Paschalia M. Mountziaris

Modulation of the Inflammatory Response for Enhanced Bone Tissue Regeneration

Proinflammatory cytokines are infamous for their catabolic effects on tissues and joints in both inflammatory diseases and following the implantation of biomedical devices. However, recent studies indicate that many of these same molecules are critical for triggering tissue regeneration following injury. This review will discuss the role of inflammatory signals in regulating bone regeneration and the impact of both immunomodulatory and antiinflammatory pharmacologic agents on fracture healing, to demonstrate the importance of incorporating rational control of inflammation into the design of tissue engineering strategies.

Dose effect of dual delivery of vascular endothelial growth factor and bone morphogenetic protein-2 on bone regeneration in a rat critical-size defect model.

The dose effect of dual delivery of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) on bone regeneration was investigated in a rat cranial critical-size defect (CSD). It was hypothesized that decreasing amounts of BMP-2 would result in a dose-dependent decrease in bone formation, and that this reduction in bone formation could be reversed by adding increasing amounts of VEGF. In vitro release kinetics of VEGF or BMP-2 were examined over 28 days. Next, scaffolds were implanted within a rat cranial CSD containing different combinations of both BMP-2 and VEGF. At 12 weeks, samples were analyzed using microcomputed tomography and histology. In vitro, VEGF and BMP-2 exhibited burst release in the first 24 h followed by a significant decrease in release rate over 27 days. Overall, BMP-2 had a more sustained release versus VEGF. An in vivo dose-dependent decrease in percentage of bone fill (PBF) was observed for BMP-2. The addition of VEGF was unable to reverse this decrease in PBF, although improvements in the number of bridged defects did occur in some groups. This suggests that for this particular model simultaneous release of BMP-2 and VEGF does not increase bone formation over BMP-2 alone at 12 weeks.

Dose effect of tumor necrosis factor-α on in vitro osteogenic differentiation of mesenchymal stem cells on biodegradable polymeric microfiber scaffolds

This study presents a first step in the development of a bone tissue engineering strategy to trigger enhanced osteogenesis by modulating inflammation. This work focused on characterizing the effects of the concentration of a pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on osteogenic differentiation of mesenchymal stem cells (MSCs) grown in a 3D culture system. MSC osteogenic differentiation is typically achieved in vitro through a combination of osteogenic supplements that include the anti-inflammatory corticosteroid dexamethasone. Although simple, the use of dexamethasone is not clinically realistic, and also hampers in vitro studies of the role of inflammatory mediators in wound healing. In this study, MSCs were pre-treated with dexamethasone to induce osteogenic differentiation, and then cultured in biodegradable electrospun poly(epsilon-caprolactone) (PCL) scaffolds, which supported continued MSC osteogenic differentiation in the absence of dexamethasone. Continuous delivery of 0.1 ng/mL of recombinant rat TNF-alpha suppressed osteogenic differentiation of rat MSCs over 16 days, which was likely the result of residual dexamethasone antagonizing TNF-alpha signaling. Continuous delivery of a higher dose, 5 ng/mL TNF-alpha, stimulated osteogenic differentiation for a few days, and 50 ng/mL TNF-alpha resulted in significant mineralized matrix deposition over the course of the study. These findings suggest that the pro-inflammatory cytokine TNF-alpha stimulates osteogenic differentiation of MSCs, an effect that can be blocked by the presence of anti-inflammatory agents like dexamethasone, with significant implications on the interplay between inflammation and tissue regeneration.

Emerging intra-articular drug delivery systems for the temporomandibular joint.

Temporomandibular joint (TMJ) disorders are a heterogeneous group of diseases that cause progressive joint degeneration leading to chronic pain and reduced quality of life. Both effective pain reduction and restoration of TMJ function remain unmet challenges. Intra-articular injections of corticosteroids and hyaluronic acid are currently used to treat chronic pain, but these methods require multiple injections that increase the risk of iatrogenic joint damage and other complications. The small and emerging field of TMJ tissue engineering aims to reduce pain and disability through novel strategies that induce joint tissue regeneration. Development of methods for sustained, intra-articular release of growth factors and other pro-regenerative signals will be critical for the success of TMJ tissue engineering strategies. This review discusses methods of intra-articular drug delivery to the TMJ, as well as emerging injectable controlled release systems with potential to improve TMJ drug delivery, to encourage further research in the development of sustained release systems for both long-term pain management and to enhance tissue engineering strategies for TMJ regeneration.

