Apoptosis is one of the main causes of chondrocyte death after mechanical injury [16–18]. It is mediated by cysteinyl aspartate-specific proteases called caspases and their inhibitors have been shown to reduce the level of apoptosis and the severity of cartilage lesion in vivo and in vitro. Intra-articular injections of the pan caspase inhibitor ZVAD-FMK (benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethylketone reduced cartilage degradation via the inhibition of caspase 3 activity and p85 fragment and prevented the development of cartilage lesions in the Anterior Cruciate Ligament (ACL) PTOA model [18, 19]. In vitro, a protective effect of Z-VAD-FMK was demonstrated on cartilage from various species (bovine, rabbit, equine, and human) subjected to a single impact, static compression or blunt trauma [17]. However, in our studies on human cartilage acute injury model the effect of caspase inhibitors (inhibitors of caspase 3 and 9 or pan-caspase inhibitors [Z-VAD-FMK or Q-VD-OPh]) [20], was not as pronounced as in studies by D’Lima et al., [18] which used a lower peak stress during impaction (25 MPa vs. 14 MPa). Yet in both studies the cells that survived impaction showed elevated proteoglycan (PG) synthesis after the treatment with caspase inhibitors resulting in better matrix preservation (low Mankin score) especially in the areas adjacent to the impact. Both pan-caspase inhibitors tested in our laboratory demonstrated similar efficacy. Despite a wide range of effects, evidence suggests that caspase inhibitors could be and should be considered for targeted therapeutic intervention in PTOA, especially if they are used immediately or soon after joint injury before the fully blown apoptotic cascade takes place. Yet no clinical investigation has been made for caspase inhibitors for the treatment of PTOA (Table 26.2).

The integrity of cellular membrane is critical in preventing the development of PTOA. Its disruption by injury alters the capacity of the cells to maintain normal homeostasis leading to cell necrosis followed by the leakage of the intracellular components with subsequent catabolic activation [21]. For instance, altered intracellular calcium homeostasis has been implicated as an upstream event in progressive chondrocyte death after mechanical injury [22]. A reduction in extracellular calcium (by chelating calcium from the culture media using EGTA) has shown to decrease chondrocyte death following single impacted load, possibly through the prevention of an increase in cytoplasmic calcium [22].

Surfactants have hydrophilic and hydrophobic centers similar to the lipid bilayer composition of the membrane. Therefore, they can fill the holes formed as a result of the membrane disruption and thus promote membrane healing and prevent cell death. A number of laboratories (including ours) focused on the use of poloxamer 188 (P188) to prevent chondrocyte death in various in vitro and in vivo PTOA models. [10, 20, 23–25]. Initially, it was shown that P188 can significantly reduce the level of apoptosis in bovine chondrocytes in the ex vivo blunt impact model [25]. Then, the same effect was documented with early P188 administration in the in vivo rabbit model [24], where P188 was effective in a short- and long-term follow-up in preventing DNA fragmentation of injured chondrocytes. This study implied that P188 acutely repaired damaged plasma membrane, which precluded further degradation of traumatized chondrocytes. Contrary, in a similar study by Martin et al. [10] P188 was shown to be ineffective. One of the major limitations of early reports on P188 is that they focused only on chondrocyte survival without looking at its overall effect on cartilage metabolism and matrix integrity.

Our laboratory chose a different approach and investigated the mechanism of action of P188 in addition to its documented effect on cell survival and metabolism. We demonstrated that P188 was superior to caspase inhibitors 3 and 9 in promoting cell survival after acute injury [20]. We also found that a single treatment with P188 added immediately after injury was able to inhibit cell death by necrosis and apoptosis and, more importantly, was able to prevent horizontal and longitudinal spread of cell death to the areas that were not directly affected by the impaction. Though P188 was present in the explant culture only for the first 48 h, the effect was sustainable for 7 out of 14 days of the experiment. Furthermore, we identified the mechanisms through which P188 exhibited its effects [23]. P188 surfactant directly or indirectly inhibited phosphorylation of the key mediators of the IL-6 signaling pathway: Stat1, Stat3, and p38. In addition, it also inhibited phosphorylation of another kinase involved in apoptosis, glycogen synthase kinase 3. Our biochemical and histological data suggested that p38 kinase may act upstream of Stats signaling and that activation of p38 kinase as result of injury may be partially responsible for initiation of IL-6/Stats mediated catabolism. The role of p38 was confirmed using specific p38 inhibitor, which not only inhibited IL-6 signaling but also reduced apoptosis. Interestingly, pretreatment with P188 or its multiple applications post injury were not superior to a single initial treatment suggesting that the protection of damaged cell membrane remains its primary function through which P188 prevents trauma-induced cell death. Together, data presented in this part of the review suggest that chondroprotective therapy should be considered as the first and the earliest step in biologic approaches to PTOA regardless which mechanism of cell death is targeted. When chondrocyte death is arrested or prevented there are more chances to trigger anti-catabolic and pro-anabolic responses in the remaining viable cells. Yet no clinical investigation has been made for P-188 for the treatment of PTOA (Table 26.3).

