Abstract
The management of symptomatic chondral defects is one of the biggest challenges in Orthopaedics. The quest for the ideal orthobiologic treatment modality, in which mechanically functional hyaline cartilage can be regenerated around and in the defect site, is ongoing. The growing understanding of the function of the tissue within joint microenvironment on the one hand is helping to clarify the reasons of the failures, on the other is introducing even more intricate perspectives where a multitude of factors should be ruled to chase therapeutic success. Microfractures (MF) and autologous chondrocyte implantation (ACI) typically result in clinical satisfactory outcome only in selected patients and we are still far from restoring native tissue biology. Preliminary experience with hyaluronic acid, platelet-rich plasma and mesenchymal stem cell as adjuvant therapies for MF and ACI has been positive but high quality prospective clinical trial are lacking. The new perspective of a tissue therapy strategy, based on more mature engineered tissues, holds promise to tackle limitations of standard cell procedures. Furthermore, current research has pointed-up the demand for more targeted therapies as the induction of tolerance with granulocyte colony-stimulating factor or preventing Interleukin-6 (IL-6) downregulation, as combined IL-4 and IL-10 local release, as selective activation of prostaglandin E2 (PGE2) signaling, as intra-articular injection of growth factors such as Sprifermin (Merck kGA, Germany), and as identification of molecules (Kartogenin) that selectively direct mesenchymal stem cells differentiation to chondrocytes. Combining cell and tissue therapies with targeted therapies could represent the next-generation cartilage regeneration strategy to answer both mechanical and environmental requirements of the joint surface.
Articular cartilage is a unique complex tissue with a highly specialized extracellular matrix (ECM) made of collagens, proteoglycans, and non-collagen proteins that guarantee mechanical properties which are located in chondrocytes, the unique cellular component. Both the former and the latter are disposed in organized fashion, and the tissue can be divided into four distinct regions [1, 2] (Fig. 51.1). Compressive resistance is bestowed by the large aggregating proteoglycan aggrecan, which is attached to hyaluronic acid polymers via link protein. The half-life of aggrecan core protein ranges from 3 to 24 years. The proteoglycans are essential for protecting the collagen network, which has a half-life of more than 100 years if not subjected to inappropriate degradation [3]. Differences in the morphologies of zonal subpopulations of chondrocytes may be due to matrix composition and are largely ascribed to differences in the mechanical environment [4]. Differences in the expression of molecules may determine the zonal differences in matrix composition and function [5–7]. How chondrocytes maintain their ECM under homeostatic conditions has remained somewhat of a mystery since they do not divide and the matrix isolates them from each other [3]. Moreover, hyaline cartilage is an avascular tissue and ECM normally shields chondrocytes, which exist at low oxygen tension within the matrix. This contributes to their low metabolic activity, which represents a deficiency, together to the absence of vascularization, in the case of damage-response necessity.
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1 Why Repairing Articular Cartilage Is So Challenging?
Articular cartilage is a unique complex tissue with a highly specialized extracellular matrix (ECM) made of collagens, proteoglycans, and non-collagen proteins that guarantee mechanical properties which are located in chondrocytes, the unique cellular component. Both the former and the latter are disposed in organized fashion, and the tissue can be divided into four distinct regions [1, 2] (Fig. 50.1). Compressive resistance is bestowed by the large aggregating proteoglycan aggrecan, which is attached to hyaluronic acid polymers via link protein. The half-life of aggrecan core protein ranges from 3 to 24 years. The proteoglycans are essential for protecting the collagen network, which has a half-life of more than 100 years if not subjected to inappropriate degradation [3]. Differences in the morphologies of zonal subpopulations of chondrocytes may be due to matrix composition and are largely ascribed to differences in the mechanical environment [4]. Differences in the expression of molecules may determine the zonal differences in matrix composition and function [5,6,7]. How chondrocytes maintain their ECM under homeostatic conditions has remained somewhat of a mystery since they do not divide and the matrix isolates them from each other [3]. Moreover, hyaline cartilage is an avascular tissue and ECM normally shields chondrocytes, which exist at low oxygen tension within the matrix. This contributes to their low metabolic activity, which represents a deficiency, together to the absence of vascularization, in the case of damage-response necessity.
In a healthy joint, normal alignment, stability provided by ligaments, and smooth motion provided by menisci and articular cartilage maintain a noninflammatory microenvironment that allows chondrocytes to reside in a quiescent state with very little turnover of the ECM. Interestingly, physiological loading may protect against cartilage loss by inhibiting TAK1 (TGF-β-activated kinase 1) phosphorylation [8] as well as inhibiting IKKβ (IƙB kinase-beta) activity in the canonical NF-ƙB cascade and attenuating NF-κB transcriptional activity [9]. When physiological factors are affected by trauma or overload, the homeostatic joint environment is disrupted, and the chondrocytes become activated with increased proliferation, production of matrix-degrading enzymes, cytokines, and cytokine receptors. In the case of chronic overload, the consequence could be an increased chondrocyte activity as mechano- and osmo-sensors, as osteocytes in bone alter cell metabolism in response to physical cues [10, 11]. In the case of trauma, the activated phenotype is likely an injury response driven by inflammation. Despite the classical view of osteoarthritis (OA) as a noninflammatory arthritis, recent literature supports the concept of OA as an inflammation-driven process where cartilage destruction is maintained and perpetuated by inflammatory mediators [10, 12, 13] (Table 50.1).
IL-1β and TNF-α are the main pro-inflammatory cytokines produced by activated chondrocytes [12]. They have a dual action: the upregulation of degradative proteases (starting from ADAMTS-5 and MMP3) and the synthesis by chondrocytes and synovial cells of other inflammatory mediators. The result is an autocatalytic process established and maintained in an autocrine/paracrine manner. Other pathways, as prostaglandin E2, are involved in the establishment of a catabolic environment, increasing the complexity of the problem [27]. In OA, degradative enzymes are produced primarily by chondrocytes, but the episodic intra-articular inflammation with synovitis indicates that the synovia may also be a source of cytokines and cartilage-degrading proteinases [28, 29]. In addition, the cartilage and bone are tightly interlinked with a cellular and molecular communication through the WNT and BMP signaling pathways [30, 31]. Consequently, when chondrocytes acquire the activated phenotype, enhanced matrix remodeling occurs in the articular cartilage and in the subchondral bone, ultimately leading to damage the articular surface, to subchondral bone sclerosis [31, 32] and osteophyte formation (Fig. 50.2).
