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Nonunion following bone fracture and segmental bone defects are challenging clinical conditions. To combat this clinical dilemma, development of new bone tissue engineering therapies using biocompatible materials to deliver bone growth factors is desirable. This aim of this study is to use a heparin/polycation coacervate sustained-release platform to compare 5 bone morphogenetic proteins (BMPs) for promoting bone defect healing in a critical sized calvarial defect model. The in vitro 3D osteogenic pellet cultures assays demonstrated that BMPs 2, 4, 6, 7 and 9 all enhanced mineralization in vitro compared to the control group. BMP2 resulted in higher mineralized volume than BMP4 and BMP6. All BMPs and the control group activated the pSMAD5 signaling pathway and expressed osterix (OSX). The binding of BMP2 with coacervate significantly increased the coacervate average particle size. BMP2, 4, 6, & 7 bound to coacervate significantly increased the Zeta potential of the coacervate while BMP9 binding showed insignificant increase. Furthermore, using a monolayer culture osteogenic assay, it was found that hMDSCs cultured in the coacervate BMP2 osteogenic medium expressed higher levels of RUNX2, OSX, ALP and COX-2 compared to the control and BMPs 4, 6, 7 & 9. Additionally, the coacervate complex can be loaded with up to 2 μg of BMP proteins for sustained release. In vivo, when BMPs were delivered using the coacervate sustained release system, BMP2 was identified to be the most potent BMP promoting bone regeneration and regenerated 10 times of new bone than BMPs 4, 6 & 9. BMP7 also stimulated robust bone regeneration when compared to BMPs 4, 6 & 9. The quality of the newly regenerated bone by all BMPs delivered by coacervate is equivalent to the host bone consisting of bone matrix and bone marrow with normal bone architecture. Although the defect was not completely healed at 6 weeks, coacervate sustain release BMPs, particularly BMP2 and BMP7, could represent a new strategy for treatment of bone defects and non-unions.

A variety of techniques have been applied to generate tissue engineered constructs, where cells are combined with degradable scaffolds followed by a period of in vitro culture or direct implantation. In the current study, a cellularized scaffold was generated by concurrent deposition of electrospun biodegradable elastomer (poly(ester urethane)urea, PEUU) and electrosprayed culture medium + skeletal muscle-derived stem cells (MDSCs) or electrosprayed culture medium alone as a control. MDSCs were obtained from green fluorescent protein (GFP) transgenic rats. The created scaffolds were implanted into allogenic strain-matched rats to replace a full thickness abdominal wall defect. Both control and MDSC-integrated scaffolds showed extensive cellular infiltration at 4 and 8 wk. The number of blood vessels was higher, the area of residual scaffold was lower, number of multinucleated giant cells was lower and area of connective tissue was lower in MDSC-integrated scaffolds (p

Myocardial infarction (MI) causes myocardial necrosis, triggers chronic inflammatory responses, and leads to pathological remodeling. Controlled delivery of a combination of angiogenic and immunoregulatory proteins may be a promising therapeutic approach for MI. We investigated the bioactivity and therapeutic potential of an injectable, heparin-based coacervate co-delivering an angiogenic factor, fibroblast growth factor-2 (FGF2), and an anti-inflammatory cytokine, Interleukin-10 (IL-10) in a spatially and temporally controlled manner. Coacervate delivery of FGF2 and IL-10 preserved their bioactivities on cardiac stromal cell proliferation in vitro. Upon intramyocardial injection into a mouse MI model, echocardiography revealed that FGF2/IL-10 coacervate treated groups showed significantly improved long-term LV contractile function and ameliorated LV dilatation. FGF2/IL-10 coacervate substantially augmented LV myocardial elasticity. Additionally, FGF2/IL-10 coacervate notably enhanced long-term revascularization, especially at the infarct area. In addition, coacervate loaded with 500 ng FGF2 and 500 ng IL-10 significantly reduced LV fibrosis, considerably preserved infarct wall thickness, and markedly inhibited chronic inflammation at the infarct area. These results indicate that FGF2/IL-10 coacervate has notably greater therapeutic potential than coacervate containing only FGF2. Overall, our data suggest therapeutically synergistic effects of FGF-2/IL-10 coacervate, particularly coacervate with FGF2 and 500 ng IL-10, for the treatment of ischemic heart disease.

