Tissue repair and regeneration are partially determined by genetic factors.
Successful regeneration requires a balanced immune cell response.
The increased understanding of molecular signals governing native joint-resident stem cell function could lead to pharmacologic interventions that trigger intrinsic repair mechanisms to treat or prevent progression of damage.
Cell-based therapeutics and their combination products are complex regarding their mechanism of action and manufacturing and have evolved into advanced therapy medicinal products with a specific regulatory path.
Tissue engineering has adopted the concept of biomimetics of in vivo tissue development. Developmental engineering is the term used to describe a novel methodology for the rational and accurate design of robust, well-controlled manufacturing processes of “biologic spare parts.”
Recent advances in regenerative medicine and tissue engineering relevant to rheumatology have entered clinical practice and include the biologic repair of joint surface defects and bone healing.
Introduction
Tissue destruction and joint failure are ultimately the disabling outcomes of most forms of inflammatory or degenerative arthritides. The need for repair and regeneration of joints and joint-associated tissues is becoming more relevant as the dramatic advances of targeted treatments and improved disease management have allowed much more efficient control of inflammation and joint destruction.
In view of this, other aspects of joint biology require more attention—notably, and most importantly, the mechanisms driving tissue response and repair. Indeed, to restore the balance between tissue destruction and tissue repair ( Fig. 7.1 ), we should be looking at the more complete picture, that is, the “systems biology” of the joint as an organ. Targeting tissue repair has entered our discipline, and investigating the potential to activate and enhance joint tissue repair mechanisms has become a prime goal. Introducing regenerative medicine approaches provides a significant opportunity to restore joint homeostasis and thus possibly provide a cure.
Regenerative medicine and tissue engineering (TE) seek to repair or regenerate damaged tissues and organs, regardless of the cause of the damage, ideally without scar tissue formation, thereby restoring both the structure and function of the damaged tissues/organs. Nature demonstrates that this goal is achievable, as successful wound healing and fracture repair are processes that happen routinely after birth. We also know, as demonstrated in fetal surgery, that scarless repair is partially dependent on age and context. Thus, it is attractive to envision that, with an in-depth understanding of the repair processes at the cellular and molecular level, we may be able to intervene quickly at the time of injury and guide the healing process more appropriately, thereby preventing scar formation. In view of this, there is increasing evidence that the role of the immune system is of importance in postnatal tissue regeneration, and that the further understanding of the crosstalk between the immune cells and the stem cells may be crucial for clinical success.
Postnatal tissue healing mimics developmental processes of tissue formation. For example, it appears that the process of rebuilding an adult limb, such as seen in the axolotl, but also during fracture healing in higher species, has many similarities with how the limb forms in the embryo. Thus both limb formation and limb regeneration appear to use the same molecular pathways. The remarkable advances in developmental biology during the past several decades have now provided the knowledge platform to advance into novel regenerative approaches in postnatal life. These advances not only include our understanding of the mechanisms of body axis formation and organogenesis but also impressive progress in stem cell biology, including the regulation of stemness, stem cell niches, lineage specification and cell differentiation, and the molecular pathways involved. Consequently, we have entered a new era in regenerative medicine and TE. In this chapter we will review the approaches that seek to repair damaged and diseased synovial joints and skeletal structures.
When we aim to repair tissues, two mechanistic approaches are possible. The first approach consists of enhancing intrinsic repair mechanisms with stimulation of local cell proliferation, differentiation and tissue metabolic activity, and recruitment of endogenous progenitor populations into the damaged tissue. The second approach becomes necessary when intrinsic repair is insufficient and entails extrinsic repair, that is, TE approaches via manufacturing of cellular and/or combination products that can contribute mostly locally to tissue repair ( Fig. 7.2 ).
Intrinsic Repair
In this section we will discuss the mechanism by which, in favorable conditions, joint tissues can repair after injury and how such mechanisms can be pharmacologically harnessed to prime and support repair in conditions in which spontaneous repair fails.
Genetic Basis of Tissue Repair
For many years it has been commonly accepted that “cartilage…when destroyed, is never recovered.” This statement probably still holds true for established defects (e.g., defects that have failed to heal and have become chronically symptomatic, and therefore come to the attention of the clinician); however, prospective imaging and arthroscopy studies have revealed that even in adult humans, acute cartilage defects are much more prevalent than suspected, even in asymptomatic subjects, and have some capacity for spontaneous healing.
Such studies showed that roughly half of adults without joint symptoms have chondral defects, and that about a third of such lesions improve spontaneously, a third remain stable, and the remainder worsen.
In addition to comorbidities and environmental factors influencing the natural history of chondral defects (age, co-existing osteoarthritis [OA], high body mass, female sex, abnormal bone geometry, bone marrow lesions), a prospective, magnetic resonance imaging (MRI)-based study in a sib-paired cohort identified a very high (>80%) heritability of the rate of progression of chondral defects.
Nonetheless, in contrast with the remarkable progress in the identification of genetic markers predisposing to OA, no quantitative trait linkage and association analysis study is available yet in humans with regard to the intrinsic capacity of repairing articular cartilage defects.