2 The Reparative Potential Rising from the Subchondral Bone: Microfractures
For these reasons, the well-known problem of the poor cartilage spontaneous healing potential is worsened by the generation of an inflammatory microenvironment by the interlinked intra-articular structures. However, the network between joint tissues is the starter point for reparation, too. In fact, the subchondral bone is in continuity/contiguity with the underlying bone, and its vascular system allows for the migration of mesenchymal progenitor cells from the bottom of the lesion and of chondrocytes from the margins of the adjacent cartilage [33]. These are the theoretical bases of the microfracture (MF) technique, which was introduced by Pride in the 1950s under the name of drilling and spread rapidly after the work of Steadman in 1998 [34]. However, in the case of an injury, the fine equilibrium between anabolic and catabolic cytokines [4] is disrupted, and the imbalance is not beneficial to cartilage repair, as it is in the bone, but typically results in a flawed regenerative process, which resembles endochondral ossification and leads to chondrocyte hypertrophy and death, cartilage calcification, and scar fibrous healing. In fact, while the role of IL-1β and TNF-α in the initiation of the regenerative process has long been known, especially for bone tissue [7], different experimental settings demonstrated the detrimental effect of the exposure of IL-1β to articular cartilage [14, 35, 36]. The limited response of articular cartilage together with the multipotent potential of the bone explains the prevalence of the bone over cartilage after bone marrow stimulation techniques such as MF [37,38,39] and the formation of a fibrous repair tissue that might not be able to preserve the articular cartilage function for the long term.
MF is a feasible, cost-effective, and simple first-line option but only when performed in young patients with small, single lesions and low postoperative demands. Deterioration of the repair tissue frequently begins between 2 and 5 years posttreatment, and degenerative changes are seen at long-term follow-up, regardless of lesion size [40]. Older athletes with large and multiple lesions typically display higher failure rates [41]. The poorer results in older patients could be explained by inflammation and gene expression. The former enhances endochondral ossification by human bone marrow-derived mesenchymal stem cell [42,43,44] and is likely present in older patients who have higher IL-1β, MMP-13, and oxidative stress levels [45]. The latter is similar in aged chondrocytes and in OA chondrocytes, suggesting an intrinsic lower healing potential of aged cartilage, independently from the inflammatory state [26].
Hyaluronic acid (HA) and platelet-rich plasma (PRP) have been tested as adjuvant therapies for MF with encouraging results [46,47,48,49,50,51]; nevertheless, a recent meta-analysis showed that when OA degenerative changes are established, viscosupplementation with HA is associated with a small and clinically irrelevant benefit [52], and HA alone may not be sufficient to restore joint environment in these patients. Besides, given the wide variability in PRP composition, it is difficult to define properly its dual anti-inflammatory and trophic effect and additional studies should be promoted.
3 Regenerative Strategies: Cellular Therapies
As far as researchers try to improve MF reparative tissue, it is still mechanically and biologically far from native articular cartilage. To bridge this gap, they focused on the cellular component, essential for the recovery and maintenance of the complex tissue structure, and they developed new treatment strategies. The main field of application of this paradigm is the autologous chondrocyte implantation, but, in the last few years, new perspectives (i.e., mesenchymal stem cells) are springing up.
3.1 Autologous Chondrocyte Implantation (ACI) Procedures
Since Peterson and Brittberg first introduced ACI technique in the clinical practice in 1994 [53], it has become popular and widespread with more than 20,000 patients treated. Yet their efficacy and cost-effectiveness are still debated [54, 55]. The technique is two-step, expensive, and complicated with short-term clinical results comparable to those of MF, but potentially has the capacity to develop a more durable hyaline-like and functional repair tissue thanks to the chondrogenic cellular component implanted. A series of clinical trials have been reported with improvement of symptoms at medium- to long-term follow-up [54], but recently published results of the longest follow-up investigation (i.e., 14–15 years) comparing MF and ACI confirmed the absence of significant difference in clinical outcomes and radiographic signs of OA between the two groups [56]. It is important to underline that more than half of the patients had radiographic evidence of OA as determined by the Kellgren and Lawrence scale. Patients who had undergone prior surgery and patients with a history of symptoms of more than 2–3 years’ duration frequently have worse results at 5-year follow-up when compared to those with traumatic/acute lesions [57,58,59]. These findings highlight how an “activated environment” could potentially lead to failure in healthy tissue regeneration, even when differentiated cells are used. Consistent with what was described for the microfracture procedure, it may be crucial to develop strategies to restore the proper balance between anabolism and catabolism before surgery and during the long process of graft maturation. A further intriguing application of this concept is the use of intra-articular PRP to establish a regenerative microenvironment to improve results of ACI, similar to that reported for the MF procedure. A crucial step forward would be represented by the identification of the most suitable PRP preparation to be used as adjuvant for cartilage repair procedure [60, 61]. Interestingly, P-PRP (a pure platelet concentrate without leukocytes with a lower amount of platelets) appears to have more anabolic and anti-inflammatory properties than L-PRP (with high concentration of both platelets and leukocytes) [60, 61] highlighting the need for studies focused on the identification of the optimal ratio of cellular and molecular components of PRP according to the different requirements. Moreover, a recent systematic review showed no evidence supporting the use of intra-articular injections of PRP for preventing OA progression. This demonstrates that further knowledge is needed for the identification of the proper use and the ideal formulation of PRP in the OA patients.