Synovial inflammation has been observed at early stages of PTOA, especially after joint injury. Innate immunity has been implicated as an active player in the development of synovitis and activation of downstream inflammatory and catabolic events in articular cartilage and other tissues of the joint that may lead to PTOA onset and progression. It is unclear whether morphological changes are primarily due to whole joint trauma, or in less severe cases to a systemic immune response or occur secondarily to menisci tear, rupture of ACL followed by subsequent cartilage degeneration and subchondral bone lesions. Soluble inflammatory mediators are detected in synovial fluid of patients with OA and PTOA, including a variety of cytokines and chemokines such as IL-1β, TNF-α, IL-6, IL-8, and IL 10. The innate immune system plays an essential role in modulating multiple forms of tissue injury and repair. The role of innate immune players, including pattern recognition receptors (PPRs) and damage-associated molecular patterns (DAMPs) is still to be understood in the progression and development of PTOA.

Anti-catabolic therapy has been primarily tested for degenerative OA. Among anti-catabolic agents currently approved for clinical use (summarized in Table 26.4) are antioxidant NAC (described in detail above), interleukin-1 (IL) receptor antagonist (IL1-Ra), and TNF-α antagonist. IL-1 and TNF-α are the most studied cytokines in OA [26]. Both are potent activators of cartilage degradation and their activity and concentrations have been significantly increased after acute injury in correlation with the disease severity [27]. In addition, many other cytokines, including IL-6, IL-8, and IL-10, are elevated early after injury and play a role in cartilage loss and progression of PTOA [3, 20] justifying anti-catabolic therapy as a potential way to counteract PTOA.

IL1-Ra has been studied as the protein or gene in both in vitro and in vivo models. In the OA equine in vivo model IL1-Ra was injected as adenoviral gene construct intra-articularly [28] and showed marked clinical improvement in treated horses characterized by significant reduction in subchondral edema, joint fibrillation, and chondrocyte necrosis. Autologous conditioned serum enriched in endogenous IL1-Ra has been developed under the name “Orthokine” [29] and initial data suggested that its intra-articular injections reduce pain and increase joint function [29]. In our laboratory using ex vivo acute trauma model on human cartilage explants IL1-Ra has been tested in two doses, low (20 ng/ml) and high (100 ng/ml). While low dose was ineffective, high dose promoted cell survival. Surprisingly, although low dose of IL1-Ra was not able to reduce chondrocyte death, it was able to increase PG synthesis by remaining viable cells. An overall effect of IL1-Ra was not sustainable and was lost soon after the agent was removed from culture.

TNFα is a second cytokine strongly associated with cartilage loss in OA and PTOA. We found TNF-α being elevated immediately after injury in the acute trauma model. Antagonist of TNF-α, PEGylated soluble TNF-α receptor I, alone and/or in combination, downregulated MMP-1, MMP-3, and MMP-13 expression and promoted cartilage preservation by reducing the release of PGs and increasing production of lubricin in the rat model of PTOA [30]. Collectively, the literature available on pro-inflammatory cytokines suggests that the inhibition of IL-1 and/or TNF-α, and perhaps IL-6 family of chemokines, may offer a useful therapeutic approach for the management of PTOA. We do think though that anti-inflammatory therapy might be secondary to chondrocyte protection in preventing PTOA, but is critical in reducing the disease progression. It is also important to recognize that acute inflammation may be necessary to trigger cellular and matrix remodeling responses, while chronic inflammation may be associated with the progression and manifestation of PTOA. Table 26.4 summarizes current clinical therapies with inhibitors of proinflammatory mediators.

Degradation of cartilage matrix constituents occurs directly due to proteolytic enzymes of various families: Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinases (ADAMs), ADAMs with Trombospondin Motif (ADAM-TSs), cathepsins, and others. Therefore, to protect the matrix, two general approaches can be considered: inhibition of matrix-degrading proteinases with inhibitors of specific or general mode of action or by affecting factors responsible for their activation, such as ROS, NO, inflammatory cytokines, and matrix fragments. Inhibition of ROS and inflammatory cytokines has been already discussed above.

