Jeong S. Hyun

In vivo directed differentiation of pluripotent stem cells for skeletal regeneration.

Pluripotent cells represent a powerful tool for tissue regeneration, but their clinical utility is limited by their propensity to form teratomas. Little is known about their interaction with the surrounding niche following implantation and how this may be applied to promote survival and functional engraftment. In this study, we evaluated the ability of an osteogenic microniche consisting of a hydroxyapatite-coated, bone morphogenetic protein-2–releasing poly-l-lactic acid scaffold placed within the context of a macroenvironmental skeletal defect to guide in vivo differentiation of both embryonic and induced pluripotent stem cells. In this setting, we found de novo bone formation and participation by implanted cells in skeletal regeneration without the formation of a teratoma. This finding suggests that local cues from both the implanted scaffold/cell micro- and surrounding macroniche may act in concert to promote cellular survival and the in vivo acquisition of a terminal cell fate, thereby allowing for functional engraftment of pluripotent cells into regenerating tissue.

Dura Mater Stimulates Human Adipose-Derived Stromal Cells to Undergo Bone Formation in Mouse Calvarial Defects†‡§

Human adipose‐derived stromal cells (hASCs) have a proven capacity to aid in osseous repair of calvarial defects. However, the bone defect microenvironment necessary for osseous healing is not fully understood. In this study, we postulated that the cell‐cell interaction between engrafted ASCs and host dura mater (DM) cells is critical for the healing of calvarial defects. hASCs were engrafted into critical sized calvarial mouse defects. The DM‐hASC interaction was manipulated surgically by DM removal or by insertion of a semipermeable or nonpermeable membrane between DM and hASCs. Radiographic, histologic, and gene expression analyses were performed. Next, the hASC‐DM interaction is assessed by conditioned media (CM) and coculture assays. Finally, bone morphogenetic protein (BMP) signaling from DM was investigated in vivo using novel BMP‐2 and anti‐BMP‐2/4 slow releasing scaffolds. With intact DM, osseous healing occurs both from host DM and engrafted hASCs. Interference with the DM‐hASC interaction dramatically reduced calvarial healing with abrogated BMP‐2–Smad‐1/5 signaling. Using CM and coculture assays, mouse DM cells stimulated hASC osteogenesis via BMP signaling. Through in vivo manipulation of the BMP‐2 pathway, we found that BMP‐2 plays an important role in DM stimulation of hASC osteogenesis in the context of calvarial bone healing. BMP‐2 supplementation to a defect with disrupted DM allowed for bone formation in a nonhealing defect. DM is an osteogenic cell type that both participates in and stimulates osseous healing in a hASC‐engrafted calvarial defect. Furthermore, DM‐derived BMP‐2 paracrine stimulation appears to play a key role for hASC mediated repair. STEM CELLS 2011;29:1241‐1255

Nonintegrating Knockdown and Customized Scaffold Design Enhances Human Adipose‐Derived Stem Cells in Skeletal Repair

An urgent need exists in clinical medicine for suitable alternatives to available techniques for bone tissue repair. Human adipose‐derived stem cells (hASCs) represent a readily available, autogenous cell source with well‐documented in vivo osteogenic potential. In this article, we manipulated Noggin expression levels in hASCs using lentiviral and nonintegrating minicircle short hairpin ribonucleic acid (shRNA) methodologies in vitro and in vivo to enhance hASC osteogenesis. Human ASCs with Noggin knockdown showed significantly increased bone morphogenetic protein (BMP) signaling and osteogenic differentiation both in vitro and in vivo, and when placed onto a BMP‐releasing scaffold embedded with lentiviral Noggin shRNA particles, hASCs more rapidly healed mouse calvarial defects. This study therefore suggests that genetic targeting of hASCs combined with custom scaffold design can optimize hASCs for skeletal regenerative medicine. STEM Cells 2011;29:2018–2029.

Isolation of Human Adipose-Derived Stromal Cells Using Laser-Assisted Liposuction and Their Therapeutic Potential in Regenerative Medicine