3.2 A New Promising Cellular Therapy: Mesenchymal Stem Cells
In most recent years, a new cellular source has been introduced as orthobiologic approach to cartilage treatment: the mesenchymal stem cells (MSC), also known as multipotent mesenchymal stromal cells. They are a non-hematopoietic adult stem cell population, which is present in various tissues (e.g., bone marrow, adipose tissue, synovial membrane), which has the ability to differentiate into mesenchymal lineages including chondrogenic, and, therefore, is a promising source in regenerative medicine. It was initially believed that engraftment and differentiation of MSC would lead to neotissue formation and tissue repair [62]. However, more recently, it has been shown that MSC can stimulate tissue repair by the secretion of potent paracrine factors, and only a limited amount, if any, of MSC actually engraft and differentiate in vivo [63]. According to recent literature, the beneficial effects of MSC are due to the release of a cocktail of trophic and immunomodulatory factors, rather than to an active participation in tissue regeneration, thus working as “medicinal signaling cells” [64]. This twofold activity has the potential to address the complexity of joint disorders, which require both immunomodulation and tissue regeneration.
Chondrocyte implantation presents some disadvantages such as a two-stage surgical procedure that may cause further cartilage damage and degeneration [53, 65, 66] and the chondrocyte dedifferentiation during culture that might result in fibrocartilage rather than hyaline cartilage [65, 67]. MSC, on the other hand, are promising, as they would eliminate the need for ex vivo chondrocyte expansion, which is necessary for the ACI procedure. Additionally, from the patient, from the treating physician, and from an economic perspective, a single-stage non-cultured cell-based therapy would represent a great advantage [68].
As a source of MSC, bone marrow aspirate concentrate (BMAC) has been tested. Gobbi et al. reported a satisfactory clinical outcome in patients with grade 4 cartilage lesions of the knee who received one-step BMAC implantation with a hyaluronan-based scaffold [69]. Interestingly, this proved to be mainly affected by lesion size and number and not by age. In addition, it allows to address the older than 45-year-old population, with results comparable to younger patients. Alternatively, intra-articular bone marrow-derived MSC injection following isolation and in vitro expansion showed positive clinical and MRI outcomes [70] as an adjuvant treatment following high tibial osteotomy (HTO) for early unicompartmental OA and genu varum, further supporting the role of MSC in the restoration of the articular surface.
As the concentration of active cells present in the bone marrow was found to be particularly low, other cell sources were tested for the same purpose and a higher concentration of MSC was founded in adipose tissue, the so-called adipose tissue-derived MSC (AD-MSC). However, to date, few clinical studies have examined this option. The injection of stromal vascular fraction containing adipose-derived MSC as an adjuvant of MF for osteochondral lesions of talus showed encouraging clinical and MRI results even in patients with poor prognostic factors [71]. Another study highlighted the benefit of injecting a high dose of in vitro expanded AD-MSC (1 × 108 cells) in OA knees with no adverse events, improvement of function, and, foremost, histological regeneration of hyaline-like articular cartilage [68].
Acknowledging their limited power, these pioneering studies support the potential of intra-articular MSC injection and highlight the need for a high number of MSC to obtain a clinical improvement, encouraging further studies involving cohorts of patients [72].
4 Shifting the Paradigm: Tissue Therapy
Despite the general clinical improvement, current strategies are far from generating a long-lasting repair tissue that matches native cartilage in terms of tissue quality and mechanical performance [73].
A tissue therapy [74] based on a more mature tissue with high cell density and a more abundant cartilaginous ECM could potentially have a better chance of coping with the stresses of an inflammatory environment and therefore regenerating hyaline tissue. This is suggested by different basic studies, which demonstrated that cytokine synthesis and response to IL-1β by human articular chondrocytes are modulated by their differentiation stage [14, 15, 36, 75], and, in particular, more developed engineered tissues are more able to preserve the deposited cartilaginous matrix and to release lower amounts of MMP and higher amounts of anabolic factors [15]. These studies prompt the idea that more mature cartilage constructs might represent a better graft for cartilage regeneration as it might be more resistant to pro-inflammatory chemokines and more effective in recruiting/committing cells involved in tissue repair processes. In MF and in ACI techniques, cells are exposed to the joint environment without the protection typically provided by the cartilaginous ECM from inflammatory and mechanical insults.
A promising proof of principle of tissue therapy was recently reported [74] showing successful alar lobule cartilage regeneration with restoration of function and aesthetic satisfaction. Despite the notable difference in terms of environment and function with the articular cartilage, it is important to highlight the mechanical resiliency of the engineered graft, which is a valuable feature also for articular cartilage repair.
A further interesting advancement, which stems from this clinical experience and from several experimental basic science studies [14, 76,77,78], is represented by the use of autologous nasal chondrocytes as cell source for engineering a cartilaginous graft. Nasal chondrocytes demonstrated capacity of self-renewal and environmental plasticity [77]. They showed positive response to dynamic compression mimicking joint loading [76], higher chondrogenic capacity than articular chondrocytes (from matched human donors) [14], more efficient recover than articular chondrocytes from exposure to IL-1β (from matched human donor) [14], and a greater survival and integration in the joint environment together with the acquisition of a mesodermal phenotype [77]. Moreover, the use of nasal chondrocytes for tissue therapies has insignificant morbidity for their harvest from the nasal septum, which represents a crucial improvement compared to the use of articular chondrocytes. Taken together, these features render nasal chondrocytes a superior cell source for cartilage tissue engineering. Consistently, a phase I clinical trial for the treatment of traumatic cartilage defects is currently ongoing with an approved extension to 25 patients (http://www.clinicaltrials.gov/ Identifier:NCT01605210), and it represents the first clinical application of “tissue therapy” using autologous nasal chondrocytes, as opposed to the standard “cell therapy” procedure of delivering articular chondrocytes by a scaffold material. Promising results about the first ten patients have recently been published. For each patient, the nasal-derived engineered tissues were stable through handling with forceps and could be secured in the injured joints. No adverse reactions were recorded and self-assessed clinical scores for pain, knee function, and quality of life were improved significantly from before surgery to 24 months after surgery. Radiological assessments indicated variable degrees of defect filling and development of repair tissue approaching the composition of native cartilage [79]. This encouraging data confirmed the value of tissue therapy strategy and the need for future clinical experimentation before offering this approach to chronic/early OA patients. Based on the experimental and preclinical studies performed [74, 77], this strategy has the potential to overcome the classical limitations of cartilage regeneration such as kissing lesions, larger defects, and older patients [72]. A randomized phase 2 clinical trial has been planned to address these issues.