Ruptured human anterior cruciate ligaments (ACL) contain vascular stem cells capable of enhancing the healing of tendon grafts. In the current study we explored the role that neo-angiogenesis plays in ACL healing. ACL-derived CD34+ cells were isolated via Fluorescence Activated Cell Sorting (FACS) from the rupture sites of human ACLs. The cells were then virally transduced to express either vascular endothelial growth factor (VEGF) or soluble FLT-1 (sFLT-1), which is an antagonist of VEGF. We established five groups: CD34+VEGF(100%), where 100% of the cells were transduced with VEGF, CD34+VEGF(25%), where only 25% of the cells were transduced with VEGF, CD34+, CD34+sFLT-1, and a No cells group. The CD34+sFLT1 group had a significant reduction in biomechanical strength compared to the CD34+ group at 4 and 8 weeks; whereas the biomechanical strength of the CD34+VEGF(25%) group was significantly greater than the CD34+ group at week 4; however, no difference was observed by week 8. Immunohistochemical staining demonstrated a significantly lower number of isolectin B4 and hCD31 positive cells, markers associated with angiogenesis, in the CD34+sFLT1 group, and a higher number of isolectin B4 and hCD31 positive cells in the CD34+VEGF(100%) and CD34+VEGF(25%) groups compared to the CD34+ group. Graft maturation was significantly delayed in the CD34+sFLT1 group and accelerated in the CD34+VEGF(25%) group compared to the CD34+ group. In conclusion, blocking VEGF reduced angiogenesis, graft maturation and biomechanical strength following ACL reconstruction. Native expression of VEGF by the CD34+ cells improved tendon graft maturation and biomechanical strength; however, over-expression of VEGF impeded improvements in biomechanical strength.

Effective delivery of exogenous angiogenic growth factors can provide a new therapy for ischemic diseases. However, clinical translation of growth factor therapies faces multiples challenges; the most significant one is the short half-life of the naked protein. We use heparin and a nontoxic polycation to form an injectable coacervate that protects growth factors and preserves their bioactivities. Here we report the effectiveness of fibroblast growth factor-2 (FGF2) coacervate in reducing scar burden in a mouse myocardial infarction model. The coacervate provides spatial and temporal control of the release of heparin-binding proteins. Coacervate treated animals show lower level of inflammation, fibrosis and cardiomyocyte death in the infarcted myocardium. Histological evaluation indicates that FGF2 coacervate significantly increases the number of endothelial and mural cells and results in stable capillaries and arterioles to at least 6 weeks post injection. Echocardiographic assessment shows that FGF2 coacervate promotes cardiac contractibility and inhibits ventricular dilation, suggesting that the improvement at the tissue level leads to better cardiac functions. On the contrary, identical dosage of free FGF2 shows no statistical difference from saline or vehicle control in histological or functional assessment. Overall, injection of FGF2 coacervate ameliorated the ischemic injury caused by myocardial infarction. The promising data in rodent warrant further examination of the potential of clinical translation of this technology.

There is a clinical need for a tissue-engineered vascular graft (TEVG), and combining stem cells with biodegradable tubular scaffolds appears to be a promising approach. The goal of this study was to characterize the incorporation of muscle-derived stem cells (MDSCs) within tubular poly(ester urethane) urea (PEUU) scaffolds in vitro to understand their interaction, and to evaluate the mechanical properties of the constructs for vascular applications. Porous PEUU scaffolds were seeded with MDSCs using our recently described rotational vacuum seeding device, and cultured inside a spinner flask for 3 or 7 days. Cell viability, number, distribution and phenotype were assessed along with the suture retention strength and uniaxial mechanical behavior of the TEVGs. The seeding device allowed rapid even distribution of cells within the scaffolds. After 3 days, the constructs appeared completely populated with cells that were spread within the polymer. Cells underwent a population doubling of 2.1-fold, with a population doubling time of 35 h. Stem cell antigen-1 (Sca-1) expression by the cells remained high after 7 days in culture (77+/-20% vs. 66+/-6% at day 0) while CD34 expression was reduced (19+/-12% vs. 61+/-10% at day 0) and myosin heavy chain expression was scarce (not quantified). The estimated burst strength of the TEVG constructs was 2127+/-900 mm Hg and suture retention strength was 1.3+/-0.3N. We conclude from this study that MDSCs can be rapidly seeded within porous biodegradable tubular scaffolds while maintaining cell viability and high proliferation rates and without losing stem cell phenotype for up to 7 days of in-vitro culture. The successful integration of these steps is thought necessary to provide rapid availability of TEVGs, which is essential for clinical translation.

Skeletal muscle has been recognized as an essential source of progenitor or satellite cells, which are primarily responsible for muscle regeneration. Recently, muscle has also been identified as a valuable source of postnatal stem cells that appear to be distinct from satellite cells and possess the ability to differentiate into other cell lineages. These cells, named muscle-derived stem cells, possess a high myogenic capacity and effectively regenerate both skeletal and cardiac muscle. Remarkably, when genetically modified ex vivo to express growth factors, these cells can differentiate into osteogenic and chondrogenic lineages and have been shown to promote the repair of bone and cartilage. Muscle stem-cell-based regenerative therapy and tissue engineering using ex vivo gene therapy, are promising approaches for the treatment of various musculoskeletal, cardiovascular, and urological disorders.

