Ana Rey-Rico

Current perspectives in stem cell research for knee cartilage repair

Protocols based on the delivery of stem cells are currently applied in patients, showing encouraging results for the treatment of articular cartilage lesions (focal defects, osteoarthritis). Yet, restoration of a fully functional cartilage surface (native structural organization and mechanical functions) especially in the knee joint has not been reported to date, showing the need for improved designs of clinical trials. Various sources of progenitor cells are now available, originating from adult tissues but also from embryonic or reprogrammed tissues, most of which have already been evaluated for their chondrogenic potential in culture and for their reparative properties in vivo upon implantation in relevant animal models of cartilage lesions. Nevertheless, particular attention will be needed regarding their safe clinical use and their potential to form a cartilaginous repair tissue of proper quality and functionality in the patient. Possible improvements may reside in the use of biological supplements in accordance with regulations, while some challenges remain in establishing standardized, effective procedures in the clinics.

N-alkylation of poloxamines modulates micellar assembly and encapsulation and release of the antiretroviral efavirenz

Poloxamines (X-shaped poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) diblocks connected to a central ethylenediamine group) were N-methylated and N-allylated with the aim of widening their versatility as drug nanocarriers. The self-aggregation properties of various derivatives, covering a wide range of molecular weights and EO/PO ratios, were thoroughly investigated. The cytocompatibility of different modified poloxamines was compared to that of the pristine counterparts by MTT and LDH assays. The most hydrophilic varieties were highly cytocompatible even at concentrations of 5%. Toward the optimization of the oral pharmacotherapy of the Human Immunodeficiency Virus (HIV) infection in pediatric patients, the encapsulation and in vitro delivery of efavirenz (EFV), a lipophilic first-line antiretroviral drug, were evaluated. Pristine and N-alkylated poloxamines behaved as highly efficient EFV solubilizers enhancing the aqueous solubility of the drug between 166 and 7426-times. EFV promotes self-micellization of poloxamines; their tiny structural modification (i.e., just one methyl- or allyl-group) being able to regulate drug/micellar core interaction. Despite the physical stability of the micelles against dilution in physiological mimicking fluids, the N-alkylated derivatives were slightly more prone to disassembly promoting EFV release from the micellar reservoir. For all the derivatives evaluated, the in vitro release fitted zero-order kinetics and was sustained for at least 24 h. These findings point out N-alkylated poloxamines as promising nanocarriers for oral or parenteral drug delivery.

Hot melt poly-ε-caprolactone/poloxamine implantable matrices for sustained delivery of ciprofloxacin.

It has been suggested that prevention and treatment of osteomyelitis could be achieved through local drug delivery using implantable devices, which provide therapeutic levels at the infection site with minimum side-effects. Physical blends of polycaprolactone (PCL) and poloxamine (Tetronic®) were prepared by applying a solvent-free hot melting approach to obtain cytocompatible implants with a tunable bioerosion rate, ciprofloxacin release profile and osteoconductive features. Differential scanning calorimetry and X-ray analysis indicate that the hydrophilic poloxamine varieties T908, T1107, and T1307 are miscible with PCL, while the hydrophobic block copolymer T1301 is immiscible. Incorporation of the block copolymer at weight ratios ranging from 25 to 75 wt.% led to matrices with viscoelastic parameters in the range of those of fresh cortical bone. Once immersed in buffer the matrices underwent a similar weight loss in the first week to the content of poloxamine, followed by a slower erosion rate due to PCL. The initial rapid erosion and the increase in porosity partially explain the observed burst of ciprofloxacin release, which is more intense in the PCL:T1301 formulation due to drug/T1301 repulsion due to polarity. The matrices sustained ciprofloxacin release for several months (<50% released after 3 months) and showed in vitro efficacy against Staphylococcus aureus, eradicating the bacteria in less than 48 h. PCL:poloxamine was cytocompatible with osteoblasts and the matrices prepared with low proportions of T908 were also compatible with mesenchymal stem cell differentiation to osteoblasts. The influence of the nature and proportion of temperature-responsive poloxamine on the performance of PCL implantable systems was determined.

