Introduction

Osteoarthritis (OA) is a major worldwide challenge for health systems, representing the most common degenerative joint disorder and the leading cause of chronic disability among older adults. Aging is the most prominent risk factor for OA, considering that the disease affects 32.5 million people in the US, and 88% of them are 45 or older. The overall burden associated with OA in the US ($136.8 billion/year) surpasses that of tobacco-related health effects, cancer, and diabetes. Post-traumatic OA (PTOA) develops after joint injury and is responsible for 12% of the cases of OA in the US. Available pharmacological treatments only provide symptomatic pain relief and fail to arrest the progressive degeneration of cartilage associated with PTOA or idiopathic OA. Surgical procedures that promote cartilage repair are expensive, require long rehabilitation, and might expose patients to the risk of complications. Thus, there is an imperative need in developing novel biologic means that either prevent or treat early onset of the disease. The majority of experimental approaches aimed at investigation of the mechanisms of the disease or the development of treatments focus either on the use of small and large animal models or cartilage obtained from patients during joint arthroplasty. The uniqueness of our work lies in the access to normal human cartilage obtained from organ donors within 24 hours of death through more than a 3-decade collaboration with the Gift of Hope Organ and Tissue Donor Network of Illinois. Since April 1998, we received about 16,000 cartilage specimens from the knee and ankle joints of more than 7500 donors, both genders and multiple races. Knee and ankle pairs of the same donor were obtained from 741 donors. Comparing the weight of donors we received the tissue from between the two decades, 1998-2009 and 2010-present, a statistically significant increase by 18% (p<0.0001) in the number of overweight donors has been observed in the latest decade suggesting that the population is getting heavier and this can further contribute to the development of OA. One of the exclusion criteria in our procurement strategy is the absence of documented history of joint diseases; however, morphological appearance of cartilage samples has ranged from absolutely normal to highly-degenerated/OA-like as assessed by Collins grading system. This allowed us to conduct multiple age- and grade-related studies with the use of donor cartilage. In addition, since the majority of cartilage specimens from both joints fall under normal grades of 0 and 1 we were able to develop various ex vivo PTOA cartilage models to study the mechanisms of cartilage degeneration and investigate potential of therapeutic mitigation and biologic orthoregeneration. In this abstract we discuss some of our key studies using human donor cartilage.

Content

Large body of work has been aimed at understanding the role growth factors, specifically BMPs, IGF-1, and FGF-2, play in human cartilage homeostasis. It was found that the expression of endogenous anabolic growth factors BMP-7, BMP-6, and IGF-1, has decreased with age and cartilage degeneration. For BMP-7, for example, promoter overmethylation was one of the reasons for such decrease; while for IGF-1, aging affected not only the expression of IGF-1 and its receptor IGF-1R, but also the ability of IGF-1 to stimulate proteoglycan (PG) synthesis and other anabolic responses due to modulation of its downstream signaling.

We then developed an inflammatory model of cartilage degeneration using concentrations of IL-1β (1 or 10ng/ml) relevant to its levels in synovial fluid of OA and RA patients. In this cartilage explant model IL-1β inhibited PGs synthesis and induced its degradation, released and degraded collagen, and caused chondrocytes death. IL-1β also had a profound effect on canonical BMP-7 signaling by reducing the number of ALK-2 and ALK-3 receptors, inhibiting Smad1 and Smad6 expression, delaying and prematurely terminating the onset of BMP-7 mediated R-Smad phosphorylation and affecting nuclear translocation of R-Smad/Smad4 complexes. In the same study we discovered that the alternative phosphorylation of R-Smad in the linker region via the MAPK pathway (primarily p38 and JNK) could be a possible mechanism through which IL-1β interfered with BMP-7 signaling and responses to BMP-7. Conversely, recombinant BMP-7 was found to directly inhibit phosphorylation of p38 and restore PG synthesis reduced by IL-1β, more so than other major BMPs. In addition to BMP-7, other potential therapeutic agents for cartilage regeneration, such as IGF-1 and Dexamethasone (Dex) , were tested in multiple collaborative studies with Drs. Loeser and Grodzinsky. Both IGF-1 and Dex alone or in combination were able not only to prevent release of PGs, but also arrest collagen degradation.

In our latest studies using the same model of human cartilage inflammation we assessed metabolism of adipose-derived stromal cells (ADSCs) +/- hydrogel and their protective effect. We found that ADSC exhibited 1) a pro-anabolic activity by restoring and stimulating above control levels PG synthesis inhibited by IL-1β (p=0.033); and 2) a continuous anti-inflammatory response by upregulating IL-1RA and its ratio to IL-1β (p<0.001).

