most cases are associated with corticosteroid use or alcohol abuse, typically occurring in young patients of working age. Collapse of the affected joint leads to pain and end-stage arthritis. Therefore, early diagnosis of osteonecrosis is essential to assess and mitigate potential inciting factors to limit its progression to late stages. Furthermore, early diagnosis and treatment may prevent or reverse disease progression, thus preserving the patient’s own anatomy and avoiding joint replacement surgery. Unfortunately, a recent study on osteonecrosis at our tertiary care center showed that 77% of cases were diagnosed at late stages of ONFH, impacting the process of hip preservation.

Relationship Between Chronic Inflammation and Osteonecrosis

Despite the many etiologies associated with osteonecrosis, ultimately, they all involve insufficient oxygen and nutrient supply to the affected area (Figure 1). These events are associated with enhanced differentiation of bone marrow mesenchymal stem cells along adipogenic pathways and insufficient osteogenesis and angiogenesis. However, to some extent, the affected anatomical regions exhibit chronic inflammation, cell death, and histological evidence of impaired repair. Real-time imaging probe analyses show that activated macrophages and neutrophils persist locally six weeks after vascular cautery-induced osteonecrosis in mice. In other studies, hormone-related osteonecrosis in rats led to upregulation of the PRR toll-like receptor 4 (TLR4), with downstream adaptor proteins MyD88 and the major transcription factors of inflammatory proteins, nuclear factor-κB (NF-κB) and monocyte chemotactic protein-1 (MCP-1) also being upregulated.

Figure 1 Pathophysiological Features of Osteonecrosis. Many etiologies are associated with osteonecrosis. However, the ultimate common pathophysiological pathway involves insufficient oxygen and nutrient supply to the affected area. These events lead to enhanced differentiation of bone marrow mesenchymal stem cells along adipogenic pathways and insufficient osteogenesis and angiogenesis. Osteonecrosis also exhibits signs of chronic inflammation: persistent accumulation of activated neutrophils, macrophages, T cells, and other cell types; sustained activation of TLR4, MyD88, and NF-kB; and increased generation of pro-inflammatory mediators.

Notably, many molecules associated with acute and chronic inflammation, osteonecrosis, and bone healing overlap and play important roles in the activation of the innate immune system and tissue repair. NF-κB is the main pro-inflammatory transcription factor induced by injurious stimuli, activating pro-inflammatory factors or licensing MSCs. TLR4 is a PRR on the cell surface that can be activated by PAMPs, DAMPs, and other substances. TLR4 has two signaling pathways: MyD88-dependent (TLR4/MyD88/NF-κB) and MyD88-independent (TLR4/TRIF/IRF3). The MyD88-dependent pathway activates NF-κB and promotes the expression of the chemokine MCP-1. MCP-1 is a chemotactic agent for monocyte-macrophage lineage and MSC osteoblast lineage cells. MCP-1 induces proliferation of monocytes/macrophages and promotes differentiation and activation of osteoclasts. In pig models, byproducts of necrotic bone have been shown to upregulate a large number of pro-inflammatory cytokines, a mechanism dependent on macrophage activation of TLR4. This observation has been confirmed in hormone-related ONFH rat models, which exhibit excessive activation of TLR4/NF-κB and inhibition of the typical Wnt/β-catenin pathway (which regulates cell cycle, cell migration, and organogenesis). In one study, serum was collected from 20 patients at different stages of ONFH and compared with serum from a normal control group, identifying eight genes including TLR4 as potential serum biomarkers for disease severity. Other biomarkers include BIRC3, CBL, CCR5, LYN, PAK1, PTEN, and RAF1, which are related to inflammation, bone and cartilage metabolism, and angiogenesis.This suggests that potential biological strategies to alleviate complications of osteonecrosis may need to reduce chronic inflammation and promote osteogenesis and angiogenesis.

Strategies to Alleviate Chronic Inflammation and Promote Osteogenesis and Angiogenesis in ONFH

Healing of chronic critical-sized bone defects caused by trauma (delayed healing, nonunion), prior infections, periprosthetic bone resorption, and other causes is similar in many ways to the defects encountered in osteonecrosis. To some extent, these etiologies are associated with chronic inflammation, including local osteonecrosis, fibrosis, insufficient osteogenesis and angiogenesis, and tissue adipose infiltration. Therefore, studies of critical-sized bone defect healing models are relevant to the treatment of osteonecrotic lesions. Our laboratory and others have reviewed strategies and approaches to address these challenging clinical scenarios.