5 Could Allogeneic Therapies Be an Option?
The standard for cell therapies for musculoskeletal condition has typically been autologous cells, while allogeneic cells were mainly used for onco-hematological disorders [80]. However, following the first studies that demonstrated the safety of this approach [81], clinical trials involving allogeneic cells increased in number [82,83,84]. A growing body of literature proving the low immunogenicity of allogeneic MSC [85] supports their use, which has the potential to tackle the classic limitations of autologous cell therapies such as interindividual variability and the need for additional surgery for cell harvest. To ensure sufficient cells, expanded allogeneic MSC could be used as on off-the-shelf cell product [86]. This shift could improve the cost-effectiveness and the sustainability of the cell-based therapies.
De Windt et al. [86] reported positive results for allogeneic MSC implantation for single-stage cartilage repair. They demonstrated that the proof of concept, in which rapidly isolated chondrons were recycled from debrided cartilage instead of harvested from a non-load-bearing site of the knee, combined with allogeneic human bone marrow MSC, is feasible, stimulates reproducible tissue regeneration, and provides clinical improvement. This study was the first showing allogeneic MSC as safe and effective in stimulating cartilage regeneration in the knee when combined with autologous chondrons. The fact that 1 year after surgery no stem cell DNA could be traced in the regenerative tissue may confirm the recent view on MSC as cellular moderators, which stimulate autologous repair through paracrine mechanisms.
Allogeneic human umbilical cord blood-derived MSC (hUCB-MSC) has been tested as a source of MSC, too. Park et al. [87] showed the safety and efficacy of articular cartilage regeneration by a stem cell-based medicinal product (a composite of culture-expanded allogeneic hUCB-MSC and hyaluronic acid hydrogel [Cartistem®]) in patients with Kellgren-Lawrence grade 3 OA and International Cartilage Repair Society (ICRS) grade 4 cartilage defects. Seven participants were enrolled. Maturing repair tissue was observed at the 12-week arthroscopic evaluation. The VAS and IKDC scores were improved at 24 weeks. The improved clinical outcomes were stable over 7 years of follow-up. The histological findings at 1 year showed hyaline-like cartilage. MRI at 3 years showed persistence of the regenerated cartilage. Only five mild to moderate treatment-emergent adverse events were observed. There were no cases of osteogenesis or tumor genesis over 7 years.
On the other hand, the increased immunogenicity and upregulation of cell surface antigenic antigens of MSC as they differentiate [85] create the need of assessing the feasibility of allogeneic cell therapies in the latest intriguing orthobiologic field of research: tissue therapy. The function of Fas ligand and induction of immune privilege in tissue-engineered cartilage have been recently studied, and the subsequent suppression of uncontrolled inflammation and macrophage activity resulted in increased formation of cartilage [88]. Although performed in a mouse model, with limited translational value, this work sheds light on a fundamental aspect of cartilage regeneration and paves the way to a broader application of regenerative therapies based on allogeneic cells. This could be achieved by treating the tissue-engineered cartilage with granulocyte colony-stimulating factor (G-CSF) [88] or by preventing IL-6 downregulation [89]. However, the pro-inflammatory activity of IL-6, which inhibits ECM synthesis, might limit its use to improve tolerance of the implanted graft.
6 Future Perspectives: Targeted Therapies
Another valuable option could be the development of more targeted therapies, capable of interacting with specific signaling pathways to alter the joint balance toward the trophic side, both modulating inflammation and enhancing cartilage anabolism.
IL-4 and IL-10 are modulatory cytokines and capable of reestablishing the joint homeostasis through inhibition of pro-inflammatory cytokines like IL-1β [16, 21, 22]. Additionally, IL-4 and IL-10 are known for their anti-inflammatory properties, and their combination is suggested to be beneficial [90], also preventing the potential harmful effects of IL-4 alone [91]. Strategies based on human recombinant IL-10 and IL-4 with a longer half-life, on autologous/allogeneic cells modified to overexpress IL-10/IL-4, or on smart scaffolds capable of controlled release of IL-10/IL-4 may be used alone or as a support to regenerative therapies. It has also been demonstrated that IL-4 increases IGF-1 expression, suggesting a cross talk between the two pathways. In addition, it possesses a synergistic effect with IGF-1 with greater type II collagen upregulation, inflammatory cytokine downregulation, and nitric oxide level decrease [92]. A recent study demonstrated that IL-4 is downregulated in human OA cartilage, compared to healthy cartilage, and directly involved in the downregulation of IL-1β-induced MMP13 [93]. These in vitro studies prompt further in vivo orthotopic studies to demonstrate the potential of these cytokines for cartilage repair in the most challenging scenarios.
A further interesting approach involved the modulation of the PGE2 signal through a specific E2 receptor agonist [5, 94]. In a first study, authors demonstrated successful cartilage tissue regeneration in a rabbit orthotopic model through microsphere-based release at 12-week follow-up. In a second study, the same authors reported a transient prevention of OA changes upon anterior cruciate ligament transection and subsequent intra-articular injection of the E2 receptor agonist [5, 94]. These studies highlighted the need for delivery systems capable of a long-term controlled release to harness the potential of a more targeted modulation of the inflammatory environment.
In a recently published paper, Johnson et al. [95] provided new insights for the control of chondrogenesis introducing kartogenin, a small molecule that promotes chondrocyte differentiation, shows chondroprotective effect in vitro, is efficacious in two OA animal models, and may ultimately lead to a stem cell-targeted therapy for OA. Most importantly, kartogenin selectively interacts with RUNX1, thus promoting hyaline cartilage formation, and does not activate RUNX2 signaling, which would lead to an undesired hypertrophic differentiation.
Dahlberg and colleagues [96] published the first in human clinical trial of sprifermin (Merck kGA, Germany), a recombinant, truncated, non-glycosylated form of human FGF18, tested as a disease-modifying OA drug. Their study revealed no serious safety concerns for intra-articular administration of single and multiple ascending doses of rhFGF18 from 3 to 300 μg in knee OA patients scheduled for total knee replacement; neither sprifermin was systemically found, nor anti-FGF18 antibodies were detected. Additionally, a 1-year proof-of-concept trial examining multiple dose regimens up to 100 μg in 192 patients with less severe OA has recently been completed with encouraging results [97].