Although the evidence for the heritability of cartilage repair capacity in humans is still circumstantial, findings of animal studies are more convincing. Comparing the capacity of different inbred mouse strains to heal full-thickness cartilage defects, one group demonstrated that different inbred mouse strains have a dramatically different capacity to heal cartilage defects and to avoid the onset of post-traumatic OA. This finding supports the concept that repair capacity has a genetic component. The development of such an animal model lends itself to genetic analysis. For instance, another group took advantage of the different healing capacity of two strains of mice: LG/J, which can efficiently heal joint surface defects and experimental wounds to the cartilage of the pinna of the ear, and SM/J, which are poor healers. A set of recombinant inbred lines were obtained by crossing the two strains. It was found that both the capacity of healing joint surface defects and the capacity to heal ear wounds were highly heritable and that they correlated with each other. This finding suggests that the capacity to heal cartilage is inheritable by itself, whether the cartilage is on the surface of a diarthrodial joint or in the pinna of the ear and is not just dependent on the resistance to develop OA or on factors extrinsic to cartilage such as bone shape/quality or synovitis. For example, the fact that homeostatic mechanisms affect the rate of OA progression suggests that the same genes associated with the predisposition to OA might affect also the capacity to repair; however, this assumption has never been rigorously tested experimentally. It must be remembered that other independent aspects come into play in the determination of OA progression, including joint shape, body mass index, inflammation, and several other aspects that are also under genetic control. Therefore, allelic variants that affect such parameters will also affect OA predisposition, independently from repair mechanisms intrinsic to the joint. Nevertheless, it is interesting that signaling molecules belonging to the same pathways have been associated with both OA and cartilage regeneration. For instance, loss of function alleles of the wingless type (Wnt) inhibitor FRZB are associated with OA predisposition, and a single nucleotide polymorphism of the Wnt ligand WNT-3A has been associated with the heritability of cartilage repair capacity in mice. Most of the genes identified in these screenings affect directly the behavior of joint resident stem cells or their interaction with joint tissues.
Signaling Pathways Orchestrating Joint Homeostasis and Surface Repair
Joint surface repair involves a number of events that are highly coordinated in space and time: when and for how long stem cells should proliferate; the path of their migration; the timing and extent of extra-cellular matrix production; and where and when, within the repair tissue, a stem cell should become an osteoblast or a chondrocyte are all processes regulated by soluble secreted molecular signals. Given the similarity between repair and embryonic joint morphogenesis, it is unsurprising that the major signaling molecules controlling these two processes are very similar: the transforming growth factor (TGF)-β superfamily, including the bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs), the Wnt family of proteins, the fibroblast growth factors (FGFs), the hedgehog proteins, parathyroid hormone (PTH)/PTH-related protein (PTHrP), and hedgehog and Notch signaling. Next, we select a few examples of how these signaling pathways are involved in joint formation and their role in postnatal joint repair, with a specific focus on those that have led to advances in early clinical development in arthritic diseases.
PTHrP Signaling
PTHrP signaling during embryonic development of the skeletal elements contributes to the positional information and regulates the rate of differentiation of the chondrocytes through the epiphysis, preventing precocious or ectopic hypertrophic differentiation and endochondral bone formation. In adulthood, the PTH/PTHrP receptor PPR is barely expressed in normal articular cartilage but is strongly upregulated in the intermediate layers of repair cartilage, just above the bottom layer of hypertrophic chondrocytes and in osteoarthritic cartilage. Because hypertrophic differentiation of articular chondrocytes is not only a feature of OA but also drives cartilage breakdown and PTH/PTHrP signaling is known to delay hypertrophy, investigators tested whether a truncated form of human recombinant PTH currently used in the clinic for osteoporosis could improve the outcome of OA in mice. Indeed, human recombinant PTH could not only stop cartilage breakdown but also induced a regenerative effect. Unfortunately, the doses required to achieve this impressive effect in mice were very high and induced osteopetrosis. A restriction of the signaling domains to the tissue of interest is therefore required before this approach can be used in the clinic. Another caveat is that this study cannot discriminate between the effects on cartilage and those on the bone. In this context, it is noteworthy that strontium ranelate, another compound used as an anabolic agent in osteoporosis, was also able to reduce the rate of joint space narrowing and to improve symptoms in patients with OA. Similar results were replicated in a rat and a dog model of instability-induced OA.
Transforming Growth Factor-β/Bone Morphogenetic Protein Signaling
Recent data highlighted the relevance and critical role of members of the TGF-β superfamily (TGF-β, BMPs, and GDFs) in the biology of articular cartilage, bone, joints and joint-associated tissues, both during development and in postnatal tissue homeostasis, repair, and tissue response to injury and aging. TGF-β is involved in the maintenance and aging of articular cartilage and OA. Despite the well-documented anabolic actions of TGF-β, excessive or sustained activation of this signaling pathway is detrimental to cartilage. In fact, overexpression of TGF-β resulted in spontaneous osteoarthritis in mice and TGF-β or TGF-β receptor blockade protected cartilage integrity in models of osteoarthritis.