Inhibiting Chronic Inflammation

Given the association of osteonecrosis with chronic inflammation, it seems prudent to consider intervening in these processes. Potential therapeutic approaches must be time- and space-sensitive, as healing of soft and hard tissues after acute injury relies on acute inflammation for a short period (usually days), followed by the licensing of mesenchymal stem cells and other cells to initiate repair.

In light of these facts, the following are potential methods to alleviate chronic inflammation (Figure 2):

(a) Interfering with or obstructing receptor engagement and sustained activation that prolong the inflammatory process;

(b) Inhibiting intracellular pro-inflammatory pathways;

(d) Interfering with the response of end organs to specific inflammatory mediators;

(e) Promoting competing biological processes by providing signals and cues to alter the local cellular microenvironment;

(f) Completely eliminating cells associated with chronic inflammation.

Inflammation, Bone Healing, and Osteonecrosis: From Clinic to Laboratory

Figure 2 Potential Therapeutic Approaches to Address Chronic Inflammation. Acute inflammation is essential for tissue healing after injury. However, chronic inflammation is detrimental and leads to loss of tissue integrity and function. This article outlines potential pathways to alleviate chronic inflammation.

Many of these strategies have been employed in the treatment of systemic chronic inflammatory diseases such as rheumatoid arthritis (RA). Pharmacological agents for RA include antimetabolites and other chemotherapeutic agents, disease-modifying drugs, and biologics that directly or indirectly interfere with specific cytokines, chemokines, and other pro-inflammatory molecules like tumor necrosis factor α (TNFα), interleukins (IL) such as IL-1β and IL-6. While these drugs are very effective for treating rheumatoid arthritis, it is impractical to administer these potentially serious side-effect-inducing drugs systemically for chronic inflammation due to osteonecrosis and significant bone defects. Therefore, local administration may be the preferred route. Regarding alleviating chronic inflammation associated with osteonecrosis and critical-sized bone defects, the following local methods show promise: inhibiting specific TLRs, especially TLR4; interfering with the following proteins: adaptor protein MyD88, transcription factor NF-кB, or chemokine MCP-1 and macrophage inhibitory factor (MIF); and polarizing macrophage states from M1 pro-inflammatory to M2 anti-inflammatory phenotypes through local delivery of IL-4 or IL-13. Our laboratory and others have used these strategies in models simulating chronic inflammation associated with wear particle disease.Infusion of IL-4 (an anti-inflammatory cytokine) is an important proposed strategy to suppress chronic inflammation in various clinical conditions. IL-4 protein can be delivered directly, via scaffolds or other devices, or through genetically modified mesenchymal stem cells overexpressing IL-4, which can also be upregulated via NF-кB. This approach is a significant direction in our laboratory for treating chronic bone defects and osteonecrosis. Other potential immunotherapeutic approaches include delivery of IL-13, IL-10, IL-1Ra, TNF receptor sR, etc.

Local Delivery of Biomolecules and/or Cells to Promote Osteogenesis and Angiogenesis (Figure 3)

Inflammation, Bone Healing, and Osteonecrosis: From Clinic to Laboratory

Figure 3 Potential Approaches for Local Delivery of Biomolecules, Cell Therapy, and Gene Therapy to Enhance Osteogenesis and Angiogenesis in Osteonecrosis. Abbreviations: BMAC, Bone Marrow Aspirate Concentrate; MSCs, Bone Marrow Mesenchymal Stem Cells; GAMs, Gene Activation Matrices.

