Combined Gene Therapy and Functional Tissue Engineering for the Treatment of Osteoarthritis

The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin-1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (hMSC) chondrogenesis. We combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in hMSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce hMSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

Following this, we modified this anti-inflammatory engineered cartilage to incorporate rabbit MSCs and evaluated this therapeutic strategy in a pilot study in vivo in rabbit osteochondral defects. Rabbits were fed a custom doxycycline diet to induce gene expression in engineered cartilage implanted in the joint. Serum and synovial fluid were collected and the levels of doxycycline and inflammatory mediators were measured. Rabbits were euthanized 3 weeks following surgery and tissues were harvested for analysis. We found that doxycycline levels in serum and synovial fluid were too low to induce strong overexpression of hIL-1Ra in the joint and hIL-1Ra was undetectable in synovial fluid via ELISA. Although hIL-1Ra expression in the first few days local to the site of injury may have had a beneficial effect, overall a higher doxycycline dose and more readily transduced cell population would improve application of this therapy.

In addition to the 3D woven PCL scaffold, cartilage-derived matrix scaffolds have recently emerged as a promising option for cartilage tissue engineering. Spatially-defined, biomaterial-mediated lentiviral gene delivery of tunable and inducible morphogenetic transgenes may enable guided differentiation of hMSCs into both cartilage and bone within CDM scaffolds, enhancing the ability of the CDM scaffold to provide chondrogenic cues to hMSCs. In addition to controlled production of anti-inflammatory proteins within the joint, in situ production of chondro- and osteo-inductive factors within tissue-engineered cartilage, bone, or osteochondral tissue may be highly advantageous as it could eliminate the need for extensive in vitro differentiation involving supplementation of culture media with exogenous growth factors. To this end, we have utilized controlled overexpression of transforming growth factor-beta 3 (TGF-β3), bone morphogenetic protein-2 (BMP-2) or a combination of both factors, to induce chondrogenesis, osteogenesis, or both, within CDM hemispheres. We found that TGF-β3 overexpression led to robust chondrogenesis in vitro and BMP-2 overexpression led to mineralization but not accumulation of type I collagen. We also showed the development of a single osteochondral construct by combining tissues overexpressing BMP-2 (hemisphere insert) and TGF-β3 (hollow hemisphere shell) and culturing them together in the same media. Chondrogenic ECM was localized in the TGF-β3-expressing portion and osteogenic ECM was localized in the BMP-2-expressing region. Tissue also formed in the interface between the two pieces, integrating them into a single construct.

Since CDM scaffolds can be enzymatically degraded just like native cartilage, we hypothesized that IL-1 may have an even larger influence on CDM than PCL tissue-engineered constructs. Additionally, anti-inflammatory engineered cartilage implanted in vivo will likely affect cartilage and the underlying bone. There is some evidence that osteogenesis may be enhanced by IL-1 treatment rather than inhibited. To investigate the effects of an inflammatory environment on osteogenesis and chondrogenesis within CDM hemispheres, we evaluated the ability of IL-1Ra-expressing or control constructs to undergo chondrogenesis and osteogenesis in the prescence of IL-1. We found that IL-1 prevented chondrogenesis in CDM hemispheres but did not did not produce discernable effects on osteogenesis in CDM hemispheres. IL-1Ra-expressing CDM hemispheres produced robust cartilage-like ECM and did not upregulate inflammatory mediators during chondrogenic culture in the presence of IL-1.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Le cordon ombilical humain, source de cellules pour le génie tissulaire : Isolement, caractérisation et production de substituts humains

Le cordon ombilical humain suscite beaucoup d’intérêt comme source de cellules à des fins de recherche et de thérapie. Quatre types cellulaires majeurs - les cellules épithéliales, stromales, musculaires lisses et endothéliales - composent les tissus solides du cordon ombilical. Quelques-uns de ces types cellulaires ont été utilisés en recherche scientifique depuis longtemps, alors que d’autres commencent à peine à dévoiler leur potentiel. Nous avons développé un protocole unique pour l’extraction séquentielle de tous ces types cellulaires d’un seul cordon ombilical, permettant ainsi la reconstruction à partir d’une même source. La combinaison des techniques de perfusion, immersion et explants a mené à la mise en culture et à l’expansion de ces cellules, dont les cellules épithéliales et les cellules stromales de la gelée de Wharton qui ont été caractérisées plus en détail par l’immunomarquage de protéines spécifiques. Leur potentiel pour la médecine régénératrice a été démontré par la production de tissus par génie tissulaire. Un vaisseau sanguin composé de cellules stromales et de cellules musculaires lisses du cordon ombilical démontra une résistance substantielle à l’éclatement. Les capacités de différenciation des cellules épithéliales ont été étudiées dans le contexte d’une peau bilamellaire reconstruite en combinaison avec des kératinocytes, des fibroblastes dermiques, et des cellules stromales de la gelée de Wharton. Les cellules épithéliales ont montré une différenciation similaire à celle des kératinocytes lorsque cultivées sur des fibroblastes dermiques et exposées à l’air, tandis que sur des cellules stromales du cordon, elles ont subi une désorganisation. Finalement, la différenciation des cellules stromales a été induite en culture vers plusieurs types cellulaires afin de compléter cette étude. L’ensemble des résultats fait ressortir l’importance non seulement de l’influence du milieu physique sur la croissance et la différenciation des cellules, mais également de l’impact de la provenance des cellules sur la qualité des tissus reconstruits.