BMPs have been reported to play major roles in articular cartilage metabolism, with BMP-7/osteogenic protein-1 (OP-1) being of particular interest. In addition, modulation of TGF-β/BMP downstream receptor-Smad signaling plays an essential role in both the regulation of chondrocyte differentiation and the development and progression of OA. Therefore, given overwhelming evidence of the regenerative potential of this family of growth and differentiation factors in pre-clinical models, it is not surprising that their therapeutic use is being explored in clinical studies and in indications such as long bone healing and OA. In addition, intense research is ongoing to identify modulators of the TGF-β/BMP receptor/Smad signaling pathways through peptide technology (peptidomimetics) or small molecule screens. Because synovial joints allow for local treatment, it is expected that some newly identified compounds will first be tested as local applications, such as joint surface repair and monoarticular OA. BMP devices are already in the clinic for orthopedic applications such as spine fusion and the healing of nonunions.
Fibroblast Growth Factor Signaling
Extensive investigations have identified fibroblast growth factor receptor (FGFR)3 signaling as a key regulator of chondrocyte and osteoblast function, both during development and postnatally. In particular, absence of signaling through FGFR3 in the joints of Fgfr3 −/− mice leads to premature cartilage degeneration and early signs of OA. One of the key ligands of FGFR3 signaling appears to be FGF18. In the postnatal joint, FGF18 has significant anabolic effects on cartilage metabolism. Intra-articular injection of FGF18 induced a dose-dependent increase in cartilage formation and a reduction in cartilage degeneration scores in the medial tibial plateau of rats with OA. Importantly, this effect was seen only in joints affected by OA, not in normal rat joints, suggesting a specific response to tissue injury. At the molecular level, this joint-protective effect may be due partially to its interaction with other signaling pathways such as BMP signaling by repressing noggin, a BMP antagonist.
Wnt Signaling
Overwhelming evidence indicates a critical role for Wnt signaling in cartilage and bone biology, with a specific relevance to osteoporosis and OA (for a review, see ref. ). Although Wnt signaling is essential for the development and homeostasis of synovial joints, genetic studies in humans and experimental data demonstrate that excessive/uncontrolled activation of Wnt/β catenin signaling leads to reprogramming of articular chondrocytes toward catabolism or loss of their stable phenotype with subsequent loss of articular cartilage tissue structure and function. In particular, loss of function allelic variants of the Wnt inhibitor FRZB were associated with increased predisposition to develop OA. Similarly, disruption of the FRZB gene in mice resulted in increased activity of the WNT signaling pathway and consequent increased bone stiffness and enhanced cartilage damage. Importantly, upon induction of experimental OA, Frzb −/− mice showed greater cartilage loss than their wild-type counterparts. Increased cartilage damage in Frzb −/− mice was associated with higher levels of β-catenin dependent Wnt signaling and with higher expression levels of matrix metalloproteinase (MMP)-3. Moreover, FRZB can directly inhibit MMP-3, probably through the netrin domain, indicating the potential complexity of the underlying mechanisms of observed phenomena.
Tightly regulated Wnt signaling is critical for the homeostasis of synovial joints. One study demonstrated that in human and experimental murine arthritis, the inflammatory cytokine tumor necrosis factor drives the excessive production of the Wnt antagonist DKK1. The consequent excessive inhibition of the Wnt pathway was found to be responsible for bone resorption (erosions), typical of this arthritis model, as well as of rheumatoid arthritis (RA). Accordingly, blocking DKK1, and therefore de-repressing Wnt signaling, resulted in reversal of bone damage. In the same model, direct activation of WNT signaling using the Wnt agonist R-Spondin 1 reversed not only the bone damage (also achieved with DKK1 blockade), but also the cartilage damage. Therefore, both uncontrolled suppression and activation of the WNT signaling may lead to catabolism and cartilage loss. It appears that a tight control, both in terms of timing and magnitude, is essential not only to preserve homeostasis but even to induce tissue regeneration. For instance, controlled and temporary activation of β catenin signaling in adult mice led to an initial reduction of extra-cellular matrix (ECM), followed by chondrocyte proliferation and thickening of the articular cartilage. Even more strikingly, although more remote from the context of joint disease, one study demonstrated that although WNT signaling is essential for the capacity of some species (i.e., axolotl or Xenopus tadpoles) to regenerate entire limbs, it was sufficient to slightly modify the spatiotemporal distribution of WNT-β catenin activation to afford the chick embryo with the capacity to regenerate an entire limb.
In the clinical context of joint disease and regeneration, the complexity of Wnt signaling and its potential downstream effects may offer the opportunity to target catabolic effects while preserving the homeostatic ones. One group demonstrated that WNT-3A drives chondrocyte proliferation through activation of the β catenin pathway and loss of differentiation through activation of CaMKII. Therefore, targeting CaMKII specifically allowed enhancement of differentiation without affecting proliferation.