Biomolecules and Drugs

Local injection of growth factors and other molecules to enhance osteogenesis and angiogenesis, or to inhibit osteoclasts for the treatment of bone defects and osteonecrosis is not a new concept. These factors include members of the transforming growth factor (TGF) superfamily, including TGFβ and bone morphogenetic proteins (BMPs), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), hepatocyte growth factor, parathyroid hormone (PTH), etc. These reagents are delivered in the form of proteins embedded in various polymers, scaffolds, or matrices. These biomolecules serve as drug delivery systems that are absorbed, embedded, immobilized, or coated, and then released through diffusion, matrix degradation, or crosslinking. Other drugs for local delivery include corticosteroids and other steroids, statins, and bisphosphonates. These biomolecules are often multifunctional, regulating multiple pathways including inflammatory cascades, osteogenesis, and angiogenesis, and have other biological targets. While some of these interventions have been used in extensive preclinical and limited clinical studies for the treatment of bone defects, they are rarely used for the clinical treatment of osteonecrosis. In fact, clinical trials have not widely accepted and implemented systemic treatment with bisphosphonates or statins as effective for ONFH, but local treatments may be effective.The challenges of the harsh biological environment of necrotic, avascular bone lesions may be too high for successful drug therapies.

Cell Therapy

Cell therapy for ONFH and other osteonecrosis is no longer experimental. In the early stages of osteonecrosis, the use of concentrated autologous iliac bone marrow aspirate concentrate combined with core decompression (CD) is evidence-based. Hernigou et al. (2018) reported on 125 patients undergoing simultaneous bilateral CD. In one of the hips treated with CD, they added bone marrow aspirate concentrate (BMAC). The number of MSCs (or colony-forming units-fibroblasts [CFU-Fs]) injected into the CD site ranged from 45,000 to 180,000 cells, with an average of 90,000±25,000 cells. After 20 to 30 years of follow-up, it was found that adding BMAC reduced the rate of femoral head collapse from 72% to 28%; the proportion of patients undergoing hip replacement surgery decreased from 76% to 24%. Using quantitative MRI analysis, the volume of the femoral head occupied by osteonecrotic lesions decreased from 44.8% to 12% in the group receiving BMAC. These are compelling data. The concept of adding BMAC to CD treatment for ONFH will benefit from a large prospective randomized multicenter study.

BMAC has undergone extensive preclinical and laboratory analysis, as reviewed in recent publications. Numerous factors influence the quantity and viability of harvested cells, including the patient’s age and sex, the presence of medical comorbidities and pharmacological treatments such as corticosteroids and other medications, smoking, obesity, etc. Notably, BMAC is not mesenchymal stem cells but an aggregate of different types of mononuclear cells, including macrophages, lymphocytes, mast cells, and other cells. In fact, only about 1 CFU-F exists among 30,000 nucleated cells harvested from the iliac crest, which equates to approximately 600 CFU-F per cc of bone marrow aspirate.

Despite the notable success of BMAC in treating osteonecrosis, several questions remain. Is BMAC the preferred cell for injection therapy for osteonecrosis? How many different lineages of cells are needed? Is MSC alone sufficient? Can MSCs be modified to alleviate inflammation and promote healing of osteonecrotic lesions? Are allogeneic bone marrow-derived mesenchymal stem cells equally effective? While these questions currently lack definitive answers, several relevant observations deserve mention. Substantial evidence suggests that the addition of macrophages enhances the osteogenic capacity of bone marrow-derived mesenchymal stem cells, potentially through licensing the latter cells and engaging in ongoing crosstalk between bone marrow-derived mesenchymal stem cells and macrophages. These findings confirm the aggregation of injected bone marrow-derived mesenchymal stem cells and hematopoietic lineage cells. In addition, isolating, expanding, and delivering autologous and allogeneic MSCs (or their byproducts, such as exosomes) for the repair of bone defects and treatment of osteonecrosis is also being explored. A recent article by the authors summarizes the phenotypic changes of bone marrow-derived mesenchymal stem cells to promote bone healing. Some of these techniques include bioreactor preconditioning, MSC exposure to hypoxic environments, and gene therapy or genetic manipulation of the cells. Other methods include optimizing the isolation, expansion, and storage techniques of MSCs, improving the physical, chemical, and other properties of the carriers or scaffolds used, and addressing defects in the host where the cells are implanted.

