Rapid Heterotrophic Ossification with Cryopreserved Poly (ethylene glycol-) Microencapsulated BMP2-Expressing MSCs (original) (raw)
Polymers for Advanced Technologies, 2002
We have previously shown that genetically engineered adult mesenchymal stem cells (AMSCs) expressing recombinant human bone morphogenetic protein ±2 (rhBMP-2), under tet-regulation, can induce bone formation and regeneration. We showed that these cells induce bone formation via paracrine and autocrine effect of the secreted rhBMP-2 protein. To distinguish between these two effects, and to develop a platform for continuous delivery of rhBMP-2 by engineered cells protected from the immune system, we have used hydrogel polymer (alginate) in order to encapsulate the AMSCs. Mixing of the cells with potassium alginate, followed by sedimentation in Ca 2 solution, results in polymerization of the alginate around the cells, forming microcapsules composed of a membrane allowing diffusion of small molecule and proteins. Encapsulated engineered AMSCs were able to survive inside the capsules in vitro and in vivo and secrete rhBMP-2 under tet-regulation. Transplantation of capsules both subcutaneously and into bone defect elicited physiological response manifested in osteogenic tissue composed of bone trabeculae and cartilage. Inside the capsules, engineered AMSCs differentiated to chondrocytes (autocrine effect), but not to osteoblasts. Newly formed bone has developed around the polymeric external layer without any observed intermediate layer of tissue. There was no evidence of immune response in the transplants area. We therefore conclude that engineered AMSCs can be efficiently encapsulated within polymeric alginate microcapsules, maintain viability, differentiate by autocrine effect, secrete rhBMP-2 under exogenous regulation, and induce bone formation by paracrine effect, with no adverse or immune response to the transplanted capsules.
Biomaterials, 2008
In this study, we investigated the in vitro and in vivo biological activities of bone morphogenetic protein 2 (BMP-2) released from four sustained delivery vehicles for bone regeneration. BMP-2 was incorporated into (1) a gelatin hydrogel, (2) poly(lactic-co-glycolic acid) (PLGA) microspheres embedded in a gelatin hydrogel, (3) microspheres embedded in a poly(propylene fumarate) (PPF) scaffold and (4) microspheres embedded in a PPF scaffold surrounded by a gelatin hydrogel. A fraction of the incorporated BMP-2 was radiolabeled with 125 I to determine its in vitro and in vivo release profiles. The release and bioactivity of BMP-2 were tested weekly over a period of 12 weeks in preosteoblast W20-17 cell line culture and in a rat subcutaneous implantation model. Outcome parameters for in vitro and in vivo bioactivities of the released BMP-2 were alkaline phosphatase (AP) induction and bone formation, respectively. The four implant types showed different in vitro release profiles over the 12-week period, which changed significantly upon implantation. The AP induction by BMP-2 released from gelatin implants showed a loss in bioactivity after 6 weeks in culture, while the BMP-2 released from the other implants continued to show bioactivity over the full 12-week period. Micro-CT and histological analysis of the delivery vehicles after 6 weeks of implantation showed significantly more bone in the microsphere/PPF scaffold composites (Implant 3, p < 0.02). After 12 weeks, the amount of newly formed bone in the microsphere/PPF scaffolds remained significantly higher than that in the gelatin and microsphere/gelatin hydrogels (p < 0.001), however, there was no statistical difference compared to the microsphere/PPF/gelatin composite. Overall, the results from this study show that BMP-2 could be incorporated into various bone tissue engineering composites for sustained release over a prolonged period of time with retention of bioactivity.
Differentiation of preosteoblasts using a delivery system with BMPs and bioactive glass microspheres
Journal of Materials Science: Materials in Medicine, 2007
Bone morphogenetic proteins (BMPs) and 45S5 Bioglass R microspheres (bioactive GM) can increase the differentiation of osteoblasts. Recombinant human BMP-2 (rhBMP-2) is presently the BMP most frequently used in delivery systems and it has already been used in clinical bone healing studies. We have developed a delivery system that combines a collagen Type I gel, BMP and bioactive GM. Since BMP-9 seems to be more osteogenic than BMP-2, we compared the differentiation of MC3T3-E1 preosteoblasts induced by our delivery system containing either a peptide derived from BMP-9 (pBMP-9), or rhBMP-2, both at 100 ng/mL. After 5 days, alkaline phosphatase staining showed that pBMP-9 induced more differentiation than rhBMP-2 in all experimental conditions. Also, bioactive GM increased this BMP effect. Since preosteoblasts secreted matrix metalloproteinases (MMPs) that can degrade collagen, we then studied the influence of the delivery system on MMPs production. We observed that MMP-2 was the major MMP involved in all experimental conditions. In addition, pBMP-9 with bioactive GM generated less MMP-2 than did rhBMP-2 on days 3 and 5. Thus, a delivery system using collagen Type I gel with pBMP-9 and bioactive GM seems to be a promising system for bone regeneration.