The formation of fibrous tissue during the healing of skeletal muscle injuries leads to incomplete recovery of the injured muscle. Platelet-rich-plasma (PRP) contains beneficial growth factors for skeletal muscle repair; however, it also contains deleterious cytokines and growth factors, such as TGF-β1, that can cause fibrosis and inhibit optimal muscle healing. Objective To test if neutralizing TGF-β1's action within PRP, through neutralization antibodies, could improve PRP's beneficial effect on skeletal muscle repair. Methods PRP was isolated from in-bred Fisher rats. TGF-β1 neutralization antibody (Ab) was used to block the TGF-β1 within the PRP prior to injection. The effects of customized PRP (TGF-β1 neutralized PRP) on muscle healing was tested on a cardiotoxin (CTX) induced muscle injury model. Results A significant increase in the numbers of regenerative myofibers was observed in the PRP and customized PRP groups compared to the untreated control. A significant decrease in collagen deposition was observed in customized PRP groups when compared to the control and PRP groups. Significantly enhanced angiogenesis and more Pax-7 positive satellite cells were found in the PRP and customized PRP groups compared to the control group. Macrophage infiltration was increased in the customized PRP groups when compared with the PRP group. More M2 macrophages were recruited to the injury site in the customized PRP groups when compared with the PRP and control groups. Conclusion Neutralizing TGF-β1 within PRP significantly promotes muscle regeneration while significantly reducing fibrosis. Not only did the neutralization reduce fibrosis, it enhanced angiogenesis, prolonged satellite cell activation, and recruited a greater number of M2 macrophages to the injury site which also contributed to the efficacy that the customized PRP had on muscle healing.

Adult multipotent stem cells have been isolated from a variety of human tissues including human skeletal muscle, which represent an easily accessible source of stem cells. It has been shown that human skeletal muscle-derived stem cells (hMDSCs) are muscle-derived mesenchymal stem cells capable of multipotent differentiation. Although hMDSCs can undergo osteogenic differentiation and form bone when genetically modified to express BMP2; it is still unclear whether hMDSCs are as efficient as human bone marrow mesenchymal stem cells (hBMMSCs) for bone regeneration. The current study aimed to address this question by performing a parallel comparison between hMDSCs and hBMMSCs to evaluate their osteogenic and bone regeneration capacities. Our results demonstrated that hMDSCs and hBMMSCs had similar osteogenic-related gene expression profiles and had similar osteogenic differentiation capacities in vitro when transduced to express BMP2. Both the untransduced hMDSCs and hBMMSCs formed very negligible amounts of bone in the critical sized bone defect model when using a fibrin sealant scaffold; however, when genetically modified with lenti-BMP2, both populations successfully regenerated bone in the defect area. No significant differences were found in the newly formed bone volumes and bone defect coverage between the hMDSC and hBMMSC groups. Although both cell types formed mature bone tissue by 6 weeks post-implantation, the newly formed bone in the hMDSCs group underwent quicker remodelling than the hBMMSCs group. In conclusion, our results demonstrated that hMDSCs are as efficient as hBMMSCs in terms of their bone regeneration capacity; however, both cell types required genetic modification with BMP in order to regenerate bone in vivo.

As a new strategy for enhancing recovery after anterior cruciate ligament (ACL) reconstruction, we developed a cell sheet wrapped graft using ACL-derived CD34+ cells. Our results from an ex vivo cell migration assay demonstrated gradual cell migration into the graft from the cell sheet, and an enzyme-linked immunosorbent assay indicated that the cell sheet cultures secreted a significantly greater amount of VEGF than typical monolayer cultures. In the in vivo study, the rats received one of three possible ACL reconstruction treatments; 1) a tendon graft wrapped in an ACL-derived CD34+ cell sheet, 2) a tendon graft and ACL-derived CD34+ cell injection, or 3) the control group that received just the tendon graft. The in vivo studies revealed that the ACL-derived CD34+ cell sheet wrapped grafts demonstrated a greater number of the cells derived from the cell sheets incorporated within the bone tunnel site and grafted tendon. We believe the incorporated CD34+ cells enhanced the healing of both the bone-tendon junction and the grafted tendon in the reconstructed rat ACLs by increasing proprioceptive recovery, graft maturation, and biomechanical strength. ACL-derived CD34+ cell sheet wrapped grafts could become a promising strategy to revitalize tendon autografts and recovery after ACL reconstruction resulting in superior and quicker recovery in patients requiring ACL reconstruction.

