In vitro and in vivo evaluation of self-mineralization and biocompatibility of injectable, dual-gelling hydrogels for bone tissue engineering.

In this study, we investigated the mineralization capacity and biocompatibility of injectable, dual-gelling hydrogels in a rat cranial defect as a function of hydrogel hydrophobicity from either the copolymerization of a hydrolyzable lactone ring or the hydrogel polymer content. The hydrogel system comprised a poly(N-isopropylacrylamide)-based thermogelling macromer (TGM) and a polyamidoamine crosslinker. The thermogelling macromer was copolymerized with (TGM/DBA) or without (TGM) a dimethyl-γ-butyrolactone acrylate (DBA)-containing lactone ring that modulated the lower critical solution temperature and thus, the hydrogel hydrophobicity, over time. Three hydrogel groups were examined: (1) 15wt.% TGM, (2) 15wt.% TGM/DBA, and (3) 20wt.% TGM/DBA. The hydrogels were implanted within an 8mm critical size rat cranial defect for 4 and 12weeks. Implants were harvested at each timepoint and analyzed for bone formation, hydrogel mineralization and tissue response using microcomputed tomography (microCT). Histology and fibrous capsule scoring showed a light inflammatory response at 4weeks that was mitigated by 12weeks for all groups. MicroCT scoring and bone volume quantification demonstrated a similar bone formation at 4weeks that was significantly increased for the more hydrophobic hydrogel formulations - 15wt.% TGM and 20wt.% TGM/DBA - from 4weeks to 12weeks. A complementary in vitro acellular mineralization study revealed that the hydrogels exhibited calcium binding properties in the presence of serum-containing media, which was modulated by the hydrogel hydrophobicity. The tailored mineralization capacity of these injectable, dual-gelling hydrogels with hydrolysis-dependent hydrophobicity presents an exciting property for their use in bone tissue engineering applications.

Mesenchymal stem cell and gelatin microparticle encapsulation in thermally and chemically gelling injectable hydrogels for tissue engineering.

In this work, we investigated the viability and osteogenic differentiation of mesenchymal stem cells encapsulated with gelatin microparticles (GMPs) in an injectable, chemically and thermally gelling hydrogel system combining poly(N-isopropylacrylamide)-based thermogelling macromers containing pendant epoxy rings with polyamidoamine-based hydrophilic and degradable diamine crosslinking macromers. Specifically, we studied how the parameters of GMP size and loading ratio affected the viability and differentiation of cells encapsulated within the hydrogel. We also examined the effects of cell and GMP co-encapsulation on hydrogel mineralization. Cells demonstrated long-term viability within the hydrogels, which was shown to depend on GMP size and loading ratio. In particular, increased interaction of cells and GMPs through greater available GMP surface area, use of an epoxy-based chemical gelation mechanism, and the tunable high water content of the thermogelled hydrogels led to favorable long-term cell viability. Compared with cellular hydrogels without GMPs, hydrogels co-encapsulating cells and GMPs demonstrated greater production of alkaline phosphatase by cells at all time-points and a transient early enhancement of hydrogel mineralization for larger GMPs at higher loading ratios. Such injectable, in situ forming hydrogels capable of delivering and maintaining populations of encapsulated mesenchymal stem cells and promoting mineralization in vitro offer promise as novel therapies for applications in tissue engineering and regenerative medicine.

Structure-property evaluation of thermally and chemically gelling injectable hydrogels for tissue engineering.

The impact of synthesis and solution formulation parameters on the swelling and mechanical properties of a novel class of thermally and chemically gelling hydrogels combining poly(N-isopropylacrylamide)-based thermogelling macromers containing pendant epoxy rings with polyamidoamine-based hydrophilic and degradable diamine cross-linking macromers was evaluated. Through variation of network hydrophilicity and capacity for chain rearrangement, the often problematic tendency of thermogelling hydrogels to undergo significant syneresis was addressed. The demonstrated ability to tune postformation dimensional stability easily at both the synthesis and formulation stages represents a significant novel contribution toward efforts to utilize poly(N-isopropylacrylamide)-based polymers as injectable biomaterials. Furthermore, the cytocompatibility of the hydrogel system under relevant conditions was established while demonstrating time- and dose-dependent cytotoxicity at high solution osmolality. Such injectable in situ forming degradable hydrogels with tunable water content are promising candidates for many tissue-engineering applications, particularly for cell delivery to promote rapid tissue regeneration in non-load-bearing defects.

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.

Synthesis and characterization of thermally and chemically gelling injectable hydrogels for tissue engineering.

Novel, injectable hydrogels were developed that solidify through a physical and chemical dual-gelation mechanism upon preparation and elevation of temperature to 37 °C. A thermogelling, poly(N-isopropylacrylamide)-based macromer with pendant epoxy rings and a hydrolytically degradable polyamidoamine-based diamine cross-linker were synthesized, characterized, and combined to produce in situ forming hydrogel constructs. Network formation through the epoxy-amine reaction was shown to be rapid and facile, and the progressive incorporation of the hydrophilic polyamidoamine cross-linker into the hydrogel was shown to mitigate the often problematic tendency of thermogelling materials to undergo significant postformation gel syneresis. The results suggest that this novel class of injectable hydrogels may be attractive substrates for tissue engineering applications due to the synthetic versatility of the component materials and beneficial hydrogel gelation kinetics and stability.

Student Award for Outstanding Research Winner in the Ph.D. Category for the 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: 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.

Controlled release of anti-inflammatory siRNA from biodegradable polymeric microparticles intended for intra-articular delivery to the temporomandibular joint.

PURPOSE: As the next step in the development of an intra-articular controlled release system to treat painful temporomandibular joint (TMJ) inflammation, we developed several biodegradable poly(DL-lactic-co-glycolic acid) (PLGA)-based microparticle (MP) formulations encapsulating a model anti-inflammatory small interfering RNA (siRNA) together with branched poly(ethylenimine) (PEI) as a transfecting agent. The effect of siRNA loading and N:P ratio on the release kinetics of siRNA-PEI polyplexes was determined, and the size and N:P ratio of the polyplexes released over time was characterized. METHODS: Polyplex-loaded PLGA MPs were prepared using an established double emulsion technique. Increasing the pH of the release samples enabled siRNA-PEI dissociation and subsequent measurement of the release of each component over 28 days. Polyplex diameter was measured for all release samples and compared to freshly prepared siRNA-PEI under simulated physiologic conditions. RESULTS: Systematic variation of siRNA loading and N:P ratio resulted in distinct siRNA and PEI release profiles. Polyplex diameter remained constant despite large variations in the relative amounts of siRNA and PEI. Excess PEI was sequestered through complexation with 500-1,000 nm diameter PLGA MP-derived particles, including small MPs and PLGA degradation products. CONCLUSIONS: These PLGA MP formulations show exciting potential as the first intra-articular TMJ controlled release system.

Dose effect of tumor necrosis factor-alpha 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.