Benjamin M. Wu

Adipose-derived adult stromal cells heal critical-size mouse calvarial defects

In adults and children over two years of age, large cranial defects do not reossify successfully, posing a substantial biomedical burden. The osteogenic potential of bone marrow stromal (BMS) cells has been documented. This study investigates the in vivo osteogenic capability of adipose-derived adult stromal (ADAS) cells, BMS cells, calvarial-derived osteoblasts and dura mater cells to heal critical-size mouse calvarial defects. Implanted, apatite-coated, PLGA scaffolds seeded with ADAS or BMS cells produced significant intramembranous bone formation by 2 weeks and areas of complete bony bridging by 12 weeks as shown by X-ray analysis, histology and live micromolecular imaging. The contribution of implanted cells to new bone formation was 84–99% by chromosomal detection. These data show that ADAS cells heal critical-size skeletal defects without genetic manipulation or the addition of exogenous growth factors.

Three-dimensional electrospun ECM-based hybrid scaffolds for cardiovascular tissue engineering.

Electrospinning using natural proteins or synthetic polymers is a promising technique for the fabrication of fibrous scaffolds for various tissue engineering applications. However, one limitation of scaffolds electrospun from natural proteins is the need to cross-link with glutaraldehyde for stability, which has been postulated to lead to many complications in vivo including graft failure. In this study, we determined the characteristics of hybrid scaffolds composed of natural proteins including collagen and elastin, as well as gelatin, and the synthetic polymer poly(epsilon-caprolactone) (PCL), so to avoid chemical cross-linking. Fiber size increased proportionally with increasing protein and polymer concentrations, whereas pore size decreased. Electrospun gelatin/PCL scaffolds showed a higher tensile strength when compared to collagen/elastin/PCL constructs. To determine the effects of pore size on cell attachment and migration, both hybrid scaffolds were seeded with adipose-derived stem cells. Scanning electron microscopy and nuclei staining of cell-seeded scaffolds demonstrated the complete cell attachment to the surfaces of both hybrid scaffolds, although cell migration into the scaffold was predominantly seen in the gelatin/PCL hybrid. The combination of natural proteins and synthetic polymers to create electrospun fibrous structures resulted in scaffolds with favorable mechanical and biological properties.

Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells

Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific α actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.

Human Adipose Derived Stromal Cells Heal Critical Size Mouse Calvarial Defects

Background Human adipose-derived stromal cells (hASCs) represent a multipotent cell stromal cell type with proven capacity to differentiate along an osteogenic lineage. This suggests that they may be used to heal defects of the craniofacial or appendicular skeleton. We sought to substantiate the use of undifferentiated hASCs in the regeneration of a non-healing mouse skeletal defect. Methodology/Principal Findings Human ASCs were harvested from female lipoaspirate. Critical-sized (4 mm) calvarial defects were created in the parietal bone of adult male nude mice. Defects were either left empty, treated with an apatite coated PLGA scaffold alone, or a scaffold with human ASCs. MicroCT scans were obtained at stratified time points post-injury. Histology, in situ hybridization, and histomorphometry were performed. Near complete healing was observed among hASC engrafted calvarial defects. This was in comparison to control groups that showed little healing (*P<0.01). Human ASCs once engrafted differentiate down an osteogenic lineage, determined by qRT-PCR and histological co-expression assays using GFP labeled cells. ASCs were shown to persist within a defect site for two weeks (shown by sex chromosome analysis and quantified using Luciferase+ ASCs). Finally, rBMP-2 was observed to increase hASC osteogenesis in vitro and osseous healing in vivo. Conclusions/Significance Human ASCs ossify critical sized mouse calvarial defects without the need for pre-differentiation. Recombinant differentiation factors such as BMP-2 may be used to supplement hASC mediated repair. Interestingly, ASC presence gradually dissipates from the calvarial defect site. This study supports the potential translation for ASC use in the treatment of human skeletal defects.