The success of small-diameter tissue engineered vascular grafts (TEVGs) greatly relies on an appropriate cell source and an efficient cellular delivery and carrier system. Pericytes have recently been shown to express mesenchymal stem cell features. Their relative availability and multipotentiality make them a promising candidate for TEVG applications. The objective of this study was to incorporate pericytes into a biodegradable scaffold rapidly, densely and efficiently, and to assess the efficacy of the pericyte-seeded scaffold in vivo. Bi-layered elastomeric poly(ester-urethane)urea scaffolds (length = 10 mm; inner diameter = 1.3 mm) were bulk seeded with 3 x 10(6) pericytes using a customized rotational vacuum seeding device in less than 2 min (seeding efficiency > 90%). The seeded scaffolds were cultured in spinner flasks for 2 days and then implanted into Lewis rats as aortic interposition grafts for 8 weeks. Results showed pericytes populated the porous layer of the scaffolds evenly and maintained their original phenotype after the dynamic culture. After implantation, pericyte-seeded TEVGs showed a significant higher patency rate than the unseeded control: 100% versus 38% (p < 0.05). Patent pericyte-seeded TEVGs revealed extensive tissue remodeling with collagen and elastin present. The remodeled tissue consisted of multiple layers of alpha-smooth muscle actin- and calponin-positive cells, and a von Willebrand factor-positive monolayer in the lumen. These results demonstrate the feasibility of a pericyte-based TEVG and suggest that the pericytes play a role in maintaining patency of the TEVG as an arterial conduit.

Injectable biomaterials have been recently investigated as a therapeutic approach for cardiac repair. Porcine-derived small intestinal submucosa (SIS) material is currently used in the clinic to promote accelerated wound healing for a variety of disorders. In this study, we hypothesized that gels derived from SIS extracellular matrix would be advantageous as an injectable material for cardiac repair. We evaluated 2 forms of SIS gel, types B (SIS-B) and C (SIS-C), for their ability to provide a therapeutic effect when injected directly into ischemic myocardium using a murine model of an acute myocardial infarction. Echocardiography analysis at both 2 and 6 weeks after infarction demonstrated preservation of end-systolic left ventricular geometry and improvement of cardiac contractility in the hearts injected with SIS-B when compared with control hearts injected with saline. However, the SIS-C gel provided no functional efficacy in comparison with control. Histological analysis revealed that SIS-B reduced infarct size and induced angiogenesis relative to control, whereas injection of SIS-C had minimal effect on these histological parameters. Characterization of both gels revealed differential growth factor content with SIS-B exhibiting higher levels of basic fibroblast growth factor than SIS-C, which may explain, at least in part, the differential histological and functional results. This study suggests that SIS gel offers therapeutic potential as an injectable material for the repair of ischemic myocardium. Further understanding of SIS gel characteristics, such as biological and physical properties, that are critical determinants of efficacy would be important for optimization of this biomaterial for cardiac repair.

Bladder wall replacement remains a challenging problem for urological surgery due to leakage, infection, stone formation, and extensive time needed for tissue regeneration. To explore the feasibility of producing a more functional biomaterial for bladder reconstitution, we incorporated muscle-derived cells (MDC) into small intestinal submucosa (SIS) scaffolds. MDC were harvested from mice hindleg muscle, transfected with a plasmid encoding for beta-galactosidase, and placed into single-layer SIS cell culture inserts. Twenty-five MDC and/or SIS specimens were incubated at 37 degrees C for either 10 or 20 days. After harvesting, mechanical properties were characterized using biaxial testing, and the areal strain under 1 MPa peak stress used to quantify tissue compliance. Histological results indicated that MDC migrated throughout the SIS after 20 days. The mean (+/-SE) areal strain of the 0 day control group was 0.182 +/- 0.027 (n=5). After 10 days incubation, the mean (+/-SE) areal strain in MDC/SIS was 0.247 +/- 0.014 (n=5) compared to 10 day control SIS 0.200 +/- 0.024 (n=6). After 20 days incubation, the mean areal strain of MDC/SIS was 0.255 +/- 0.019 (n=5) compared to control SIS 0.170 +/- 0.025 (n=5). Both 10 and 20 days seeded groups were significantly different (p=0.027) than that of incubated SIS alone, but were not different from each other. These results suggest that MDC growth was supported by SIS and that initial remodeling of the SIS ECM had occurred within the first 10 days of incubation, but may have slowed once the MDC had grown to confluence within the SIS.