Determination of the chondrogenic differentiation processes in human bone marrow-derived mesenchymal stem cells genetically modified to overexpress transforming growth factor-β via recombinant adeno-associated viral vectors.

Genetic modification of bone marrow-derived mesenchymal stem cells (MSCs) for use in transplantation settings may be a valuable strategy to enhance the repair processes in articular cartilage defects. Here, we evaluated the potential of overexpressing the transforming growth factor (TGF)-β via recombinant adeno-associated viral (rAAV) vector-mediated gene transfer to promote the chondrogenic differentiation of human MSCs (hMSCs). A human TGF-β sequence was delivered to undifferentiated and chondrogenically induced primary hMSCs, using rAAV vectors to test the efficacy and duration of transgene expression and its effects on the chondrogenic, osteogenic, and adipogenic differentiation patterns of the cells compared with control (lacZ) treatment after 21 days in vitro. Significant, durable TGF-β expression was noted both in undifferentiated and chondrogenically induced hMSCs transduced with the candidate rAAV-hTGF-β vector for up to 21 days compared with rAAV-lacZ treatment, allowing for increased proliferative, metabolic, and chondrogenic activities via stimulation of the critical SOX9 (SRY [sex-determining region Y]-related HMG [high-mobility group] box 9) chondrogenic pathway. Overexpression of TGF-β under the conditions applied here also activated the hypertrophic and osteogenic differentiation processes in the treated cells. Such effects were noted in association with enhanced levels of β-catenin and Indian hedgehog and decreased parathyroid hormone-related protein expression. The current findings show that rAAV vectors provide advantageous vehicles for gene- and stem cell-based approaches to treat articular cartilage defects, requiring tight regulation of TGF-β expression to avoid hypertrophy as candidate treatment for future applications in clinically relevant animal models in vivo.

Effective and durable genetic modification of human mesenchymal stem cells via controlled release of rAAV vectors from self-assembling peptide hydrogels with a maintained differentiation potency.

Controlling the release of recombinant adeno-associated virus (rAAV) vectors from biocompatible materials is a novel, attractive approach to increase the residence time and effectiveness of a gene carrier at a defined target site. Self-assembling peptides have an ability to form stable hydrogels and encapsulate cells upon exposure to physiological pH and ionic strength. Here, we examined the capacity of the peptide hydrogel RAD16-I in a pure (RAD) form or combined with hyaluronic acid (RAD-HA) to release rAAV vectors as a means to genetically modify primary human bone marrow-derived mesenchymal stem cells (hMSCs), a potent source of cells for regenerative medicine. Specifically, we demonstrate the ability of the systems to efficiently encapsulate and release rAAV vectors in a sustained, controlled manner for the effective transduction of hMSCs (up to 80%) without deleterious effects on cell viability (up to 100%) or on their potential for chondrogenic differentiation over time (up to 21days). The present study demonstrates that RAD16-I is an advantageous material with tunable properties to control the release of rAAV vectors as a promising tool to develop new, improved therapeutic approaches for tissue engineering in vivo.

Doxorubicin-loaded micelles of reverse poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers as efficient "active" chemotherapeutic agents.