Another opportunity concerns molecules that “buffer” Wnt signaling at homeostatic levels. For instance, WNT16 is a weak Wnt activator, which, however, prevents overactivation when more potent canonical Wnt molecules are present. Genetic disruption of the Wnt16 gene resulted in higher susceptibility to experimental osteoarthritis in mice and to a depletion of articular cartilage-residing progenitor cells.
Better understanding of the downstream signaling mechanism and its regulation will be essential to target only the pathogenic effect of these pleiotropic signaling pathways in cartilage, bone, and the osteochondral junction.
Growth Hormone/Insulin-like Growth Factor Axis
Other targets are activated by signaling molecules that play more prominent roles in postnatal skeletal growth. These proteins/pathways can be regarded as potential “anabolic” agents and could contribute to the restoration of joint homeostasis. In this regard, the growth hormone (GH)/insulin-like growth factor (IGF) axis is of interest. IGF-1 has been reported to be critical in the maintenance of the homeostasis of articular cartilage explants ex vivo. Further evidence of its anabolic effect in in vivo models has led to the early clinical development of intra-articular treatments with IGF-1 in knee OA, although reports of clinical trials have not been published. Furthermore, it was reported that systemic administration of GH in horses may be beneficial to joint/articular cartilage biology because it increases IGF-1 levels in synovial fluid. Improved formulations of GH have been explored to improve the duration and effect size in synovial joints. Some evidence exists of a relationship between levels of IGF-1 and OA, further suggesting a potential benefit of targeting the GH/IGF-1 axis, particularly in OA. However, data thus far are inconclusive, and further systematic analysis of the hypothalamic-pituitary axis, including GH, IGF-1, and somatostatin, is required.
As for all growth factor technologies, further studies are needed to address critical issues such as the relevant genetic background of the patients, stage of the disease process, and tissue specificity to restrict an effect to the targeted tissues, thereby avoiding systemic adverse effects.
Joint Resident Stem Cells
Stem cells persist in adult life to mediate tissue maintenance and regeneration. They have the capacity to self-renew (that is, to divide and produce more stem cells), thereby preserving a constant pool of stem cells, and to differentiate to replace the mature cells that are lost to physiologic turnover, injury, disease, or aging. Self-renewal and differentiation are regulated by stem cell intrinsic factors and signals from the surrounding microenvironment in which the stem cells reside, called the stem cell niche .
Stem cell niches have been described for a number of tissue types such as the hair follicle, intestine, and bone marrow. In bone marrow the niches of hematopoietic stem cells (HSCs) have received considerable attention, and the understanding of HSCs has led to improvements of bone marrow transplantation outcomes in hematology. The HSC niches include the endosteal niche, where HSCs are in close contact with osteoblasts residing at the bone surface of the trabeculae, and the perivascular niche, where the HSCs are close to the sinusoids in the bone marrow.
Mesenchymal stromal/stem cells (MSCs), which are derived from bone marrow and other connective tissues, including the synovium, have the ability to differentiate into chondrogenic and osteogenic lineage cells. Therefore, MSCs are of interest for their potential clinical use for joint tissue repair.
Evidence indicates that pericytes may be the native cells of the ex vivo MSCs. Pericytes are located on the abluminal side of small blood vessels and are in close connection with the endothelial cells of the vessels. The proximity to vessels would allow MSCs to enter the bloodstream and migrate to sites of injury, but it is not clear to what degree this happens and, importantly, if this phenomenon has clinical relevance. However, the notion that cells with MSC characteristics are derived from pericytes is challenged by the retrieval of MSC-like cells in articular cartilage, notoriously an avascular tissue. Thus, pericytes are not the only source of MSCs, and such a relationship is likely to be tissue specific and context dependent.
The lack of an exclusive MSC marker in the joint has impeded studies of joint-resident MSCs. One group adopted a double-nucleoside-analog labeling method in a mouse model of joint surface injury to identify functional stem cells (i.e., long-term label-retaining cells that after injury undergo proliferation and differentiation). This was based on the administration of two nucleoside analogs; iododeoxyuridine (IdU) was given before the injury followed by a washout period to identify label-retaining (slow-cycling) cells, and chlorodeoxyuridine (CIdU) was given after the injury to label cells proliferating in response to the injury. In uninjured knees, IdU-label-retaining cells were detected in the synovium and were largely negative for CldU. Phenotypic analyses showed that these cells were nonhematopoietic and nonendothelial cells that expressed known MSC markers. After articular cartilage injury, there was marked accumulation of IdU and CIdU double-positive cells, indicating that label-retaining cells proliferated to generate a pool of transit-amplifying cells. Furthermore, they formed ectopic cartilage, indicating that these cells can function as chondroprogenitors in their native environment.
The label-retaining MSCs in the mouse synovium as identified by one group were located in two niches, the lining niche and the sublining perivascular niche, the latter distinct from pericytes. In these two niches, MSCs could have distinct functions, but a hierarchy remains to be demonstrated ( Fig. 7.3 ).