To investigate early responses to injury-induced PTOA we developed an acute injury model to human normal ankle cartilage in collaboration with Drs. Wimmer and Pascual-Garrido. Within the first two weeks post-injury three phases of immediate cellular responses were identified: 1) early phase, accompanied by cell death, apoptosis, and elevation of pro-inflammatory cytokines IL-6, TNF-a, FGF-2, and their signaling pathways; 2) intermediate, with initial anabolic responses characterized by elevated PG synthesis and increased expression of BMPs and superficial zone protein; and 3) late stage, characterized by attempted repair. If remained untreated, cell death by apoptosis in this model spread out horizontally and longitudinally to the adjacent, un-impacted, cartilage areas. However, treatment with P188 surfactant was not only able to arrest expansion of apoptosis, but also modulate IL-6 signaling via the inhibition of Stat1 and P38.

This model was further developed to introduce a number of co-culture systems: 1) impacted cartilage +/- healthy synovium; 2) cartilage +/- IL-1β +/- healthy synovium; 3) impacted cartilage +/- IL-6 and TNF-α, cytokines being elevated in response to injury; and 4) impacted cartilage +/- synovium and subchondral bone. In all these models, cell death, IL-6 and TNF-α were the first to be generated in response to injury. In the injury model, in which cartilage, subchondral bone and synovium were co-cultured, pro-inflammatory response was observed through elevation of multiple cartilage and bone inflammatory markers and metabolic mediators, as well as generation of the extracellular matrix fragments.

Finally, using cadaveric knee and ankle joints we created a model of chondral and subchondral joint injury, in which cartilage or cartilage/subchondral bone explants were prepared in a donut-like shape with central core being carved out. This model was utilized to study orthoregenerative potential of Agili-C implant. It demonstrated that chondrocytes penetrated a porous structure of Agili-C, filled empty defects with extracellular matrix constituents aggrecan and type II collagen, and formed a surface layer that contained progenitor-like cells. Agili-C is currently being tested for osteochondral and chondral repair in multi-center phase II and III clinical trials.

In conclusion, many years of research on human cadaveric joint tissues proved that the ex vivo models of PTOA/OA-like disease with the use of human fresh normal cartilage are relevant in highlighting the role of injury and inflammation in earliest stages of PTOA. They also provided a unique opportunity for translational research aimed to identify targets for potential therapeutic interventions based on the mechanisms of the disease. Finally, human cartilage provided a perfect platform for fine-tuning of innovative approaches to orthoregeneration and enabled a rapid transition to clinical trials and bedside.

References

References:

Chubinskaya et al, BBA, 2002; OA&C, 2007; Int Orthop, 2007; JKS, 2008; Growth factors, 2008; AR&T, 2011; JKS, 2018; Merrihew et al, JOR, 2003; JBJS, 2003; Loeser et al, A&R, 2003; 2005; 2014; OA&C, 2009; Söder et al, A&R, 2005; Ng et al, Matrix J Biomech, 2007; Elshaier et al, A&R, 2009; Hurtig et al, JOR, 2009; Pascual Garrido et al, OA&C, 2009; Bajaj et al, JOT, 2010; Anderson et al, JOR, 2011; Kokebie et al, AR&T, 2011; Chubinskaya and Wimmer, Cartilage, 2013; Li et al, OA&C, 2013; 2015; Moran et al, JBJS, 2014; Long et al, OA&C, 2015; Yanke and Chubinskaya, 2015; vanderman et al, OA&C, 2016; Collins et al, JBC, 2016; OA&C, 2019; Free Radic Biol Med, 2019; 2021; De Girolamo et al, JKS, 2016; 2019; Gitelis et al, 2018; Kraeutler et al, Cartilage, 2017; Wang et al, Matrix Biol, 2017; Shekhawat et al, JOR, 2017; Trevino et al, Biotribology, 2017; Cartilage, 2017; Nazli et al, OA&C, 2017; Cotter et al, Cartilage, 2018; Li et al, Science Transl Med, 2019; Wimmer et al, J Mech Behav Biomed Mater, 2020; Warner et al, Front Immunol 2020; Stone et al, Cartilage, 2021; Copp et al, Cartilage, 2021; Yuh et al, J Mech Behav Biomed Mater, 2021; Reed et al, OA&C, 2021.

Acknowledgments

Acknowledgements: I would like to acknowledge my collaborators and members of my lab, who contributed to the research described in this abstract: Drs. Pascual-Garrido, Yanke, Elshaier, Soeder, Merrihew, Margulis, Rappoport, Mishra, Bajaj, Kirk, Rueger, Loeser, Wimmer, Grodzinsky, Maher, Scanzello, Cole, Katz, Altschuler, Kon, Spagnoli, and Mrs. Hakimiyan. I also would like to acknowledge all funding sources (NIH, Cartiheal, JointechLabs, Klaus Kuettner Endowed Chair), Gift of Hope and donor’s family for making this journey possible and so exciting.

Introduction

Joint preservation has been an area of intense research in recent years.^1-4 Arthroplasty is an option only for certain joints in the event of symptomatic osteoarthritis, with modest evidence to date.⁵ However, the younger patients may also be better suited to delaying joint replacement surgeries to negate the possibility of multiple joint revision surgeries in their lifetime.⁶ To preserve the joint, modern strategies have arrived at the use of biologics, exosomes, as well as osteotomies.

