A controlled release bupivacaine-alginate construct: Effect on chondrocyte hypertrophy conversion.

Objective: Osteoarthritis is a degenerative disease of the joint, affecting over 30 million people in the US1. A key characteristic of OA is chondrocyte hypertrophy, characterized by chondrocyte changes to a more rounded and osteoblastic phenotype, characterized by increased IL-6 and IL-8 secretion2. While there are no cures for OA, treatments focus on mitigating pain and inflammation, the two main symptoms of OA. However, the analgesics, NSAIDS and corticosteroids commonly used, do not target regeneration and have negative side effects. Local anesthetics (LA) can be used as a pain management alternative but are usually short lasting and therefore, not suited for chronic conditions such as OA. Our engineered sustained release local anesthetic construct successfully delivers bupivacaine for an extended period of time3-5. This study is designed to evaluate the effect of the LA system on chondrocytes in an inflammatory OA-like environment. Design: Chondrocytes were cultured with bolus, liposomal, or construct LA and either untreated or treated with TNF-α and IL-1α for 24 hrs, 48 hrs, or 96 hrs. Chondrocyte viability, interleukin-8 (IL-8), interleukin-6 (IL-6), collagenase activity and proteoglycan deposition were assessed. Results: In the presence of the engineered construct, the chondrocytes retained viability and regenerative function. Moreover, the construct allowed for higher initial doses to be used, which promoted more regeneration and decreased inflammation without compromising cellular viability. Conclusions: The construct promotes a less hypertrophic chondrocyte environment while promoting a more anti-inflammatory environment. These two factors are consistent with a less OA progressive environment when using the engineered construct, compared to bolus LA.

Alginate encapsulation for bupivacaine delivery and mesenchymal stromal cell immunomodulatory cotherapy.

PURPOSE: Mesenchymal stromal cells (MSCs) are used to treat various inflammatory conditions. In parallel, to mitigate pain associated with inflammation, analgesics or opioids are prescribed, often with significant side effects. Local anesthetics (LAs) offer a promising alternative to these medications. However, their short duration and negative effects on anti-inflammatory MSCs have limited their therapeutic effectiveness. To mitigate these negative effects and to move toward developing a cotherapy, we engineered a sustained release bupivacaine alginate-liposomal construct that enables up to 4 days of LA release. By encapsulating MSC in alginate (eMSC), we demonstrate that we can further increase drug concentration to clinically relevant levels, without compromising eMSC viability or anti-inflammatory function. MATERIALS AND METHODS: MSCs were freely cultured or encapsulated in alginate microspheres ± TNFα/IFN-γ and were left untreated or dosed with bolus, liposomal, or construct bupivacaine. After 24, 48, and 96 hours, the profiles were assessed to quantify secretory function associated with LA-MSC interactions. To approximate LA exposure over time, a MATLAB model was generated. RESULTS: eMSCs secrete similar levels of IL-6 and prostaglandin E2 (PGE2) regardless of LA modality, whereas free MSCs secrete larger amounts of IL-6 and lower amounts of anti-inflammatory PGE2. Modeling the system indicated that higher doses of LA can be used in conjunction with eMSC while retaining eMSC viability and function. In general, eMSC treated with higher doses of LA secreted similar or higher levels of immunomodulatory cytokines. CONCLUSION: eMSCs, but not free MSC, are protected from LA, regardless of LA modality. Increasing the LA concentration may promote longer and stronger pain mitigation while the protected eMSCs secrete similar, if not higher, immunomodulatory cytokine levels. Therefore, we have developed an approach, using eMSC and the LA construct that can potentially be used to reduce pain as well as improve MSC anti-inflammatory function.

Alginate-liposomal construct for bupivacaine delivery and MSC function regulation.

Mesenchymal stromal cell (MSC) therapies have become potential treatment options for multiple ailments and traumatic injuries. In the clinical setting, MSC are likely to be co-administered with local anesthetics (LA) which have been shown to have dose- and potency-dependent detrimental effects on the viability and function of cells. We previously developed and characterized a sustained-release LA delivery formulation comprised of alginate-encapsulated liposomal bupivacaine. The current studies were designed to evaluate the effect of this formulation on the secretion of three key MSC regulatory molecules, interleukin 6 (IL-6), prostaglandin E2 (PGE2), and transforming growth factor-beta 1 (TGF-ß1). MSCs were treated with several bupivacaine formulations-bolus, liposome, or alginate-liposome construct (engineered construct)-in the presence or absence of inflammatory stimulus to stimulate an injured tissue environment. Our results indicated that compared to bolus or liposomal bupivacaine, the engineered construct preserved or promoted MSC anti-inflammatory PGE2 secretion; however, the engineered construct did not increase TGF-ß1 secretion. Bupivacaine release profile analyses indicated that mode of drug delivery controlled the LA concentration over time and pathway analysis identified several shared and cytokine-specific molecular mediators for IL-6, PGE2, and TGF-ß1 which could explain differential MSC secretion responses in the presence of bupivacaine. Collectively, these studies support the potential utility of alginate-encapsulated LA constructs for anti-inflammatory cell therapy co-administration and indicate that mode of local anesthetic delivery can significantly alter MSC secretome function.