In recent years, lineage-tracing studies in mice have provided insights into the MSC populations that reside in the bone marrow, including the so-called skeletal stem cells (SSCs). In mouse bone marrow, perivascular MSCs are marked by Pdgfrα and Sca1, Nestin (Nes)-GFP, and Leptin-receptor (LepR), with partial overlap between these cell populations. They support hematopoiesis and contribute to osteogenic and adipogenic lineages. In addition, one study identified a population of osteochondroreticular stem cells expressing Gremlin (Grem)1, distinct from Nes-GFP+ cells, which contribute to early postnatal growing bones and fracture repair in adult mice. Another study proposed a model in mice whereby skeletogenesis would proceed through a hierarchy of lineage-restricted progenitors, and one primitive skeletal stem cell gives rise to all the cartilage, bone, and stromal lineages. The same team reported the identification in the human growth plate of a population of nonhematopoietic, nonendothelial cells enriched in SSCs able to self-renew and generate progenitors of bone, cartilage, and stroma, but not adipose tissue.
Recently, by combining lineage tracing studies with single-cell RNA-sequencing analyses, investigators identified a population of SSCs in mouse periosteum that is present in the long bones and calvarium, is distinct from other known SSCs, displays self-renewal ability, and forms bone via direct intramembranous ossification. These cells acquire the capacity to undergo endochondral bone formation during fracture healing after injury. A cell analogous to the mouse periosteal SSC appears to be present in the human periosteum.
These studies provide evidence that growing bones, bone and bone marrow, and periosteum contain multiple pools of SSCs/MSCs, each with distinct functions.
Similarly, lineage tracing studies have provided important insights on the joint resident stem/progenitor cells and their involvement in joint development, maintenance, and repair. Synovial joint tissues, including articular cartilage and synovium, develop from progeny of cells of the embryonic joint interzone, a stripe of mesenchymal tissue that appears in the limb bud during development and is marked by the expression of growth and differentiation factor 5 (Gdf5). The Gdf5-Cre mice developed by one group contain a modified BAC that encompasses the GDF5 gene inactivated by a knockin of Cre in the first exon, together with upstream regulatory elements driving Cre expression that are active in the knee joint interzone during development but not in adult knees. These mice were used to show that Gdf5-lineage cells persisted in adult synovium as a subpopulation of the Pdgfrα-expressing MSCs, present predominantly in the synovial lining with a minor population in the sublining tissue. They barely overlapped with SSC/MSC populations originally identified in bone marrow and also detected in the mouse synovium, marked by expression of Nestin ( Fig. 7.3 ), Leptin-receptor or Gremlin-1, suggesting that they constitute a distinct lineage extending from embryonic development into adult life.
After cartilage injury, Gdf5-lineage cells proliferated to underpin synovial lining hyperplasia, colonized the cartilage defect, and underwent chondrogenic differentiation in the repair tissue. Conditional ablation of the transcriptional cofactor Yes-associated protein (YAP) in the Gdf5 lineage prevented the synovial lining hyperplasia and markedly reduced the contribution of Gdf5-lineage cells to cartilage repair. After isolation and culture expansion, Gdf5-lineage cells retained ability to form cartilage and a synovial-lining-like layer. By comparison, other MSCs isolated from mouse knee joints (i.e., non-Gdf5-lineage) performed poorly in the chondrogenesis and synoviogenesis assays but showed osteogenic ability while the Gdf5-lineage MSCs were not osteogenic. These findings demonstrated a functional heterogeneity of joint-resident MSCs, with Gdf5-lineage MSCs showing joint-relevant progenitor activity.
The Gdf5 lineage in adult life also includes articular cartilage cells, and there is evidence that progenitor cells are present in the cartilage. Archer reported the isolation of an MSC-like population from the superficial zone of the articular cartilage. MSC properties in culture-expanded cells from articular cartilage were also described. More recently, progenitor cells have been described in the articular cartilage in vivo. One group generated mice carrying the tamoxifen-inducible CreERT2 knocked into the endogenous Prg4 locus (encoding proteoglycan 4 [Prg4], also known as lubricin ), which is expressed by the superficial cells of the articular cartilage and cells in the lining layer of the synovium. Genetic lineage tracing showed that the progeny of Prg4+ cells differentiated into articular chondrocytes. Prg4+ cells in the superficial layer of the cartilage divided slowly and express progenitor/stem cell markers. Similar findings were observed using a different, BAC-based Prg4-CreERT2 model. These studies demonstrated that postnatally the surface of the articular cartilage contains stem/progenitor cells.
In keeping with the study by one group, another group showed that after cartilage injury, the Prg4 lineage expanded in the synovial lining and contributed to the cartilage repair tissue. Because both Gdf5-lineage cells and Prg4-lineage cells are present in the synovium and in the superficial layer of articular cartilage, the tissue of origin of the chondroprogenitors that repair articular cartilage remains to be clarified. The lack of detectable proliferation of Gdf5-lineage and Prg4-lineage cells in the cartilage and their expansion in the synovium, contributing to synovial lining hyperplasia after injury, suggest the prospect that the chondroprogenitors that repair cartilage originate from the synovium. The mechanisms are unclear and could include direct synovial attachment and migration of synovial cells along the cartilage surface or derived from the synovial fluid. Indeed, studies have reported a raised number of MSCs in the synovial fluid and bone marrow lesions of patients with OA. An obvious question is why MSCs would fail to repair damaged cartilage in humans. It is possible that the MSC reparative function would be ineffective due to senescence or adverse environmental conditions.