Content

Biologics can be offered as intra-articular injections to directly facilitate the osteochondral repair.^1-4 Notably, biologics can be categorised into three broad categories: 1) extracellular matrices, 2) growth factors, and 3) stem cells. Extracellular matrices such as hyaluronic acid restore viscoelasticity and can act as a tissue network promoting cell interaction.⁴ Growth factors such as platelet-rich plasma can stimulate cellular migration, cellular proliferation, and matrix deposition.⁷ Stem cells are pluripotent and have the potential to differentiate into the desired tissue required for the damaged osteochondral construct.⁸ The mechanisms of these classical biologics are theoretically favourable for osteochondral repair. However, its supporting clinical evidence has remained limited as a whole to date.^1-4 There may be several reasons for this: 1) the optimal facilitation of osteochondral repair is of a multipronged synergistic approach of a combination of extracellular matrices with growth factors as well as possibly stem cells, 2) cellular therapies may incite immunogenic responses, and 3) pluripotent cells may generate inappropriate cell types.

Exosomes are 50 to 200 nm small extracellular vesicles most often extracted from mesenchymal stem cells.^9,10 These vesicles can carry proteins, membrane lipids, and miRNA that can be instrumental for osteochondral repair. In in vitro and animal studies to date, the postulated mechanism of exosomes for osteochondral repair has included immunomodulation, anti-apoptosis, proliferation, cellular migration, and matrix synthesis.¹⁰ Exosomes pose a breath of fresh air for osteochondral repair because of their inherent ability as a vesicle. Therefore, they can potentially allow selective loading of the favoured cytokines or scaffolding. In addition, exosome therapy negates the cellular aspect to only possess the key component for osteochondral repair and may dismiss the inflammatory effects otherwise possible in whole cellular therapy constructs.¹⁰ As exosomes are no longer associated with their stem cell construct, pluripotency is negated as well as the possibility to generate inappropriate cell types. In preclinical studies to date, exosomes appear efficacious in the regeneration of both the cartilage and subchondral bone.¹¹ However, the evidence of this for clinical models has yet to be affirmed.

The optimal administration protocol for biologics and in particular, exosomes is key for prime clinical application. Besides classical biologics, exosome concentration and volume employed for osteochondral repair have been largely variable in the literature to date.¹¹ This is especially concerning because of the possibility of a dose-response relationship.¹² The lack of preclinical and clinical studies coupled with the heterogeneity of outcomes reported has hindered further investigation of this matter to date. Specifically, regarding exosome research, the translatability of the current preclinical studies for clinical applications has also been an area of debate. Future studies must determine the bioavailability/biodistribution of biologics/exosomes.

Osteotomies can also offer to alter the biomechanics in the malaligned joint to offload stress points and therefore, alleviate pain and provide an improved environment for osteochondral repair. To date, surgical alignment correction has been mainly employed for knee osteoarthritis (high tibial osteotomy) and ankle osteoarthritis (low tibial osteotomy), both with favourable outcomes.^13,14 During surgery, the observation of degenerated cartilage has prompted adjuvant strategies to further aid osteochondral repair. Mesenchymal stem cells augmentation is of such, where its addition has demonstrated modestly improved functional outcomes compared to high tibial osteotomies alone.¹⁵

There has been a continuous stream of joint preservation research. However, due to the limitations inherent in classical biologics, pronounced by the limited evidence across the various joints to date, exosome therapy may be the future direction for the biological facilitation of osteochondral repair.¹⁰ The use of exosomes for osteochondral repair has been currently limited to preclinical models, with the transition to clinical models a necessity for safe clinical application. These studies must also aid in verifying the efficacy, optimal treatment protocol (exosome source, concentration, volume, number, and frequency of injections), and side effect profiles. Nonetheless, tibial osteotomies appear a safe and effective joint preserving strategy.

References

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12. Zhou Q, Cai Y, Jiang Y, Lin X. Exosomes in osteoarthritis and cartilage injury: advanced development and potential therapeutic strategies. Int J Biol Sci. 2020;16(11):1811-1820. doi: 10.7150/ijbs.41637.

13. Santoso MB, Wu L. Unicompartmental knee arthroplasty, is it superior to high tibial osteotomy in treating unicompartmental osteoarthritis? A meta-analysis and systemic review. J Orthop Surg Res. 2017;12(1):50. doi: 10.1186/s13018-017-0552-9.

14. Aujla RS, Perianayagam G, Siddiqui BM, Divall P, Bhatia M. Distal tibial osteotomy for varus ankle arthritis: A meta-analysis and systematic review. J Arthrosc Jt Surg. 2021;8(3):238-245

15. Tan SHS, Kwan YT, Neo WJ, Chong JY, Kuek TYJ, See JZF, et al. Outcomes of high tibial osteotomy with versus without mesenchymal stem cell augmentation: a systematic review and meta-analysis. Orthop J Sports Med. 2021;9(6):23259671211014840. doi: 10.1177/23259671211014840.

Acknowledgments

None