The effect of local anesthetic on pro-inflammatory macrophage modulation by mesenchymal stromal cells.

Administering local anesthetics (LAs) peri- and post-operatively aims to prevent or mitigate pain in surgical procedures and after tissue injury in cases of osteoarthritis (OA) and other degenerative diseases. Innovative tissue protective and reparative therapeutic interventions such as mesenchymal stromal cells (MSCs) are likely to be exposed to co-administered drugs such as LAs. Therefore, it is important to determine how this exposure affects the therapeutic functions of MSCs and other cells in their target microenvironment. In these studies, we measured the effect of LAs, lidocaine and bupivacaine, on macrophage viability and pro-inflammatory secretion. We also examined their effect on modulation of the macrophage pro-inflammatory phenotype in an in vitro co-culture system with MSCs, by quantifying macrophage tumor necrosis factor (TNF)-α secretion and MSC prostaglandin E2 (PGE2) production. Our studies indicate that both LAs directly attenuated macrophage TNF-α secretion, without significantly affecting viability, in a concentration- and potency-dependent manner. LA-mediated attenuation of macrophage TNF-α was sustained in co-culture with MSCs, but MSCs did not further enhance this anti-inflammatory effect. Concentration- and potency-dependent reductions in macrophage TNF-α were concurrent with decreased PGE2 levels in the co-cultures further indicating MSC-independent macrophage attenuation. MSC functional recovery from LA exposure was assessed by pre-treating MSCs with LAs prior to co-culture with macrophages. Both MSC attenuation of TNF-α and PGE2 secretion were impaired by pre-exposure to the more potent bupivacaine and high dose of lidocaine in a concentration-dependent manner. Therefore, LAs can affect anti-inflammatory function by both directly attenuating macrophage inflammation and MSC secretion and possibly by altering the local microenvironment which can secondarily reduce MSC function. Furthermore, the LA effect on MSC function may persist even after LA removal.

Effect of Local Anesthetics on Human Mesenchymal Stromal Cell Secretion.

Anti-fibrotic and tissue regenerative mesenchymal stromal cell (MSC) properties are largely mediated by secreted cytokines and growth factors. MSCs are implanted to augment joint cartilage replacement and to treat diabetic ulcers and burn injuries simultaneously with local anesthetics, which reduce pain. However, the effect of anesthetics on therapeutic human MSC secretory function has not been evaluated. In order to assess the effect of local anesthetics on the MSC secretome, a panel of four anesthetics with different potencies - lidocaine, procaine, ropivacaine and bupivacaine - was evaluated. Since injured tissues secrete inflammatory cytokines, the effects of anesthetics on MSCs stimulated with tumor necrosis factor (TNF)-α and interferon (IFN)-γ were also measured. Dose dependent and anesthesia specific effects on cell viability, post exposure proliferation and secretory function were quantified using alamar blue reduction and immunoassays, respectively. Computational pathway analysis was performed to identify upstream regulators and molecular pathways likely associated with the effects of these chemicals on the MSC secretome. Our results indicated while neither lidocaine nor procaine greatly reduced unstimulated cell viability, ropivacaine and bupivacaine induced dose dependent viability decreases. This pattern was exaggerated in the simulated inflammatory environment. The reversibility of these effects after withdrawal of the anesthetics was attenuated for TNF-α/IFN-γ-stimulated MSCs exposed to ropivacaine and bupivacaine. In addition, secretome analysis indicated that constitutive secretion changes were clearly affected by both anesthetic alone and anesthetic plus TNFα/IFNγ cell stimulation, but the secretory pattern was drug specific and did not necessarily coincide with viability changes. Pathway analysis identified different intracellular regulators for stimulated and unstimulated MSCs. Within these groups, ropivacaine and bupivacaine appeared to act on MSCs similarly via the same regulatory mechanisms. Given the variable effect of local anesthetics on MSC viability and function, these studies underscore the need to evaluate MSC in the presence of medications, such as anesthetics, that are likely to accompany cell implantation.