Noggin Suppression Enhances in Vitro Osteogenesis and Accelerates in Vivo Bone Formation

Several investigations have demonstrated a precise balance to exist between bone morphogenetic protein (BMP) agonists and antagonists, dictating BMP signaling and osteogenesis. We report a novel approach to manipulate BMP activity through a down-regulation of the potent BMP antagonist Noggin, and examined the effects on the bone forming capacity of osteoblasts. Reduction of noggin enhanced BMP signaling and in vitro osteoblast bone formation, as demonstrated by both gene expression profiles and histological staining. The effects of noggin suppression on in vivo bone formation were also investigated using critical-sized calvarial defects in mice repaired with noggin-suppressed osteoblasts. Radiographic and histological analyses revealed significantly more bone regeneration at 2 and 4 weeks post-injury. These findings strongly support the concept of enhanced osteogenesis through a down-regulation in Noggin and suggest a novel approach to clinically accelerate bone formation, potentially allowing for earlier mobilization of patients following skeletal injury or surgical resection.

NF-κB inhibits osteogenic differentiation of mesenchymal stem cells by promoting β-catenin degradation

Mesenchymal stem cell (MSC)-based transplantation is a promising therapeutic approach for bone regeneration and repair. In the realm of therapeutic bone regeneration, the defect or injured tissues are frequently inflamed with an abnormal expression of inflammatory mediators. Growing evidence suggests that proinflammatory cytokines inhibit osteogenic differentiation and bone formation. Thus, for successful MSC-mediated repair, it is important to overcome the inflammation-mediated inhibition of tissue regeneration. In this study, using genetic and chemical approaches, we found that proinflammatory cytokines TNF and IL-17 stimulated IκB kinase (IKK)–NF-κB and impaired osteogenic differentiation of MSCs. In contrast, the inhibition of IKK–NF-κB significantly enhanced MSC-mediated bone formation. Mechanistically, we found that IKK–NF-κB activation promoted β-catenin ubiquitination and degradation through induction of Smurf1 and Smurf2. To translate our basic findings to potential clinic applications, we showed that the IKK small molecule inhibitor, IKKVI, enhanced osteogenic differentiation of MSCs. More importantly, the delivery of IKKVI promoted MSC-mediated craniofacial bone regeneration and repair in vivo. Considering the well established role of NF-κB in inflammation and infection, our results suggest that targeting IKK–NF-κB may have dual benefits in enhancing bone regeneration and repair and inhibiting inflammation, and this concept may also have applicability in many other tissue regeneration situations.

The enhancement of VEGF-mediated angiogenesis by polycaprolactone scaffolds with surface cross-linked heparin

This study investigates the effect of surface cross-linked heparin on vascular endothelial growth factor (VEGF)-mediated angiogenesis in porous polycaprolactone (PCL) scaffolds in vivo. We tested the hypothesis that VEGF delivered by scaffolds coated with a sub-micron thick layer of immobilized heparin would accelerate angiogenesis. The bioactivity of retained VEGF was confirmed by its phosphorylation of VEGF receptor-2. After 7 and 14 days of subcutaneous implantation in mice, the heparin-PCL scaffolds loaded with VEGF displayed significantly higher infiltration of blood vessels which traversed the entire scaffold thickness (2 mm). The stability and function of the newly formed vessels were confirmed by smooth muscle cell coverage and vessel perfusability, respectively. The contribution of individual components was assessed by varying the VEGF dose and heparin thickness. Prolonging the cross-linking reaction on PCL scaffolds resulted in higher heparin content, thicker heparin layer, and higher VEGF retention. While a dose dependent angiogenic response was observed with VEGF, higher amount of cross-linked heparin did not translate into additional improvement in angiogenesis for a given dose of VEGF. The synergism of immobilized heparin and VEGF in stimulating angiogenesis was observed in vivo.

Biomimetic apatite-coated alginate/chitosan microparticles as osteogenic protein carriers.