Material-related effects of BMP2 delivery systems on bone regeneration
Acta Biomaterialia
Material-related effects of a brushite and a PLGA controlled release system loaded with two distinct doses of bone morphogenetic protein-2 (BMP-2) (3.5 and 17.5 lg), pre-encapsulated in poly(lactic-co-glycolic acid) (PLGA), were investigated in an intramedullary femur defect model in rabbits. The systems were characterized in vitro and in vivo over 12 weeks in terms of morphology, release kinetics, porosity, molecular weight, and composition using scanning electron microscopy, mercury porosimetry, radioactivity counting, X-ray diffractometry, differential scanning calorimetry, and gel permeation chromatography. During the experimental period the investigated systems underwent significant changes in vitro as well as in vivo. It should be stressed that the two in vitro release patterns were similar, however in vivo parallel profiles were observed with a higher burst effect for BMP-2 in the PLGA system. The PLGA system degraded and disintegrated significantly faster than the brushite system, which suffered slowly progressing external erosion and, additionally, material resorption by osteoclasts in vivo. The consequences of this were reflected in the degree of bone regeneration. Although a sustained delivery of BMP-2 was achieved with both systems, the brushite construct, independent of the loaded growth factor dose, failed to consistently induce defect repair, a result attributed to its slow resorption rate. In contrast, the PLGA system resulted in complete regeneration with mature trabecular bone formation 8 weeks after implantation.
Advanced Pharmaceutical Bulletin
Introduction: Insoluble fibronectin as an extracellular matrix (ECM) protein has the potential to promote proliferation, differentiation, and migration of mesenchymal stem cells (MSCs). However, there is limited information about the effects of fibronectin various concentrations on bone marrow-derived MSCs (BMMSCs) function and differentiation. Materials and Methods: In this experimental study, using a gel injection device, BMMSCs were encapsulated in sodium alginate microcapsules containing 1.25% alginate, 1% gelatin, and four different concentrations of fibronectin (0.01, 0.05, 0.1, and 0.2 µg/ml). MTT assay was used to examine the proliferation of BMMSCs in des. Also, BMMSCs apoptosis rates were calculated using Annexin-V/PI staining and FACS analysis within 48 hours of exposure. Alkaline phosphatase (ALP) test was conducted to assess BMMSCs osteogenic differentiation. Finally, mRNA expression levels of the SP7, osteocalcin (OCN), Twist Family BHLH Transcription Factor 1 (Twist1)...
Osteogenic molecules for clinical applications: improving the BMP-collagen system
Biological Research, 2013
Among the osteogenic growth factors used for bone tissue engineering, bone morphogenetic proteins (BMPs) are the most extensively studied for use in orthopaedic surgery. BMP-2 and BMP-7 have been widely investigated for developing therapeutic strategies and are the only two approved for use in several clinical applications. Due to the chemical and biological characteristics of these molecules, their authorised uses are always in combination with a carrier based on collagen type I. Although the use of these growth factors is considered safe in the short term, the very high doses needed to obtain signifi cant osteoinduction make these treatments expensive and their longterm safety uncertain, since they are highly pleiotropic and have the capacity to induce ectopic ossifi cation in the surrounding tissues. Therefore it is necessary to improve the currently used BMP-collagen system in terms of effi ciency, biosecurity and costs. There are several strategies to increase the clinical eff ectiveness of these treatments. In this review we summarize the most promising results and our related work focused on this fi eld through two diff erent approaches: i) the development of recombinant BMPs with additional features, and ii) complementing these systems with other growth factors or molecules to enhance or accelerate osteogenesis.