Cell-Derived Polymer/Extracellular Matrix Composite Scaffolds for Cartilage Regeneration, Part 2: Construct Devitalization and Determination of Chondroinductive Capacity

This work examined the chondrogenic potential of chondrocyte and mesenchymal stem cell (MSC) coculture generated poly(ɛ-caprolactone) (PCL)/extracellular matrix (ECM) hybrid scaffolds. Five different ratios of chondrocytes and MSCs were cocultured to generate cartilage-like ECM within electrospun fibrous scaffolds for 7, 14, and 21 days. These constructs were then devitalized to isolate the chondrogenic effects of the ECM alone. Devitalization was successful at removing cellular matter from the scaffolds, yet did reduce the amount of matrix present in the scaffolds. Following devitalization, the PCL/ECM scaffolds were then cultured with MSCs in serum-free conditions with or without TGF-β3 treatment for 21 days. TGF-β3 supplemented culture caused an induction of chondrogenesis in each scaffold type, but also somewhat masked the subtle differences of the different ECM coatings. Without TGF-β3, the cartilaginous matrix generated by 1:1 cocultures of chondrocytes to MSCs for 14 days supported similar chondrogenic gene expression patterns of MSCs cultured on scaffolds generated by chondrocytes alone. These scaffold formulations had a positive chondrogenic effect on aggrecan, collagen type II, and collagen II/I expression when compared to PCL controls. This study demonstrates that it is possible to utilize cocultures of chondrocytes and MSCs to coat a polymer scaffold with cartilage-like ECM capable of supporting chondrogenic differentiation of MSCs.

Effect of Temporally Patterned TNF-α Delivery on In Vitro Osteogenic Differentiation of Mesenchymal Stem Cells Cultured on Biodegradable Polymer Scaffolds

Recent insight into the critical role of pro-inflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), in bone regeneration has heralded a new direction in the design of tissue engineering constructs. Previous studies have demonstrated that continuous delivery of 50 ng/ml TNF-α to mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) biodegradable electrospun poly(ϵ-caprolactone) (PCL) microfiber meshes stimulates mineralized matrix deposition, a marker of osteogenic differentiation. Since TNF-α exhibits a biphasic pattern of expression following bone fracture in vivo, this study aimed to investigate the effects of temporal patterns of TNF-α delivery on in vitro osteogenic differentiation of MSCs cultured on 3D electrospun PCL scaffolds. MSCs were cultured for 16 days and exposed to continuous, early, intermediate, or late TNF-α delivery. To further elucidate the effects of TNF-α on osteogenic differentiation, the study design included MSCs precultured both in the presence and absence of typically required osteogenic supplement dexamethasone. Mineralized matrix deposition was not observed in constructs with dexamethasone-naïve MSCs, suggesting that TNF-α is not sufficient to trigger in vitro osteogenic differentiation of MSCs. For MSCs precultured with dexamethasone, TNF-α suppressed alkaline phosphatase activity, an early marker of osteogenic differentiation, and stimulated mineralized matrix deposition, a late stage marker of MSC osteogenic differentiation. By elucidating the impact of temporal variations in TNF-α delivery on MSC osteogenic differentiation, our results offer insight into the regenerative mechanism of TNF-α and provide the design parameters for a novel tissue engineering strategy that rationally controls TNF-α signaling to stimulate bone regeneration.

Intra-articular controlled release of anti-inflammatory siRNA with biodegradable polymer microparticles ameliorates temporomandibular joint inflammation

We investigated the in vivo therapeutic efficacy of an intra-articular controlled release system consisting of biodegradable poly(dl-lactic-co-glycolic acid) (PLGA) microparticles (MPs) encapsulating anti-inflammatory small interfering RNA (siRNA), together with branched poly(ethylenimine) (PEI) as a transfecting agent, in a rat model of painful temporomandibular joint (TMJ) inflammation. The in vivo effects of PLGA MP dose and siRNA-PEI polyplex delivery were examined via non-invasive meal pattern analysis and by quantifying the protein level of the siRNA target as well as of several downstream inflammatory cytokines. Controlled release of siRNA-PEI from PLGA MPs significantly reduced inflammation-induced changes in meal patterns compared to untreated rats with inflamed TMJs. These changes correlated to decreases in tissue-level protein expression of the siRNA target to 20-50% of the amount present in the corresponding control groups. Similar reductions were also observed in the expression of downstream inflammatory cytokines, e.g. interleukin-6, whose tissue levels in the siRNA-PEI PLGA MP groups were 50% of the values for the corresponding controls. This intra-articular sustained release system has significant implications for the treatment of severe TMJ pain, and also has the potential to be readily adapted and applied to mitigate painful, chronic inflammation in a variety of conditions.