Conclusions
All these experimental studies represent feasible and clinically compliant steps forward that can be easily implemented as an adjuvant to currently available procedures. Combining cell and tissue therapies with targeted therapies represents a valuable paradigm for next-generation cartilage regeneration strategies capable of reestablishing both a normal joint environment and a functional articular surface to extend the good results of cartilage restoration procedures both to chronic cartilage defects and to early OA.
References
Poole AR. Cartilage in health and disease. In: Koopman WS, editor. Arthritis and allied conditions: a textbook of rheumatology. 15th ed. Philadelphia: Lippincott, Williams, and Wilkins; 2005. p. 223–69.
Goldring MB. Cartilage and chondrocytes. In: Firestein GS, Budd RC, McInnes IB, Sergent JS, Harris ED, Ruddy S, editors. Kelley’s textbook of rheumatology. 8th ed. Philadelphia: WB Saunders; 2008. p. 37–69. Chapter 3.
Goldring MB, Marcu KB. Cartilage homeostasis in health and rheumatic diseases. Arthritis Res Ther. 2009;11(3):224.
Goldring SR, Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res. 2004;427(Suppl):S27.
Otsuka S, Aoyama T, Furu M, Ito K, Jin Y, Nasu A, Fukiage K, Kohno Y, Maruyama T, Kanaji T, Nishiura A, Sugihara H, Fujimura S, Otsuka T, Nakamura T, Toguchida J. PGE2 signal via EP2 receptors evoked by a selective agonist enhances regeneration of injured articular cartilage. Osteoarthr Cartil. 2009;17(4):529.
Martel-Pelletier J, Pelletier JP, Fahmi H. Cyclooxygenase-2 and prostaglandins in articular tissues. Semin Arthritis Rheum. 2003;33(3):155.
Gerstenfeld LC, Cullinane DM, Barnes GL, Graves DT, Einhorn TA. Fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. J Cell Biochem. 2003;88(5):873.
Madhavan S, Anghelina M, Sjostrom D, Dossumbekova A, Guttridge DC, Agarwal S. Biomechanical signals suppress TAK1 activation to inhibit NF-κB transcriptional activation in fibrochondrocytes. J Immunol. 2007;179(9):6246.
Dossumbekova A, Anghelina M, Madhavan S, He L, Quan N, Knobloch T, Agarwal S. Biomechanical signals inhibit IKK activity to attenuate NF-κB transcription activity in inflamed chondrocytes. Arthritis Rheum. 2007;56(10):3284.
Abramson SB, Attur M. Developments in the scientific understanding of osteoarthritis. Arthritis Res Ther. 2009;11(3):227.
Fermor B, Weinberg JB, Pisetsky DS, Misukonis MA, Banes AJ, Guilak F. The effects of static and intermittent compression on nitric oxide production in articular cartilage explants. J Orthop Res. 2001;19(4):729.
Pelletier JP, Martel-Pelletier J, Abramson SB. Osteoarthritis, an inflammatory disease: potential implication for the selection of new therapeutic targets. Arthritis Rheum. 2001;44(6):1237.
Lianxu C, Hongti J, Changlong Y. NF-κBp65-specific siRNA inhibits expression of genes of COX-2, NOS-2 and MMP-9 in rat IL-1beta-induced and TNF-alpha-induced chondrocytes. Osteoarthr Cartil. 2006;14(4):367.
Scotti C, Osmokrovic A, Wolf F, Miot S, Peretti GM, Barbero A, Martin I. Response of human engineered cartilage based on articular or nasal chondrocytes to interleukin-1β and low oxygen. Tissue Eng Part A. 2012;18(3–4):362.
Francioli S, Cavallo C, Grigolo B, Martin I, Barbero A. Engineered cartilage maturation regulates cytokine production and interleukin-1β response. Clin Orthop Relat Res. 2011;469(10):2773.
van Meegeren ME, Roosendaal G, van Veghel K, Mastbergen SC, Lafeber FP. A short time window to profit from protection of blood-induced cartilage damage by IL-4 plus IL-10. Rheumatology (Oxford). 2013;52(9):1563.
Martel-Pelletier J, Boileau C, Pelletier JP, Roughley PJ. Cartilage in normal and osteoarthritis conditions. Best Pract Res Clin Rheumatol. 2008;22(2):351.
Khokha R, Murthy A, Weiss A. Metalloproteinases and their natural inhibitors in inflammation and immunity. Nat Rev Immunol. 2013;13(9):649.
Ringe J, Strassburg S, Neumann K, Endres M, Notter M, Burmester GR, Kaps C, Sittinger M. Towards in situ tissue repair: human mesenchymal stem cells express chemokine receptors CXCR1, CXCR2 and CCR2, and migrate upon stimulation with CXCL8 but not CCL2. J Cell Biochem. 2007;101(1):135.
Fahy N, Farrell E, Ritter T, Ryan AE, Murphy JM. Immune modulation to improve tissue engineering outcomes for cartilage repair in the osteoarthritic joint. Tissue Eng Part B Rev. 2015;21(1):55.
Joosten LA, Lubberts E, Helsen MM, Saxne T, Coenen-de Roo CJ, Heinegård D, van den Berg WB. Protection against cartilage and bone destruction by systemic interleukin-4 treatment in established murine type II collagen-induced arthritis. Arthritis Res. 1999;1(1):81.
van Meegeren ME, Roosendaal G, Coeleveld K, Nieuwenhuizen L, Mastbergen SC, Lafeber FP. A single intra-articular injection with IL-4 plus IL-10 ameliorates blood-induced cartilage degeneration in haemophilic mice. Br J Haematol. 2013;160(4):515.
Wehling N, Palmer GD, Pilapil C, Liu F, Wells JW, Muller PE, Evans CH, Porter EM. Interleukin-1beta and tumor necrosis factor alpha inhibit chondrogenesis by human mesenchymal stem cells through NF-κB-dependent pathways. Arthritis Rheum. 2009;60(3):801.
Legendre F, Dudhia J, Pujol JP, Bogdanowicz P. JAK/STAT but not ERK1/ERK2 pathway mediates interleukin (IL)-6/soluble IL-6R down-regulation of type II collagen, aggrecan core, and link protein transcription in articular chondrocytes. Association with a down-regulation of SOX9 expression. J Biol Chem. 2003;278(5):2903.