Five reverse poly(butylene oxide)-poly(ethylene oxide)-poly(butylene oxide) block copolymers, BOnEOmBOn, with BO ranging from 8 to 21 units and EO from 90 to 411 were synthesized and evaluated as efficient chemotherapeutic drug delivery nanocarriers and inhibitors of the P-glycoprotein (P-gp) efflux pump in a multidrug resistant (MDR) cell line. The copolymers were obtained by reverse polymerization of poly(butylene oxide), which avoids transfer reaction and widening of the EO block distribution, commonly found in commercial poly(ethylene oxide)-poly(propylene oxide) block copolymers (poloxamers). BOnEOmBOn copolymers formed spherical micelles of 10-40 nm diameter at lower concentrations (one order of magnitude) than those of equivalent poloxamers. The influence of copolymer block lengths and BO/EO ratios on the solubilization capacity and protective environment for doxorubicin (DOXO) was investigated. Micelles showed drug loading capacity ranging from ca. 0.04% to 1.5%, more than 150 times the aqueous solubility of DOXO, and protected the cargo from hydrolysis for more than a month due to their greater colloidal stability in solution. Drug release profiles at various pHs, and the cytocompatibility and cytotoxicity of the DOXO-loaded micelles were assessed in vitro. DOXO loaded in the polymeric micelles accumulated more slowly inside the cells than free DOXO due to its sustained release. All copolymers were found to be cytocompatible, with viability extents larger than 95%. In addition, the cytotoxicity of DOXO-loaded micelles was higher than that observed for free drug solutions in a MDR ovarian NCI-ADR-RES cell line which overexpressed P-gp. The inhibition of the P-gp efflux pump by some BOnEOmBOn copolymers, similar to that measured for the common P-gp inhibitor verapamil, favored the retention of DOXO inside the cell increasing its cytotoxic activity. Therefore, poly(butylene oxide)-poly(ethylene oxide) block copolymers offer interesting features as cell response modifiers to complement their role as efficient nanocarriers for cancer chemotherapy.

Current Progress in Stem Cell-Based Gene Therapy for Articular Cartilage Repair

Administration of mesenchymal stem cells (MSCs) that have a reliable potential for chondrogenic differentiation is a promising approach currently employed to treat articular cartilage lesions (focal defects and osteoarthritis) in patients as a mean to enhance the poor intrinsic capabilities of this specialized tissue for self-repair. However, there is still a critical need for improved designs, as reproduction of a native structural and functional unit in sites of cartilage damage is not occurring upon implantation of such cells. With the availability of optimized gene transfer systems, gene therapy offers powerful tools to stimulate the chondrogenic process in MSCs via the effective, safe, and durable delivery of candidate sequences with chondroprotective and/or chondroregenerative properties, both in vitro and in experimental models of cartilage lesions in vivo. In the present article, we provide an overview of the current advances in gene- and stem cell-based treatments employed to promote cartilage repair in focal defects and for osteoarthritis, and discuss the challenges that remain to be addressed for a safe translation of such procedures into the clinics.

Poly(styrene oxide)-poly(ethylene oxide) block copolymers: From "classical" chemotherapeutic nanocarriers to active cell-response inducers.

Two poly(styrene oxide)-poly(ethylene oxide) (PSO-PEO) triblock copolymers with different chain lengths were analyzed as potential chemotherapeutic nanocarriers, and their ability to inhibit the P-glycoprotein (P-gp) efflux pump in a multidrug resistant (MDR) cell line were measured in order to establish possible cell-responses induced by the presence of the copolymer molecules. Thus, EO33SO14EO33 and EO38SO10EO38 polymeric micelles were tested regarding doxorubicin (DOXO) entrapment efficiency (solubilization test), physical stability (DLS), cytocompatibility (fibroblasts), release profiles at various pHs (in vitro tests), as well as P-gp inhibition and evasion and cytotoxicity of the DOXO-loaded micelles in an ovarian MDR NCI-ADR/RES cell line and in DOXO-sensitive MCF-7 cells. EO33SO14EO33 and EO38SO10EO38 formed spherical micelles (~13nm) at lower concentration than other copolymers under clinical evaluation (e.g. Pluronic®), exhibited 0.2% to 1.8% loading capacity, enhancing more than 60 times drug apparent solubility, and retained the cargo for long time. The copolymer unimers inhibited P-gp ATPase activity in a similar way as Pluronic P85, favoring DOXO accumulation in the resistant cell line, but not in the sensitive cell line. DOXO loaded in the micelles accumulated more slowly inside the cells, but caused greater cytotoxicity than free drug solutions in the NCI-ADR-RES cell line, which overexpressed P-gp. Hence, PSO-PEO block copolymers offer interesting features as new biological response modifiers to be used in the design of efficient nanocarriers for cancer chemotherapy.