Intra-articular Microparticles for Drug Delivery to the TMJ

This study describes the in vivo biocompatibility of intra-articular poly(DL-lactic-co-glycolic acid) (PLGA) microparticle (MP) formulations in the rat temporomandibular joint (TMJ). To our knowledge, this is the first intra-articular microparticle-based drug delivery system for the TMJ. The impact of PLGA MP concentration on rat TMJ function was quantified via computerized meal pattern analysis; in this non-invasive technique, previously validated markers of TMJ pain or nociception (specifically, meal duration and food intake) were recorded by computer-monitored pellet feeders. Bilateral intra-articular injection of 15, 30, or 50 mg/mL PLGA MPs had no impact on meal duration or food intake over 6 days, compared with controls that did not receive injections. Histological analysis showed that the MPs were retained within the synovial lining. These findings indicate that the PLGA MPs described herein are biocompatible and suitable for intra-articular delivery to the rat TMJ, a finding that has significant implications for the improvement of TMJ therapeutics.

Modulation of Polyplex Release from Biodegradable Microparticles through Poly(ethylenimine) Modification and Varying Loading Concentration

ABSTRACTPurposeThis work investigates the effects of hyaluronic acid (HA) conjugated onto branched poly(ethylenimine) (bPEI) and varying loading concentrations of these polymers complexed with DNA on their release from poly(DL-lactic-co-glycolic acid) (PLGA) microparticles and the transfection of target cells.MethodsTo examine the effect of alteration of the gene delivery polymer on the system, we observed the morphology, size, loading efficiency, polymer and DNA release, and the transfection efficiency for the microparticles formed with three internal phase loading concentrations during microparticle formation.ResultsAddition of HA to this vector allowed for increased loading concentration within these systems and significantly altered release kinetics without changing the morphology of the particles. The incorporation of HA onto the bPEI backbone significantly increased the transfection efficiency of the complexes released from the corresponding microparticle formulation.ConclusionsThe results show that the modification of bPEI with HA and the concentration of loaded polymer/DNA complexes can significantly alter the entrapment and release profiles from PLGA microparticles. This is significant in that it offers insight into the effects of modification of gene delivery vectors on a controlled release system designed to achieve a sustained therapeutic response.

The Interplay of Bone-Like Extracellular Matrix and TNF-α Signaling on In Vitro Osteogenic Differentiation of Mesenchymal Stem Cells

As an initial step in the development of a bone tissue engineering strategy to rationally control inflammation, we investigated the interplay of bone-like extracellular matrix (ECM) and varying doses of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) on osteogenically differentiating mesenchymal stem cells (MSCs) cultured in vitro on 3D poly(ε-caprolactone) (PCL) microfiber scaffolds containing pregenerated bone-like ECM. To generate the ECM, PCL scaffolds were seeded with MSCs and cultured in medium containing the typically required osteogenic supplement dexamethasone. However, since dexamethasone antagonizes TNF-α, the interplay of ECM and TNF-α was investigated by culturing naïve MSCs on the decellularized scaffolds in the absence of dexamethasone. MSCs cultured on ECM-coated scaffolds continued to deposit mineralized matrix, a late stage marker of osteogenic differentiation. Mineralized matrix deposition was not adversely affected by exposure to TNF-α for 4-8 days, but was significantly reduced after continuous exposure to TNF-α over 16 days, which simulates the in vivo response, where brief TNF-α signaling stimulates bone regeneration, while prolonged exposure has damaging effects. This underscores the exciting potential of PCL/ECM constructs as a more clinically realistic in vitro culture model to facilitate the design of new bone tissue engineering strategies that rationally control inflammation to promote regeneration.