NO has been long implicated in cartilage degradation and patients with OA show elevated levels of nitrites in their biological fluids [31]. The increased NO production has been reported to inhibit aggrecan and total PG synthesis [31] and increase MMP and iNOS activity. The use of the iNOS inhibitor L-NIL has slowed the progression of PTOA in canine experimental OA model suggesting that iNOS can be a good target for matrix protection in PTOA [32].

Specific inhibitors of MMPs have been on the wish list as the disease modifying OA drugs for a long time, yet selective inhibitors are not widely available as of to date. Therefore, the number of studies that address their utility in PTOA is very limited and the majority of them focus on the inhibition of either MMP-13 or aggrecanases. To compensate for the lack of effective synthetic inhibitors often transgenic modifications are used to prove the importance of the inhibition of specific proteinases in preventing disease development. For instance, Little et al. [33] using MMP-13 knockout mice demonstrated cartilage protection in surgically induced OA model in the absence of MMP-13 gene. This was similar to the results obtained with an oral administration of the synthetic MMP-13 inhibitor in a rabbit PTOA model [34]. Inhibition of aggrecanases or ADAMTSs also received attention in experimental OA studies, especially after ADAMTS5 knockout mice have shown not to develop OA. Therefore, inhibitors of aggrecanases and cartilage specific MMPs with high specificity and low toxicity are clearly among future therapeutic agents for the treatment of PTOA.

One of the most developed directions in biologic approaches to PTOA is the use of growth factors to stimulate production of cartilage matrix and induce pro-anabolic responses. Amongst the mainly studied in vitro and in vivo growth factors are the members of the Transforming Growth Factor-β (TGF-β) superfamily, especially bone morphogenetic proteins (BMPs), Fibroblast Growth Factors (FGF)-2 and 18, and Insulin-Like Growth Factor-1 (IGF-1) [35]. BMP-2 and BMP-7 appear to be extremely potent in cartilage and bone repair. Furthermore, Tissue Gen. Inc has recently developed TG-C (cartilage), which consists of allogeneic chondrocytes cells that have been genetically modified to produce the therapeutic growth factor (TGB1). At the moment there is a Phase II study in the USA being conducted for the treatment of knee OA with the use of this product (@clinical trials.gov/NCT01221441). BMP-7, also known as osteogenic protein-1 (OP-1), has been studied most extensively in vitro in our laboratory using human cartilage (reviewed in Chubinskaya et al.) [36, 37] as well as in OA and PTOA animal models [37, 38]. The results suggest that for adult articular cartilage BMP-7 may be the best candidate so far as a disease-modifying OA and even PTOA drug due to its pro-anabolic and anti-catabolic properties. Unlike TGF-β and other BMPs, BMP-7 upregulates chondrocyte metabolism and protein synthesis without creating uncontrolled cell proliferation and formation of osteophytes. BMP-7 prevents chondrocyte catabolism induced by pro-inflammatory cytokines or fragments of cartilage matrix components. It can induce synergistic anabolic responses with other growth factors, IGF-1, in normal and OA, young and old chondrocytes. It also regulates production of other growth factors (stimulates IGF-1 expression and inhibits BMP-2 expression) and their signaling pathways [39, 40]. In terms of IGF-1, BMP-7 restores the responsiveness of human chondrocytes to IGF-1 lost with ageing through the regulation of IGF-1, its receptor IGF-R1, binding proteins and downstream signaling mediators [36]. BMP-7 has been also extensively studied in various PTOA animal models in dogs, sheep, goats, and rabbits. In all these PTOA models (ACL transaction, osteochondral defect, and impaction), BMP-7 regenerated articular cartilage, increased repair tissue formation and improved integrative repair between new cartilage and the surrounding articular surface. In the impaction model [41], a window of opportunity for BMP-7 treatment has been identified. BMP-7 was most effective in arresting progression of cartilage degeneration if administered twice at weekly intervals either immediately after trauma or delayed by 3 weeks. If delayed by 3 months, the treatment was ineffective, suggesting that the development and progression of PTOA could be arrested and maybe even prevented if the right treatment is administered at the right time. Phase I OA clinical study (Table 26.5) produced very encouraging results by showing tolerability to the treatment, absence of toxic response, and a greater symptomatic improvement in patients that received a single injection of BMP-7.[42]