Lotz M. Cytokines in cartilage injury and repair. Clin Orthop Relat Res. 2001;391(Suppl):S108.
Acosta CA, Izal I, Ripalda P, Douglas-Price AL, Forriol F. Gene expression and proliferation analysis in young, aged, and osteoarthritic sheep chondrocytes effect of growth factor treatment. J Orthop Res. 2087;24(11):2006.
Shimomura K, Kanamoto T, Kita K, Akamine Y, Nakamura N, Mae T, Yoshikawa H, Nakata K. Cyclic compressive loading on 3D tissue of human synovial fibroblasts upregulates prostaglandin E2 via COX-2 production without IL-1β and TNF-α. Bone Joint Res. 2014;3(9):280.
Benito MJ, Veale DJ, Fitz Gerald O, van den Berg WB, Bresnihan B. Synovial tissue inflammation in early and late osteoarthritis. Ann Rheum Dis. 2005;64(9):1263.
Pearle AD, Scanzello CR, George S, Mandl LA, DiCarlo EF, Peterson M, Sculco TP, Crow MK. Elevated high-sensitivity C-reactive protein levels are associated with local inflammatory findings in patients with osteoarthritis. Osteoarthr Cartil. 2007;15(5):516.
Luyten FP, Tylzanowski P, Lories RJ. Wnt signaling and osteoarthritis. Bone. 2009;44(4):522.
Lories RJ, Luyten FP. The bone-cartilage unit in osteoarthritis. Nat Rev Rheumatol. 2011;7(1):43.
Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012;64(6):1697.
Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthr Cart. 2002;10(6):432.
Blevins FT, Steadman JR, Rodrigo JJ, Silliman J. Treatment of articular cartilage defects in athletes: an analysis of functional outcome and lesion appearance. Orthopedics. 1998;21(7):761.
Hooiveld MJ, Roosendaal G, van den Berg HM, Bijlsma JW, Lafeber FP. Haemoglobin-derived iron-dependent hydroxyl radical formation in blood-induced joint damage: an in vitro study. Rheumatology (Oxford). 2003;42(6):784.
Lima EG, Tan AR, Tai T, Bian L, Stoker AM, Ateshiam GA, Cook JL, Hung CT. Differences in interleukin-1 response between engineered and native cartilage. Tissue Eng Part A. 2008;14(10):1721.
Mithoefer K, Williams 3rd RJ, Warren RF, Potter HG, Spock CR, Jones EC, Wickiewicz TL, Marx RG. The microfracture technique for the treatment of articular cartilage lesions in the knee. A prospective cohort study. J Bone Joint Surg Am. 2005;87(9):1911.
Kreuz PC, Steinwachs MR, Erggelet C, Krause SJ, Konrad G, Uhl M, Sudkamp N. Results after microfracture of full-thickness chondral defects in different compartments in the knee. Osteoarthr Cartil. 2006;14(11):1119.
Henderson IJ, La Valette DP. Subchondral bone overgrowth in the presence of full-thickness cartilage defects in the knee. Knee. 2005;12(6):435.
Goyal D, Keyhani S, Lee EH, Hui JH. Evidence-based status of microfracture technique: a systematic review of level I and II studies. Arthroscopy. 2013;29(9):1579.
Gobbi A, Karnatzikos G, Kumar A. Long-term results after microfracture treatment for full-thickness knee chondral lesions in athletes. Knee Surg Sports Traumatol Arthrosc. 1986;22(9):2014.
Gobbi A, Karnatzikos G, Sankineani SR. One-step surgery with multipotent stem cells for the treatment of large full-thickness chondral defects of the knee. Am J Sports Med. 2014;42(3):648.
Scotti C, Piccinini E, Takizawa H, Todorov A, Bourgine P, Papadimitropoulos A, Barbero A, Manz MG, Martin I. Engineering of a functional bone organ through endochondral ossification. Proc Natl Acad Sci U S A. 2013;110(10):3997.
Hashimoto S, Creighton-Achermann L, Takahashi K, Amiel D, Coutts RD, Lotz M. Development and regulation of osteophyte formation during experimental osteoarthritis. Osteoarthr Cartil. 2002;10(3):180.
McGeer PL, McGeer EG. Inflammation and the degenerative diseases of aging. Ann N Y Acad Sci. 2004;1035(12):104.
Strauss E, Schachter A, Frenkel S, Rosen J. The efficacy of intra-articular hyaluronan injection after the microfracture technique for the treatment of articular cartilage lesions. Am J Sports Med. 2009;37(4):720.
Doral MN, Bilge O, Batmaz G, Donmez G, Turhan E, Demirel M, Atay OA, Uzumcugil A, Atesok K, Kaya D. Treatment of osteochondral lesions of the talus with microfracture technique and postoperative hyaluronan injection. Knee Surg Sports Traumatol Arthrosc. 2012;20(7):1398.
Milano G, Sanna Passino E, Deriu L, Careddu G, Manunta L, Manunta A, Saccomanno MF, Fabbriciani C. The effect of platelet rich plasma combined with microfractures on the treatment of chondral defects: an experimental study in a sheep model. Osteoarthr Cartil. 2010;18(7):971.
Milano G, Deriu L, Sanna Passino E, Masala G, Manunta A, Postacchini R, Saccomanno MF, Fabbriciani C. Repeated platelet concentrate injections enhance reparative response of microfractures in the treatment of chondral defects of the knee: an experimental study in an animal model. Arthroscopy. 2012;28(5):688.
Lee GW, Son JH, Kim JD, Jung GH. Is platelet-rich plasma able to enhance the results of arthroscopic microfracture in early osteoarthritis and cartilage lesion over 40 years of age? Eur J Orthop Surg Traumatol. 2013;23(5):581.
Gobbi A, Karnatzikos G, Mahajan V, Malchira S. Platelet-rich plasma treatment in symptomatic patients with knee osteoarthritis: preliminary results in a group of active patients. Sports Health. 2012;4(2):162.
Rutjes AW, Jüni P, da Costa BR, Trelle S, Nüesch E, Reichenbach S. Viscosupplementation for osteoarthritis of the knee: a systematic review and meta-analysis. Ann Intern Med. 2012;157(3):180.
Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med. 1994;331(14):889.
Goyal D, Goyal A, Keyhani S, Lee EH, Hui JH. Evidence-based status of second- and third-generation autologous chondrocyte implantation over first generation: a systematic review of level I and II studies. Arthroscopy. 2013;29(11):1872.
McCormick F, Harris JD, Abrams GD, Frank R, Gupta A, Hussey K, Wilson H, Bach Jr B, Cole B. Trends in the surgical treatment of articular cartilage lesions in the United States: an analysis of a large private-payer database over a period of 8 years. Arthroscopy. 2014;30(2):222.
Knutsen G, Drogset JO, Engebretsen L, Grøntvedt T, Ludvigsen TC, Løken S, Solheim E, Strand T, Johansen O. A randomized multicenter trial comparing autologous chondrocyte implantation with microfracture: long-term follow-up at 14 to 15 years. J Bone Joint Surg Am. 2016;98(16):1332.
Saris DB, Vanlauwe J, Victor J, Almqvist KF, Verdonk R, Bellemans J, Luyten FP, TIG/ACT/01/2000&EXT Study Group. Treatment of symptomatic cartilage defects of the knee: characterized chondrocyte implantation results in better clinical outcome at 36 months in a randomized trial compared to microfracture. Am J Sports Med. 2009;37(Suppl 1):10S.
Gobbi A, Kon E, Berruto M, Filardo G, Delcogliano M, Boldrini L, Bathan L, Marcacci M. Patellofemoral full-thickness chondral defects treated with second-generation autologous chondrocyte implantation: results at 5 years’ follow-up. Am J Sports Med. 2009;37(6):1083.
Krishnan SP, Skinner JA, Bartlett W, Carrington RW, Flanagan AM, Briggs TW, Bentley G. Who is the ideal candidate for autologous chondrocyte implantation? J Bone Joint Surg Br. 2006;88(1):61.
Assirelli E, Filardo G, Mariani E, Kon E, Roffi A, Vaccaro F, Marcacci M, Facchini A, Pulsatelli L. Effect of two different preparations of platelet-rich plasma on synoviocytes. Knee Surg Sports Traumatol Arthrosc. 2014;23(9):2690.
Cavallo C, Filardo G, Mariani E, Kon E, Marcacci M, Pereira Ruiz MT, Facchini A, Grigolo B. Comparison of platelet-rich plasma formulations for cartilage healing: an in vitro study. J Bone Joint Surg Am. 2014;96(5):423.
Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med. 2000;6(11):1282.
Iso Y, Spees JL, Serrano C, et al. Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment. Biochem Biophys Res Commun. 2014;354(3):700.
Caplan AI, Correa D. The MSC: an injury drugstore. Cell Stem Cell. 2011;9(1):11.
Knutsen G, Drogset JO, Engebretsen L, et al. A randomized trial comparing autologous chondrocyte implantation with microfracture. Findings at five years. J Bone Joint Surg Am. 2007;89(10):2105.
Lee CR, Grodzinsky AJ, Hsu HP, et al. Effects of harvest and selected cartilage repair procedures on the physical and biochemical properties of articular cartilage in the canine knee. J Orthop Res. 2000;18(5):790.
von der Mark K, Gauss V, von der Mark H, et al. Relationship between cell shape and type of collagen synthesised as chondrocytes lose their cartilage phenotype in culture. Nature. 1977;267(5611):531.
Jo CH, Lee YG, Yoon KS, et al. Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial. Stem Cells. 2014;32(5):1254.
Gobbi A, Scotti C, Karnatzikos G, Mudhigere A, Castro M, Peretti GM. One-step surgery with multipotent stem cells and Hyaluronan-based scaffold for the treatment of full-thickness chondral defects of the knee in patients older than 45 years. Knee Surg Sports Traumatol Arthrosc. 2016. doi:10.1007/s00167-016-3984-6.
Wong KL, Lee KB, Tai BC, Law P, Lee EH, Hui JH. Injectable cultured bone marrow-derived mesenchymal stem cells in varus knees with cartilage defects undergoing high tibial osteotomy: a prospective, randomized controlled clinical trial with 2 years’ follow-up. Arthroscopy. 2013;29(12):2020.
Kim YS, Lee HJ, Choi YJ, Kim YI, Koh YG. Does an injection of a stromal vascular fraction containing adipose-derived mesenchymal stem cells influence the outcomes of marrow stimulation in osteochondral lesions of the talus? A clinical and magnetic resonance imaging study. Am J Sports Med. 2014;42(10):2424.
Scotti C, Gobbi A, Karnatzikos G, Martin I, Shimomura K, Lane JG, Peretti GM, Nakamura N. Cartilage repair in the inflamed joint: considerations for biological augmentation toward tissue regeneration. Tissue Eng Part B Rev. 2016;22(2):149.
Steinert AF, Ghivizzani SC, Rethwilm A, Tuan RS, Evans CH, Nöth U. Major biological obstacles for persistent cell-based regeneration of articular cartilage. Arthritis Res Ther. 2007;9(3):213.
Fulco I, Miot S, Haug MD, Barbero A, Wixmerten A, Feliciano S, Wolf F, Jundt G, Marsano A, Farhadi J, Heberer M, Jakob M, Schaefer DJ, Martin I. Engineered autologous cartilage tissue for nasal reconstruction after tumour resection: an observational first-in-human trial. Lancet. 2014;384(9940):337.
Luo X, Zhou G, Liu W, Zhang WJ, Cen L, Cui L, Cao Y. In vitro precultivation alleviates post-implantation inflammation and enhances development of tissue-engineered tubular cartilage. Biomed Mater. 2009;4(2):025006.
Candrian C, Vonwil D, Barbero A, Bonacina E, Miot S, Farhadi J, Wirz D, Dickinson S, Hollander A, Jakob M, Li Z, Alini M, Heberer M, Martin I. Engineered cartilage generated by nasal chondrocytes is responsive to physical forces resembling joint loading. Arthritis Rheum. 2008;58(1):197.