An understanding of the native MSCs and how signals at the niche sites are orchestrated toward joint homeostasis, remodeling, and repair will be essential to provide guidance regarding clinical applications that use cell-based approaches. The restoration of a functional niche will safeguard durable repair by ensuring lifelong replacement of mature cells. An exciting prospect is the pharmacologic targeting of MSCs and niche repair signals to promote joint surface repair and influence outcomes of joint disorders such as OA, with the ultimate goal of restoring joint homeostasis.
The Role of Inflammation and the Immune System
The immune system appears to play a critical role in the process of postnatal tissue regeneration after tissue damage. The immune system interferes in many ways including in debris clearance; in the regulation of local progenitor cell proliferation, cell specification and differentiation; and in the promotion of angiogenesis and tissue integration. Almost every cell type of the immune system is involved and includes the innate and adaptive immune system, depending on the phase of the regeneration process. The initial post-traumatic event activates the neutrophil defense system that triggers and modulates the post-traumatic inflammatory response (for a review, see ref. ). Although neutrophils have always been considered as mediators of tissue damage, new data are revealing their role in activating post-traumatic homeostasis. For instance, Annexin 1-containing extra-cellular vesicles released by neutrophils in arthritis appear to protect cartilage from breakdown in models of inflammatory arthritis.
Persistent and excessive inflammation is considered a pathogenic event that hampers repair; however, at the appropriate time and in the right amount, inflammation is essential to prime tissue repair. For instance, TNF is expressed briefly at the site of bone fractures where it recruits neutrophils and monocytes through CC motif ligand 2 (CCL2). The local administration of low dose human recombinant TNF at the fracture site enhanced bone repair whereas inhibition of TNF or depletion of neutrophils impaired fracture repair. Crucially, systemic administration of TNF impaired fracture healing.
Macrophages are key players in the early phase of the wound healing process, particularly M1 macrophages in debris clearance. M1 macrophages, also known as pro-inflammatory macrophages, secrete inflammatory cytokines such as IL6, TNF, IL1β, and G-CSF, affecting the inflammatory response and initial cell proliferation, while M2 macrophages produce anti-inflammatory molecules such as IGF1 and TGFβ and are more involved in cell specification and differentiation. Although there is abundant evidence for the innate immune system being involved in tissue regeneration, the adaptive immune system has recently emerged as a key player. T-cells, in particular regulatory T-cells (T-reg), have been demonstrated to be involved in the repair and regeneration of various organs systems, including skeletal and heart muscle, skin, and long bones (for a review, see ref. ). T-reg cells control the level of inflammation by promoting the M1 to M2 polarization. They activate tissue progenitors and tissue growth.
Conversely, adult stem cells interact with the immune system and can suppress and modulate the immune response to favor the repair response (for a review, see ref. ).
Exploring the function of the immune system in regeneration has gained major attention, yet there is so much still to be investigated on how immunity controls the process. However, immune cells can also have a negative influence on regeneration, and a balanced immune response is key for a successful repair process.
Understanding how both the innate and adaptive immune system interact with the tissue resident progenitors and stem cells remains a key area of investigation (for a review, see ref. ). This may in turn lead to fine tuning of the immune system to support regeneration and targeted therapeutic approaches with successful clinical outcomes.
Events Leading to Joint Surface Repair
In resting condition, articular cartilage has an extremely low rate of turnover. For instance, the half-life of type II collagen has been estimated as being around 117 years, and articular chondrocytes are all essentially in the G0 of the cell cycle. After acute traumatic injury, however, chondrocytes deploy strong adaptive responses that ultimately result in a coordinated sequence of activation and chemotaxis of progenitors within the cartilage itself and in other joint tissues, along with cell proliferation and matrix turnover. In a number of cases, such adaptive responses are sufficient to repair damage and re-establish homeostasis. However, if the injury exceeds the adaptive capacity or if the adaptive responses are hampered in some way, cartilage loss leads to OA and ultimately joint failure. In OA the “injury” (i.e., excessive mechanical load) is continuous, and no immediate loss of tissue occurs. In such a situation the homeostatic responses are more subtle and are heralded by the upregulation of the transcription factor SOX9 and its direct targets, type II collagen and aggrecan, as well as a limited degree of chondrocyte proliferation. It is interesting to notice that such types of homeostatic responses persist even at late stages of OA.