Gene Therapy

Cell gene therapy and genetic manipulation for the repair of musculoskeletal tissues is an exciting concept that has been reviewed elsewhere. Gene therapy can be accomplished using chemical and physical methods, without using viruses to transport DNA or particles into the cells. By utilizing gene activation matrices (GAMs) or other platforms, genetic material can be released to surrounding cells based on predetermined time and spatial parameters. Gene transfer can be achieved by using viral vectors to genetically modify autologous or allogeneic cells in vitro, followed by injection of these cells in vivo, or directly transferring genes into cells in vivo.Gene therapy has also been applied to treat osteonecrosis, primarily in preclinical studies. We have injected BMAC, bone marrow mesenchymal stem cells, preconditioned bone marrow mesenchymal stem cells, and gene-modified bone marrow mesenchymal stem cells overexpressing IL-4 into the core decompression bone canal, with or without the use of novel 3D printed customized functionally graded scaffolds as treatment for rabbit ONFH. In preclinical studies, the addition of IL-4 overexpressing MSCs during the acute phase of osteonecrosis may hinder bone regeneration by suppressing the acute inflammatory response.

Summary

Utilizing biomolecules, drugs, cells, and gene therapies to treat osteonecrosis is very exciting. However, apart from BMAC treatment, these therapies are generally in the preclinical stage and must weigh multiple potential risks, including adverse effects on adjacent cells, as well as issues of immunogenicity, mutagenicity, and carcinogenicity. Furthermore, considerations of timing, dosage, and optimal platforms for administration, as well as cost-effectiveness must be addressed.

Discussion

Chronic inflammation often leads to normal host tissue being replaced by detrimental fibrotic vascular stroma, filled with acute and chronic inflammatory cells. This replacement tissue lacks the anatomical, physiological, metabolic, and functional integrity of the host tissue. In clinical cases, much of the organ is affected by chronic inflammation, potentially compromising vital life processes, such as chronic hepatitis, nephritis, diabetes, cardiopulmonary diseases, rheumatoid arthritis, aging, and other diseases. The associated morbidity and mortality rates are high.In this regard, bones and joints are no different. Chronic inflammation is common in inflammatory arthritis, chronic osteomyelitis, nonunion fractures, and osteonecrosis. This is characterized by a sustained hyperactivity of the innate immune system, and in some cases, hyperactivity of the adaptive immune system. In osteonecrosis, despite various related susceptibility factors, chronic inflammation driven by DAMPs impedes neovascularization and osteogenesis. If unchecked, this condition will progress to joint collapse and end-stage arthritis. The situation is even more severe in cases of multifocal osteonecrosis.

Optimal treatment for early osteonecrosis includes strategies to alleviate chronic inflammation, as well as promoting osteogenesis and angiogenesis before joint collapse. In these cases, preserving the joint is a better option than joint replacement, especially due to the young age of the patients. However, the exact treatment for these complex cases often involves persistent susceptibility factors (e.g., ongoing high-dose corticosteroid treatment for SLE), which can sometimes limit the physician’s options.

Systemic pharmacological methods seem ineffective in treating and preventing adult osteonecrosis. Early diagnosis is crucial so that treatment regimens can be reviewed and implemented. This indicates the need to identify and screen high-risk patients, at least through a comprehensive medical history and possibly selective non-invasive imaging such as MRI.

In the pre-collapse stage, local treatment with core decompression, possibly adding biological adjuncts like BMAC, seems reasonable. Research should address the components and dosages of BMAC to optimize reconstruction of osteonecrotic defects. Specific biological approaches may focus on accompanying chronic inflammation and its detrimental effects on osteogenesis and angiogenesis. Customized designs of cells and biological agents extracted from the patient’s own tissues, although currently not FDA-approved due to the involved procedures exceeding minimal limits, may further improve the goals of regenerative medicine for osteonecrosis. Customized mechanical implants may delay the physical collapse of bone and cartilage and provide important signaling cues for tissue regeneration. Such 3D printed and biodegradable implants are currently undergoing preclinical testing in our laboratory. It is hoped that some of these technologies will prove safe, effective, and economical. In this way, the pain, disability, and morbidity of millions of osteonecrosis patients worldwide may be alleviated.

This article represents the author’s personal views and does not reflect the official position of Bone Today. We hope everyone can make rational judgments and apply accordingly.

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