Mesenchymal stem cells at the intersection of cell and gene therapy

Mesenchymal stem cells have the ability to differentiate into osteoblasts, chondrocytes and adipocytes. Along with differentiation, MSCs can modulate inflammation, home to damaged tissues and secret bioactive molecules. These properties can be enhanced through genetic-modification that would combine the best of both cell and gene therapy fields to treat monogenic and multigenic diseases

Use of glycol chitosan modified by 5β-cholanic acid nanoparticles for the sustained release of proteins during murine embryonic limb skeletogenesis

Murine embryonic limb cultures have invaluable roles in studying skeletogenesis. Substance delivery is an underdeveloped area in developmental biology that has primarily relied on Affi-Gel-Blue-Agarose-beads. However, the lack of information about the efficiency of agarose-bead loading and release and difficulties for a single-bead implantation represent significant limitations. We optimized the use of glycol-chitosan–5β-cholanic-acid-conjugates (HGC) as a novel protein delivery system in mouse embryonic limbs. To this purpose, we loaded HGC either with recombinant Noggin, or bovine serum albumin (BSA). The size, morphology and stability of the protein-loaded-HGC were determined by transmission-electron-microscopy and dynamic-light-scattering. HGC-BSA and HGC-Noggin loading efficiencies were 80-90%. Time-course study revealed that Noggin and BSA were 80-90% of released after 48-hours. We developed several techniques to implant protein-loaded-HGC into murine embryonic joints from embryonic age (E)13.5 to E15.5, including a micro-injection system dispensing nanoliters. HGC did not interfere with skeletogenesis. Using CBR-3BA staining, we detected HGC-nanoparticles within implanted tissues. Furthermore, a sustained release of BSA and Noggin was demonstrated in HGC-BSA and HGC-Noggin injected regions. HGC-released Noggin was biologically active in blocking the BMP signaling in in vitro mesenchyme limb micromasses as well as in ex-vivo limb cultures. Results reveal that HGC is a valuable protein-delivery system in developmental biology

Systemically delivered insulin-like growth factor-I enhances mesenchymal stem cell-dependent fracture healing

In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I + MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I + MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I + MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I + MSC could be a novel therapeutic approach in patients who have inadequate fracture repair

Quantifying Mechanical Properties in a Murine Fracture Healing System Using Inverse Modeling: Preliminary Work

ABSTRACT Understanding bone remodeling and mechanical property characteristics is important for assessing treatments to accelerate healing or in developing diagnostics to evaluate successful return to function. The murine system whereby mid-diaphaseal tibia fractures are imparted on the subject and fracture healing is assessed at different time points and under different therapeutic conditions is a particularly useful model to study. In this work, a novel inverse geometric nonlinear elasticity modeling framework is proposed that can reconstruct multiple mechanical properties from uniaxial testing data. To test this framework, the Lame' constants were reconstructed within the context of a murine cohort (n=6) where there were no differences in treatment post tibia fracture except that half of the mice were allowed to heal 4 days longer (10 day, and 14 day healing time point, respectively). The properties reconstructed were a shear modulus of G=511.2 ± 295.6 kPa, and 833.3± 352.3 kPa for the 10 day, and 14 day time points respectively. The second Lame' constant reconstructed at =1002.9 ±42.9 kPa, and 14893.7 ± 863.3 kPa for the 10 day, and 14 day time points respectively. An unpaired Student t-test was used to test for statistically significant differences among the groups. While the shear modulus did not meet our criteria for significance, the second Lame' constant did at a value p<0.0001. Traditional metrics that are commonly used within the bone fracture healing research community were not found to be statistically significant

Culture of human bone marrow-derived mesenchymal stem cells on of poly(L-lactic acid) scaffolds: potential application for the tissue engineering of cartilage

Due to the attractive properties of poly(l-lactic acid) (PLLA) for tissue engineering, the aim was to determine the growth and differentiation capacity of mesenchymal stromal cells (MSCs) in PLLA scaffolds and their potential use in the treatment of cartilage diseases. MSCs were cultured in PLLA films and thin porous membranes to study adherence and proliferation. Permeability and porosity were determined for the different scaffolds employed. The optimal conditions for cell seeding were first determined, as well as cell density and distribution inside the PLLA. Scaffolds were then maintained in expansion or chondrogenic differentiation media for 21 days. Apoptosis, proliferation and chondrogenic differentiation was assessed after 21 days in culture by immunohistochemistry. Mechanical characteristics of scaffolds were determined before and after cell seeding. MSCs uniformly adhered to PLLA films as well as to porous membranes. Proliferation was detected only in monolayers of pure PLLA, but was no longer detected after 10 days. Mechanical characterization of PLLA scaffolds showed differences in the apparent compression elastic modulus for the two sizes used. After determining high efficiencies of seeding, the production of extracellular matrix (ECM) was determined and contained aggrecan and collagens type I and X. ECM produced by the cells induced a twofold increase in the apparent elastic modulus of the composite. Biocompatible PLLA scaffolds have been developed that can be efficiently loaded with MSCs. The scaffold supports chondrogenic differentiation and ECM deposition that improves the mechanics of the scaffold. Although this improvement does not met the expectations of a hyaline-like cartilage ECM, in part due to the lack of a mechanical stimulation, their potential use in the treatment of cartilage pathologies encourages to improve the mechanical component.This work has been supported by the Spanish Ministry of Science and Innovation DPI2010-20399-C04-00 project and Instituto de Salud Carlos III RETIC RD06/0014.Izal, I.; Aranda, P.; Sanz Ramos, P.; Ripalda, P.; Mora, G.; Granero Molto, F.; Deplaine, H.... (2013). Culture of human bone marrow-derived mesenchymal stem cells on of poly(L-lactic acid) scaffolds: potential application for the tissue engineering of cartilage. 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Periosteum-derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical-size defect rat model

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical‐size defect was created in Sprague–Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP‐2 in collagen scaffold (CSBMP2); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum‐derived MSCs (PCLPMSCs) and PCL containing bone marrow‐derived MSCs (PCLBMSCs). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)‐transgenic rats. CTL group did not show any signs of healing during the radiological follow‐up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2, and PCLPMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design