The sequence of events that leads to an adaptive response in cartilage is difficult to study in OA, which is a slow-progressing disease, but such study has been facilitated by the development of in vitro and in vivo models of acute injury. The first evidence of adaptive responses consisting of chondrocyte proliferation and new deposition of ECM dates back nearly one century (reviewed in Dell’accio and Vincent). More accurate molecular analyses have identified several other components that are amenable for targeting. These components include an immediate molecular response, the activation and attraction of progenitors, breakdown of the residual mature ECM, the filling of the defect, tissue patterning, and, finally, tissue maturation—which, in humans, can take several years. In the entire joint, inflammation is an integral part of a natural response to injury, providing molecular signals and cascades that are likely to have distinct context and tissue-specific beneficial or detrimental effects. Studies aimed at teasing apart the “good” from the “bad” pathways will help modulate repair processes and restore/maintain joint homeostasis.
In therapeutic terms, therefore, it may be possible to target any of these phases, but it is likely that a stratified approach will be necessary to ensure efficacy and consistency. Such an approach will start with the understanding of which step of the homeostatic response has failed in groups of individual patients so that the approach will be targeted to the appropriate patients ( Fig. 7.4 ).
Immediate Molecular Response
Scarification of the articular cartilage in vitro or in vivo induces a strong early molecular response dominated by the activation of signaling pathways known to play important roles in embryonic skeletogenesis. Although this response is very rapid and transient, it primes a cascade of downstream steps. Probably the best known pathways involved in this phase are the WNT, FGF, and TGF-β/BMP pathways, which are discussed elsewhere in this chapter. It is important to say here, however, that although proof of concept for efficacy with each of these pathways has been provided in some way in different models, the disparate functions that these pleiotropic signaling molecules have in each different cell type remain a challenge. Tissue-targeted treatments will be of importance, including the use of novel smart delivery systems to confine the signaling domain of such powerful morphogens and avoid off-target effects.
Activation and Attraction of Mesenchymal Progenitors
There is increasing evidence that mesenchymal progenitors are implicated in the repair of joint surface defects. The understanding of the pathophysiologic mechanisms driving stem cell commitment and differentiation is paving the way to promising therapeutic interventions that exploit homeostatic mechanisms, as discussed previously. In 2012, one study identified a small compound, kartogenin, which, by interacting with filamin A, regulates the activity of the CBFβ-RUNX1 transcriptional program in bone marrow–derived mesenchymal cells, inducing their chondrogenic differentiation. The delivery of kartogenin to mice subjected to instability-induced OA improved pain and resulted not only in the arrest of disease progression but even in regeneration of the cartilage tissue to some degree. Although kartogenin could induce chondrocytic differentiation of bone marrow stromal cells in vitro, it is not clear whether this effect on bone marrow stromal cells contributed to the chondroprotective effect of kartogenin in vivo. In fact, kartogenin could support harmonious skeletal growth, joint morphogenesis, and the resorption of the interdigital mesenchyme, which are otherwise stunted in ex vivo cultures of embryonic mouse limbs. These effects were associated with the activation of several signaling pathways and particularly the TGF-β pathway. The effect of kartogenin is likely to be more complex than the simple capacity to induce chondrogenic differentiation of MSCs.
Patterning, Differentiation, Integration, and Remodeling
After the joint surface defect is filled with mesenchymal cells, the repair tissue needs patterning tissue maturation, integration, and remodeling to acquire the final architectural structure, with a properly layered cartilage and osteochondral junction. Interestingly, both morphologically and molecularly, the repair tissue in an osteochondral defect resembles the epiphysis of a developing skeletal element, with flat cells at the surface, then chondrocytes in different stages of maturation, and hypertrophic chondrocytes at the boundary with the bone front. With time the bone front advances and, for successful repair, must arrest to leave a layer of permanent stable articular cartilage. Failure of this process results in failure of repair both in animal models and also in humans, where excessive advancement of the bone front can lead to intra-articular osteophytes after surgical procedures such as bone marrow stimulation by microfracture.
Emerging Clinical Applications Targeting Endogenous Repair
The successes in targeting homeostatic pathways to achieve cartilage repair in animal models stirred the interest of the pharmaceutical industry and led to the development of compounds currently in advanced states of clinical testing. Hereafter we summarize two of the most advanced examples of such a strategy.
Targeting FGF Signaling
FGF receptor 3–dependent signaling is essential for cartilage homeostasis and promotes cartilage anabolism. FGF-18 is a member of the FGF family of growth factors, which is highly specific for FGF receptor 3. The efficacy of intra-articular FGF-18 delivery was tested in a proof-of-concept placebo-controlled, double-blinded randomized clinical trial in which safety and efficacy were assessed at 6 and 12 months. No safety issues were identified. The patients had moderate osteoarthritis (Kellgren-Lawrence score 2-3). Although no statistically significant difference was found in the primary efficacy endpoint (change in central medial femorotibial compartment cartilage thickness), the loss of cartilage thickness in the less severely affected lateral compartment was dose-dependently reduced. Patients who received FGF-18 also had less pain. In addition to a possible underpowering of the study (for instance, the actual reduction of the cartilage thickness in the medial compartment in the placebo group was much smaller than expected from a cohort used for powering and an ambitious assumption of an expected 75% reduction in joint space narrowing compared with placebo), this study suggests that, at least with FGF-18, the best chances of success are with modest levels of cartilage loss, such as that present on the lateral condyle of the patients in this study. This raises the need for criteria to identify patients with early symptomatic knee OA who are at high risk of progression for inclusion in clinical trials and, ultimately, for treatment.