PEO–PPO–PEO micelles as effective rAAV-mediated gene delivery systems to target human mesenchymal stem cells without altering their differentiation potency

UNLABELLED Recombinant adeno-associated viral (rAAV) vectors are clinically adapted gene transfer vectors for direct human cartilage regenerative medicine. Their appropriate use in patients is still limited by a relatively low efficacy of vector penetration inside the cells, by the pre-existing humoral immune responses against the viral capsid proteins in a large part of the human population, and by possible inhibition of viral uptake by clinical compounds such as heparin. The delivery of rAAV vectors to their targets using optimized vehicles is therefore under active investigation. Here, we evaluated the possibility of providing rAAV to human bone marrow-derived mesenchymal stem cells (hMSCs), a potent source of cartilage regenerative cells, via self-assembled poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) triblock copolymers as linear poloxamers or X-shaped poloxamines. Encapsulation in poloxamer PF68 and poloxamine T908 polymeric micelles allowed for an effective, durable, and safe modification of hMSCs via rAAV to levels similar to or even higher than those noted upon direct vector application. The copolymers were capable of restoring the transduction of hMSCs with rAAV in conditions of gene transfer inhibition, i.e. in the presence of heparin or of a specific antibody directed against the rAAV capsid, enabling effective therapeutic delivery of a chondrogenic sox9 sequence leading to an enhanced chondrocyte differentiation of the cells. The present findings highlight the value of PEO-PPO copolymers as powerful tools for rAAV-based cartilage regenerative medicine. STATEMENT OF SIGNIFICANCE While recombinant adeno-associated viral (rAAV) vectors are adapted vectors to treat a variety of human disorders, their clinical use is still restricted by pre-existing antiviral immune responses, by a low efficacy of natural vector entry in the target cells, and by inhibition of viral uptake by clinically used compounds like heparin. The search for alternative routes of rAAV delivery is thus becoming a new field of investigation. In the present study, we describe the strong benefits of providing rAAV to human mesenchymal stem cells, a potent source of cells for regenerative medicine, encapsulated in polymeric micelles based on poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) triblock copolymers as novel, effective and safe delivery systems for human gene therapy.

Controlled release strategies for rAAV-mediated gene delivery.

UNLABELLED The development of efficient and safe gene transfer vectors capable of achieving appropriate levels of therapeutic gene expression in a target is one of the most challenging issues in clinical gene therapy. Diverse nonviral and viral gene vehicles have been developed to modify human cells and tissues that may be affected in a variety of diseases, among which the nonpathogenic, effective, and relatively safe recombinant adeno-associated viral (rAAV) vectors that make them a preferred gene delivery system to treat human disorders. Yet, their adapted clinical application is still limited by several hurdles including the presence of immune responses in the host organism and the existence of rate-limiting steps associated with physiological barriers. The use of controlled release strategies to deliver gene vectors such as rAAV may provide powerful tools to enhance the temporal and spatial presentation of therapeutic agents in a defined target and to overcome such obstacles in vivo. The goal of this review is to provide an overview of the most recent advances in gene therapy with a focus on rAAV vectors for clinical translation based on the controlled release from adapted biomaterials as a means to improve the performance of the gene transfer procedure. We also discuss the challenges that remain to be addressed for a safe and efficient adaptation and use of such approaches in the patient. STATEMENT OF SIGNIFICANCE The development of effective gene vectors to achieve suitable levels of a therapeutic agent in a target is a critical issue in clinical gene therapy and regenerative medicine. Diverse vehicles are currently available among which the nonpathogenic recombinant adeno-associated virus (rAAV) vectors, a preferred system to effectively treat human disorders. Yet, the clinical use of rAAV is impaired by the host immune responses and by rate-limiting steps of transgene expression. Controlled rAAV delivery systems may provide workable approaches to overcome such obstacles. Here, we give an overview of the most recent advances on the controlled release of vectors with a focus on rAAV using adapted biomaterials and discuss the key challenges for a safe translation in patients.