Bone morphogenetic proteins (BMPs) are currently approved for spinal fusion, tibial fracture repair, and maxillofacial bone regeneration. However, BMP pleiotropism, paradoxical activities on precursor cells, and unexpected side effects at local and ectopic sites may limit their usage. Thus, the need remains for alternative osteoinductive factors that provide more bone-specific activities with fewer adverse effects. Nell-1 [Nel-like molecule-1; Nel (a protein highly expressed in neural tissue encoding epidermal growth factor like domain)] is a novel osteogenic protein believed to specifically target cells committed to the osteogenic lineage. The objective of this project is to incorporate Nell-1 into a moldable putty carrier that can adapt to bony defects and deliver Nell-1 to the local microenvironment. We show here that moldability can be achieved by mixing hyaluronan hydrogel with two types of particles: demineralized bone powder for osteoconductivity, and biomimetic apatite-coated alginate/chitosan microparticles for controlled Nell-1 delivery. Besides enhancing overall osteoconductivity of the carrier, the biomimetic apatite coating also provides a more sustained release (approximately 15% cumulative release over 30 days) and greatly reduces the initial burst release that is observed with non-coated alginate/chitosan microparticles (approximately 40% release after 1 day). The efficacy of Nell-1 delivery from these carriers was evaluated in a rat spinal fusion model against Nell-free carriers as controls. At 4 weeks post-implantation, Nell-1 enhanced spinal fusion rates as assessed by manual palpation, radiographs, high-resolution micro-computerized tomography (microCT), and histology. This moldable putty carrier system appears to be a suitable carrier for promoting osteogenesis, and will be further evaluated in larger animal models over longer periods to follow the remodeling of the regenerated bone.

Evolving Concepts in Bone Tissue Engineering

The field of tissue engineering integrates the latest advances in molecular biology, biochemistry, engineering, material science, and medical transplantation. Researchers in the developing field of regenerative medicine have identified bone tissue engineering as an attractive translational target. Clinical problems requiring bone regeneration are diverse, and no single regeneration approach will likely resolve all defects. Recent advances in the field of tissue engineering have included the use of sophisticated biocompatible scaffolds, new postnatal multipotent cell populations, and the appropriate cellular stimulation. In particular, synthetic polymer scaffolds allow for fast and reproducible construction, while still retaining biocompatible characteristics. These criteria relate to the immediate goal of determining the ideal implant. The search is becoming a reality with widespread availability of biocompatible scaffolds; however, the desired parameters have not been clearly defined. Currently, most research focuses on the use of bone morphogenetic proteins (BMPs), specifically BMP-2 and BMP-7. These proteins induce osteogenic differentiation in vitro, as well as bone defect healing in vivo. Protein-scaffold interactions that enhance BMP binding are of the utmost importance, since prolonged BMP release creates the most osteogenic microenvironment. Transition into clinical studies has had only mild success and relies on large doses of BMPs for bone formation. Advances within the field of bone tissue engineering will likely overcome these challenges and lead to more clinically relevant therapies.

Effect of scaffold architecture and pore size on smooth muscle cell growth

Tissue engineering has the potential to replace damaged tissues and organs. Diffusion limitation of cell growth in three-dimensional (3D) scaffolds is a significant constraint in most tissue engineering applications. This study describes a scaffold architecture that improves mass transfer. Scaffolds with three different geometries of villi architecture (0.5, 1, 0.5; 0.5, 1, 1; 1, 1, 1 mm; villus diameter, height, intervillus spacing, respectively) were fabricated by indirect 3D printing technique. The ability of these scaffolds to support smooth muscle cell growth was investigated in vitro. Smooth muscle cells attached to the scaffolds uniformly after 1 day of culture, and the cell density in the scaffold with small villi feature (0.5 mm) was significantly higher as compared to that for the scaffold with large villi features (1 mm) after 14 days of culture. To evaluate the effect of scaffold pore size on cell growth, scaffolds with three different pore size ranges (50-100, 100-150, and 150-200 microm) were fabricated by the solvent casting and particulate leaching technique. Scaffold pore size did not significantly affect cell growth after 14 days of culture. Optimization in the architectural design of scaffolds provides an alternative method to improve diffusion limitation in the 3D constructs.