Targeting Wnt Signaling
Excessive activation of Wnt signaling is a major driver of cartilage breakdown, but a physiologic level of Wnt activation is required to maintain joint stem cell populations and activate repair. Therefore, inhibition of Wnt signaling has been pursued as a strategy. SB04690 is a small compound identified for its capacity to inhibit Wnt signaling in vitro. The compound induced chondrogenesis in vitro and protected from cartilage loss in a severe model of osteoarthritis in rats. In patients, a single intra-articular injection of SB04690 resulted in durable symptomatic relief and even radiologic evidence of thickening of the articular cartilage over 24 weeks. The remarkable long-term persistence of the compound within the tissue is possibly the reason for the long-term effect. These data need confirmation in an adequately powered phase III trial, and the precise mechanism of action needs to be clarified; nevertheless, such data, considered together with the success in pre-clinical models, may represent a major step forward towards pharmacologic cartilage regeneration.
Extrinsic Repair: Current Therapeutic Interventions
Cell-based therapies and TE for joint surface repair using expanded articular chondrocytes have entered clinical practice with long-term follow-up data. At an earlier stage of clinical development are the use of adult stem cells (such as MSCs), which have shown promising pre-clinical and early clinical safety and efficacy data. The mechanisms whereby cellular therapies and combination products contribute to tissue repair are multiple and involve direct engraftment, proliferation, and differentiation to tissue-specific cell types but also include paracrine actions such as the secretion of growth and differentiation factors that enhance the local tissue response. In addition, adult stem cells may have immunomodulatory properties, which have been clinically explored in graft-versus-host disease and autoimmune diseases.
Joint Surface Defects
A variety of articular cartilage repair techniques have been developed, typically for the treatment of (sub)acute focal joint surface defects that are often associated with excessive joint loading, either as a result of high-impact trauma or cumulative low-impact repetitive overload. Joint surface lesions can extend to the subchondral bone (osteochondral defects) or be limited to the cartilage (chondral defects). Most chondral defects do not reach to the bone (partial thickness), and only about 5% are full thickness ; nevertheless, because most partial-thickness lesions are debrided at the time of surgery to full thickness, including the removal of the calcified layer ( Fig. 7.5 ), repair strategies focus on the treatment of full-thickness defects. The best-established cell-based technology for the treatment of these lesions is autologous chondrocyte transplantation and variations thereof.
Autologous Chondrocyte Transplantation
Regeneration or repair of symptomatic articular cartilage defects has been at the forefront of regenerative medicine ever since 1994 when one group reported a remarkably good clinical and structural outcome using a procedure called autologous chondrocyte transplantation/implantation (ACI). Briefly, cell populations were prepared by enzymatic release from a biopsy specimen of articular cartilage taken from an unloaded area in the symptomatic joint. The chondrocytes were subsequently expanded in vitro, and after six to eight population doublings, they were reimplanted in the joint surface defect under a periosteal flap taken from the tibia from the same patient. This procedure was then followed by a long rehabilitation period to reach its optimal outcome at 18 to 24 months. This high-profile publication attracted considerable interest and triggered a wave of basic, translational, and clinical research activities in the field. It also evolved quite quickly in 1997 in the marketing of Carticel, an autologous cell product for the repair of symptomatic condylar defects of the knee, by Genzyme Corporation (Cambridge, Mass.).
Progress has been made since then with studies aiming to improve and standardize the autologous chondrocyte preparation (the cell product), the development of other delivery systems for the chondrocytes and the replacement of the periosteal flap with a membrane of diverse composition, and a series of clinical studies. After a number of mostly open studies with diverse outcomes (for a review, see ref. ), one study —the first multicenter, prospective, randomized trial—failed to demonstrate that ACI was superior to microfracture, a technique mostly considered to be the standard of care for small (<2 to 3 cm 2 ) symptomatic joint surface defects. Microfracture is a bone marrow stimulation procedure and is based on the puncturing of the subchondral bone plate into the bone marrow, the generation of a blood clot containing precursor cell populations derived from the subchondral bone marrow, and the spontaneous transformation of the repair clot into a fibrocartilaginous repair tissue. Microfracture is clearly associated with a clinical benefit and a filling of the joint surface defect with repair tissue, but it is generally accepted that this repair is not durable, resulting in a consistent decline in clinical outcome over the long term. It is the aim of ACI to repair the cartilage defect with better quality tissue, mostly hyaline cartilage with tissue characteristics matching those of the neighboring tissue, thereby resulting in improved long-term outcomes ( Fig. 7.6 ). Although one study did not indicate clinical superiority of ACI over microfracture, there were some indications that good structural repair was associated with durable clinical outcome.