Pelttari K, Pippenger B, Mumme M, Feliciano S, Scotti C, Mainil-Varlet P, Procino A, von Rechenberg B, Schwamborn T, Jakob M, Cillo C, Barbero A, Martin I. Adult human neural crest-derived cells for articular cartilage repair. Sci Transl Med. 2014;6(251):251ra119.
Vinatier C, Gauthier O, Fatimi A, Merceron C, Masson M, Moreau A, Moreau F, Fellah B, Weiss P, Guicheux J. An injectable cellulose-based hydrogel for the transfer of autologous nasal chondrocytes in articular cartilage defects. Biotechnol Bioeng. 2009;102(4):1259.
Mumme M, Barbero A, Miot S, Wixmerten A, Feliciano S, Wolf F, Asnaghi AM, Baumhoer D, Bieri O, Kretzschmar M, Pagenstert G, Haug M, Schaefer DJ, Martin I, Jakob M. Nasal chondrocyte-based engineered autologous cartilage tissue for repair of articular cartilage defects: an observational first-in-human trial. Lancet. 1985;388(10055):2016.
Martin IPD, Ireland H, Baldomero H, Passweg J. The survey on cellular and engineered tissue therapies in Europe in 2012. Tissue Eng Part A. 2015;21(1–2):1.
Almqvist KF, Dhollander AA, Verdonk PC, Forsyth R, Verdonk R, Verbruggen G. Treatment of cartilage defects in the knee using alginate beads containing human mature allogenic chondrocytes. Am J Sports Med. 2009;37(10):1920.
Vangsness Jr CT, Farr 2nd J, Boyd J, Dellaero DT, Mills CR, LeRoux-Williams M. Adult human mesenchymal stem cells delivered via intra-articular injection to the knee following partial medial meniscectomy: a randomized, double-blind, controlled study. J Bone Joint Surg Am. 2014;96(2):90.
Evans CH, Kraus VB, Setton LA. Progress in intra-articular therapy. Nat Rev Rheumatol. 2014;10(1):11.
US National Institutes of Health. Reparation of cartilage injuries in the human knee by implantation of fresh human allogeneic chondrocytes. 2013. ClinicalTrials.gov. Identifier: NCT00263432.
Lohan P, Coleman CM, Murphy JM, Griffin MD, Ritter T, Ryan AE. Changes in immunological profile of allogeneic mesenchymal stem cells after differentiation: should we be concerned? Stem Cell Res Ther. 2014;5(4):99.
de Windt TS, Vonk LA, Slaper-Cortenbach IC, van den Broek MP, Nizak R, van Rijen MH, de Weger RA, Dhert WJ, Saris DB. Allogeneic mesenchymal stem cells stimulate cartilage regeneration and are safe for single-stage cartilage repair in humans upon mixture with recycled autologous chondrons. Stem Cells. 2016;35(1):256–64.
Park YB, Ha CW, Lee CH, Yoon YC, Park YG. Cartilage Regeneration in Osteoarthritic Patients by a Composite of Allogeneic Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronate Hydrogel: Results from a Clinical Trial for Safety and Proof-of-Concept with 7 Years of Extended Follow-Up. Stem Cells Transl Med. 2017;6(2):613–21.
Fujihara Y, Takato T, Hoshi K. Macrophage-inducing FasL on chondrocytes forms immune privilege in cartilage tissue engineering, enhancing in vivo regeneration. Stem Cells. 2014;32(5):1208.
Li P, Li SH, Wu J, Zang WF, Dhingra S, Sun L, Weisel RD, Li RK. Interleukin-6 downregulation with mesenchymal stem cell differentiation results in loss of immunoprivilege. J Cell Mol Med. 2013;17(9):1136.
van Roon JA, Lafeber FP, Bijlsma JW. Synergistic activity of interleukin-4 and interleukin-10 in suppression of inflammation and joint destruction in rheumatoid arthritis. Arthritis Rheum. 2001;44(1):3.
Lubberts E, Joosten LA, van Den Bersselaar L, Helsen MM, Bakker AC, van Meurs JB, Graham FL, Richards CD, van Den Berg WB. Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction. J Immunol. 1999;163(8):4546.
Manning K, Rachakonda PS, Rai MF, Schmidt MF. Co-expression of insulin-like growth factor-1 and interleukin-4 in an in vitro inflammatory model. Cytokine. 2010;50(3):297.
Assirelli E, Pulsatelli L, Dolzani P, Platano D, Olivotto E, Filardo G, Trisolino G, Facchini A, Borzì RM, Meliconi R. Human osteoarthritic cartilage shows reduced in vivo expression of IL-4, a chondroprotective cytokine that differentially modulates IL-1β-stimulated production of chemokines and matrix-degrading enzymes in vitro. PLoS One. 2014;9(5):e96925.
Mitsui H, Aoyama T, Furu M, Ito K, Jin Y, Maruyama T, Kanaji T, Fujimura S, Sugihara H, Nishiura A, Otsuka T, Nakamura T, Toguchida J. Prostaglandin E2 receptor type 2-selective agonist prevents the degeneration of articular cartilage in rabbit knees with traumatic instability. Arthritis Res Ther. 2011;13(5):R146.
Johnson K, Zhu S, Tremblay MS, et al. A stem cell-based approach to cartilage repair. Science. 2012;336(6082):717.
Dahlberg LE, Aydemir A, Muurahainen N, Gühring H, Fredberg Edebo H, Krarup-Jensen N, Ladel CH, Jurvelin JS. A first-in-human, double-blind, randomised, placebo-controlled, dose ascending study of intra-articular rhFGF18 (sprifermin) in patients with advanced knee osteoarthritis. Clin Exp Rheumatol. 2016;34(3):445.
Lohmander LS, Hellot S, Dreher D, et al. Intraarticular sprifermin (recombinant human fibroblast growth factor 18) in knee osteoarthritis: a randomized, double-blind, placebo-controlled trial. Arthritis Rheumatol. 2014;66(7):1820.
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Brambilla, L., Scotti, C., Gobbi, A., Peretti, G.M. (2017). Evolving Perspectives in Orthobiologic Approaches to Articular Cartilage Regeneration. In: Gobbi, A., Espregueira-Mendes, J., Lane, J., Karahan, M. (eds) Bio-orthopaedics. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-54181-4_50
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