Tissue Engineering Part A, 2013
The repair of craniofacial bone defects is surgically challenging due to the complex anatomical structure of the craniofacial skeleton. Current strategies for bone tissue engineering using a preformed scaffold have not resulted in the expected clinical regeneration due to difficulty in seeding cells into the deep internal space of scaffold, and the inability to inject them in minimally invasive surgeries. In this study, we used the osteoconductive and mechanical properties of nano-scale calcium sulfate (nCS) and the biocompatibility of alginate to develop the injectable nCS/alginate (nCS/A) paste, and characterized the effect of this nCS/A paste loaded with bone morphogenetic protein 2 (BMP2) gene-modified rat mesenchymal stem cells (MSCs) on bone and blood vessel growth. Our results showed that the nCS/A paste was injectable under small injection forces. The mechanical properties of the nCS/A paste were increased with an increased proportion of alginate. MSCs maintained their viability after the injection, and MSCs and BMP2 gene-modified MSCs in the injectable pastes remained viable, osteodifferentiated, and yielded high alkaline phosphatase activity. By testing the ability of this injectable paste and BMP2-gene-modified MSCs for the repair of critical-sized calvarial bone defects in a rat model, we found that BMP2-gene-modified MSCs in nCS/A (nCS/A + M/B2) showed robust osteogenic activity, which resulted in consistent bone bridging of the bone defects. The vessel density in nCS/A + M/B2 was significantly higher than that in the groups of blank control, nCS/A alone, and nCS/A mixed with MSCs (nCS/A + M). These results indicate that BMP2 promotes MSCs-mediated bone formation and vascularization in nCS/A paste. Overall, the results demonstrated that the combination of injectable nCS/A paste and BMP2-gene-modified MSCs is a new and effective strategy for the repair of bone defects.
Title Osteogenic molecules for clinical applications: improving the BMP-collagen system
Among the osteogenic growth factors used for bone tissue engineering, bone morphogenetic proteins (BMPs) are the most extensively studied for their use in orthopaedic surgery. BMP-2 and BMP-7 have been widely investigated for developing therapeutic strategies and are the only two approved for their use in several clinical applications. Due to the chemical and biological characteristics of these molecules, their authorised uses are always in combination with a carrier based on collagen type I. Although the use of these growth factors is considered safe in short terms, the very high doses needed to obtain significant osteoinduction make these treatments expensive and their safety uncertain at long term, since they are highly pleiotropic and have the capacity to induce ectopic ossification in the surrounding tissues. Therefore it is necessary to improve the currently used BMP-collagen system in terms of efficiency, biosecurity and costs. There are several strategies to increase the clinical effectiveness of these treatments. In this review, we summarize the most promising and our related work focused on this field, through two different approaches: i) the development of recombinant BMPs with additional features, and ii) complementing these systems with other growth factors or molecules to enhance or accelerate osteogenesis.
PLoS ONE, 2013
Current clinical therapies for critical-sized bone defects (CSBDs) remain far from ideal. Previous studies have demonstrated that engineering bone tissue using mesenchymal stem cells (MSCs) is feasible. However, this approach is not effective for CSBDs due to inadequate vascularization. In our previous study, we have developed an injectable and porous nano calcium sulfate/alginate (nCS/A) scaffold and demonstrated that nCS/A composition is biocompatible and has proper biodegradability for bone regeneration. Here, we hypothesized that the combination of an injectable and porous nCS/A with bone morphogenetic protein 2 (BMP2) gene-modified MSCs and endothelial progenitor cells (EPCs) could significantly enhance vascularized bone regeneration. Our results demonstrated that delivery of MSCs and EPCs with the injectable nCS/ A scaffold did not affect cell viability. Moreover, co-culture of BMP2 gene-modified MSCs and EPCs dramatically increased osteoblast differentiation of MSCs and endothelial differentiation of EPCs in vitro. We further tested the multifunctional bone reconstruction system consisting of an injectable and porous nCS/A scaffold (mimicking the nano-calcium matrix of bone) and BMP2 genetically-engineered MSCs and EPCs in a rat critical-sized (8 mm) caviarial bone defect model. Our in vivo results showed that, compared to the groups of nCS/A, nCS/A+MSCs, nCS/A+MSCs+EPCs and nCS/A+BMP2 gene-modified MSCs, the combination of BMP2 gene -modified MSCs and EPCs in nCS/A dramatically increased the new bone and vascular formation. These results demonstrated that EPCs increase new vascular growth, and that BMP2 gene modification for MSCs and EPCs dramatically promotes bone regeneration. This system could ultimately enable clinicians to better reconstruct the craniofacial bone and avoid donor site morbidity for CSBDs.