A new approach to heart valve tissue engineering: mimicking the heart ventricle with a ventricular assist device in a novel bioreactor

The `biomimetic` approach to tissue engineering usually involves the use of a bioreactor mimicking physiological parameters whilst supplying nutrients to the developing tissue. Here we present a new heart valve bioreactor, having as its centrepiece a ventricular assist device (VAD), which exposes the cell-scaffold constructs to a wider array of mechanical forces. The pump of the VAD has two chambers: a blood and a pneumatic chamber, separated by an elastic membrane. Pulsatile air-pressure is generated by a piston-type actuator and delivered to the pneumatic chamber, ejecting the fluid in the blood chamber. Subsequently, applied vacuum to the pneumatic chamber causes the blood chamber to fill. A mechanical heart valve was placed in the VAD`s inflow position. The tissue engineered (TE) valve was placed in the outflow position. The VAD was coupled in series with a Windkessel compliance chamber, variable throttle and reservoir, connected by silicone tubings. The reservoir sat on an elevated platform, allowing adjustment of ventricular preload between 0 and 11 mmHg. To allow for sterile gaseous exchange between the circuit interior and exterior, a 0.2 mu m filter was placed at the reservoir. Pressure and flow were registered downstream of the TE valve. The circuit was filled with culture medium and fitted in a standard 5% CO(2) incubator set at 37 degrees C. Pressure and flow waveforms were similar to those obtained under physiological conditions for the pulmonary circulation. The `cardiomimetic` approach presented here represents a new perspective to conventional biomimetic approaches in TE, with potential advantages. Copyright (C) 2010 John Wiley & Sons, Ltd.

Stromal vascular fraction cell sheets angiogenic potential for tissue engineering and regenerative medicine applications

One of the biggest concerns in the Tissue Engineering field is the correct vascularization of engineered constructs. Strategies involving the use of endothelial cells are promising but adequate cell sourcing and neo-vessels stability are enduring challenges. In this work, we propose the hypoxic pre-conditioning of the stromal vascular fraction (SVF) of human adipose tissue to obtain highly angiogenic cell sheets (CS). For that, SVF was isolated after enzymatic dissociation of adipose tissue and cultured until CS formation in normoxic (pO2=21%) and hypoxic (pO2=5%) conditions for 5 and 8 days, in basal medium. Immunocytochemistry against CD31 and CD146 revealed the presence of highly branched capillary-like structures, which were far more complex for hypoxia. ELISA quantification showed increased VEGF and TIMP-1 secretion in hypoxia for 8 days of culture. In a Matrigel assay, the formation of capillary-like structures by endothelial cells was more prominent when cultured in conditioned medium recovered from the cultures in hypoxia. The same conditioned medium increased the migration of adipose stromal cells in a scratch assay, when compared with the medium from normoxia. Histological analysis after implantation of 8 days normoxic- and hypoxic-conditioned SVF CS in a hindlimb ischemia murine model showed improved formation of neo-blood vessels. Furthermore, Laser Doppler results demonstrated that the blood perfusion of the injured limb after 30 days was enhanced for the hypoxic CS group. Overall, these results suggest that SVF CS created under hypoxia can be used as functional vascularization units for tissue engineering and regenerative medicine.

Development of advanced cell/tissue culture systems, based on enhanced polymeric scaffolds and sophisticated bioreactors, for tissue engineering applications

Programa Doutoral em Engenharia Biomédica

Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm x 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O(2)) or hypoxic (5% O(2)) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10-20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (+/-25 degrees; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro.

Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60°C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2×105/cm2 on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6×104/cm2 at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair. © 2003 Elsevier Ltd. All rights reserved.

Regulation of Bone Marrow Stem Cells through Oscillatory Shear Stresses - A Heart Valve Tissue Engineering Perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range.

Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering

Cardiac tissue engineering (CTE) is currently a prime focus of research due to an enormous clinical need. In this work, a novel functional material, Poly(3-hydroxyoctanoate), P(3HO), a medium chain length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Our results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of the P(3HO) neat patches, assessed using Neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated to the patches. Also, no deleterious effect was observed on the adults cardiomyocytes’ contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO) based multifunctional cardiac patches are promising constructs for efficient CTE. This work will provide a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials.

Dynamic Coculture of a Prevascularized Engineered Bone Construct

The generation of functional, vascularized tissues is a key challenge for the field of tissue engineering. Before clinical implantations of tissue engineered bone constructs can succeed, in vitro fabrication needs to address limitations in large-scale tissue development, including controlled osteogenesis and an inadequate vasculature network to prevent necrosis of large constructs. The tubular perfusion system (TPS) bioreactor is an effective culturing method to augment osteogenic differentiation and maintain viability of human mesenchymal stem cell (hMSC)-seeded scaffolds while they are developed in vitro. To further enhance this process, we developed a novel osteogenic growth factors delivery system for dynamically cultured hMSCs using microparticles encapsulated in three-dimensional alginate scaffolds. In light of this increased differentiation, we characterized the endogenous cytokine distribution throughout the TPS bioreactor. An advantageous effect in the ‘outlet’ portion of the uniaxial growth chamber was discovered due to the system’s downstream circulation and the unique modular aspect of the scaffolds. This unique trait allowed us to carefully tune the differentiation behavior of specific cell populations. We applied the knowledge gained from the growth profile of the TPS bioreactor to culture a high-volume bone composite in a 3D-printed femur mold. This resulted in a tissue engineered bone construct with a volume of 200cm3, a 20-fold increase over previously reported sizes. We demonstrated high viability of the cultured cells throughout the culture period as well as early signs of osteogenic differentiation. Taking one step closer toward a viable implant and minimize tissue necrosis after implantation, we designed a composite construct by coculturing endothelial cells (ECs) and differentiating hMSCs, encouraging prevascularization and anastomosis of the graft with the host vasculature. We discovered the necessity of cell to cell proximity between the two cell types as well as preference for the natural cell binding capabilities of hydrogels like collagen. Notably, the results suggested increased osteogenic and angiogenic potential of the encapsulated cells when dynamically cultured in the TPS bioreactor, suggesting a synergistic effect between coculture and applied shear stress. This work highlights the feasibility of fabricating a high-volume, prevascularized tissue engineered bone construct for the regeneration of a critical size defect.

Regulation of bone marrow stem cells through oscillatory shear stresses - A heart valve tissue engineering perspective

Heart valve disease occurs in adults as well as in pediatric population due to age-related changes, rheumatic fever, infection or congenital condition. Current treatment options are limited to mechanical heart valve (MHV) or bio-prosthetic heart valve (BHV) replacements. Lifelong anti-coagulant medication in case of MHV and calcification, durability in case of BHV are major setbacks for both treatments. Lack of somatic growth of these implants require multiple surgical interventions in case of pediatric patients. Advent of stem cell research and regenerative therapy propose an alternative and potential tissue engineered heart valves (TEHV) treatment approach to treat this life threatening condition. TEHV has the potential to promote tissue growth by replacing and regenerating a functional native valve. Hemodynamics play a crucial role in heart valve tissue formation and sustained performance. The focus of this study was to understand the role of physiological shear stress and flexure effects on de novo HV tissue formation as well as resulting gene and protein expression. A bioreactor system was used to generate physiological shear stress and cyclic flexure. Human bone marrow mesenchymal stem cell derived tissue constructs were exposed to native valve-like physiological condition. Responses of these tissue constructs to the valve-relevant stress states along with gene and protein expression were investigated after 22 days of tissue culture. We conclude that the combination of steady flow and cyclic flexure helps support engineered tissue formation by the co-existence of both OSS and appreciable shear stress magnitudes, and potentially augment valvular gene and protein expression when both parameters are in the physiological range. ^

Seeding Osteoblastic Cells into a Macroporous Biodegradable CaP/PLGA Scaffold by a Centrifugal Force

This study aims to construct a hybrid biomaterial by seeding osteoblastic cells into a CaP/PLGA scaffold by a centrifugal force. Constructs are evaluated with respect to potential application in bone tissue engineering. Cells adher, spread, and form a layer of tissue lining the scaffold and are capable of migrating, proliferating, and producing mineralized matrix. We have demonstrated that the centrifugal force is highly efficient for constructing a hybrid biomaterial, which acts similarly to bone explants in a cell culture environment. In this way, these constructs could mimic an autogenous bone graft in clinical circumstances. Such a strategy may be useful for bone tissue engineering.

Mechanical fatigue performance of PCL-chondroprogenitor constructs after cell culture under bioreactor mechanical stimulus

In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.