Harvesting adipose‐derived stromal cells (ASCs) for tissue engineering is frequently done through liposuction. However, several different techniques exist. Although third‐generation ultrasound‐assisted liposuction has been shown to not have a negative effect on ASCs, the impact of laser‐assisted liposuction on the quality and differentiation potential of ASCs has not been studied. Therefore, ASCs were harvested from laser‐assisted lipoaspirate and suction‐assisted lipoaspirate. Next, in vitro parameters of cell yield, cell viability and proliferation, surface marker phenotype, osteogenic differentiation, and adipogenic differentiation were performed. Finally, in vivo bone formation was assessed using a critical‐sized cranial defect in athymic nude mice. Although ASCs isolated from suction‐assisted lipoaspirate and laser‐assisted lipoaspirate both successfully underwent osteogenic and adipogenic differentiation, the cell yield, viability, proliferation, and frequency of ASCs (CD34+CD31−CD45−) in the stromal vascular fraction were all significantly less with laser‐assisted liposuction in vitro (p < .05). In vivo, quantification of osseous healing by micro‐computed tomography revealed significantly more healing with ASCs isolated from suction‐assisted lipoaspirate relative to laser‐assisted lipoaspirate at the 4‐, 6‐, and 8‐week time points (p < .05). Therefore, as laser‐assisted liposuction appears to negatively impact the biology of ASCs, cell harvest using suction‐assisted liposuction is preferable for tissue‐engineering purposes.

Enhancing stem cell survival in vivo for tissue repair.

The ability to use progenitor cells for regenerative medicine remains an evolving but elusive clinical goal. A serious obstacle towards widespread use of stem cells for tissue regeneration is the challenges that face these cells when they are placed in vivo into a wound for therapy. These environments are hypoxic, acidic, and have an upregulation of inflammatory mediators creating a region that is hostile towards cellular survival. Within this environment, the majority of progenitor cells undergo apoptosis prior to participating in lineage differentiation and cellular integration. In order to maximize the clinical utility of stem cells, strategies must be employed to increase the cells ability to survive in vivo through manipulation of both the stem cell and the surrounding environment. This review focuses on current advances and techniques being used to increase in vivo stem cell survival for the purpose of tissue regeneration.

Studies in fat grafting: Part I. Effects of injection technique on in vitro fat viability and in vivo volume retention.

Background: Fat grafting has become increasingly popular for the correction of soft-tissue deficits at many sites throughout the body. Long-term outcomes, however, depend on delivery of fat in the least traumatic fashion to optimize viability of the transplanted tissue. In this study, the authors compare the biological properties of fat following injection using two methods. Methods: Lipoaspiration samples were obtained from five female donors, and cellular viability, proliferation, and lipolysis were evaluated following injection using either a modified Coleman technique or an automated, low-shear device. Comparisons were made to minimally processed, uninjected fat. Volume retention was also measured over 12 weeks after injection of fat under the scalp of immunodeficient mice using either the modified Coleman technique or the Adipose Tissue Injector. Finally, fat grafts were analyzed histologically. Results: Fat viability and cellular proliferation were both significantly greater with the Adipose Tissue Injector relative to injection with the modified Coleman technique. In contrast, significantly less lipolysis was noted using the automated device. In vivo fat volume retention was significantly greater than with the modified Coleman technique at the 4-, 6-, 8-, and 12-week time points. This corresponded to significantly greater histologic scores for healthy fat and lower scores for injury following injection with the device. Conclusion: Biological properties of injected tissues reflect how disruptive and harmful techniques for placement of fat may be, and the authors’ in vitro and in vivo data both support the use of the automated, low-shear devices compared with the modified Coleman technique.

Enhancement of Human Adipose-Derived Stromal Cell Angiogenesis through knockdown of a BMP-2 inhibitor

Background: Previous studies have demonstrated the role of noggin, a bone morphogenetic protein-2 inhibitor, in vascular development and angiogenesis. The authors hypothesized that noggin suppression in human adipose-derived stromal cells would enhance vascular endothelial growth factor secretion and angiogenesis in vitro and in vivo to a greater extent than bone morphogenetic protein-2 alone. Methods: Human adipose-derived stromal cells were isolated from human lipoaspirate (n = 6) noggin was knocked down using lentiviral techniques. Knockdown was confirmed and angiogenesis was assessed by tubule formation and quantitative real-time polymerase chain reaction. Cells were seeded onto scaffolds and implanted into a 4-mm critical size calvarial defect. In vivo angiogenic signaling was assessed by immunofluorescence and immunohistochemistry. Results: Human adipose-derived stromal cells with noggin suppression secreted significantly higher amounts of angiogenic proteins, expressed higher levels of angiogenic genes, and formed more tubules in vitro. In vivo, calvarial defects seeded with noggin shRNA human adipose-derived stromal cells exhibited a significantly higher number of vessels in the defect site than controls by immunohistochemistry (p < 0.05). In addition, bone morphogenetic protein-2–releasing scaffolds significantly enhanced vascular signaling in the defect site. Conclusions: Human adipose-derived stromal cells demonstrate significant increases in angiogenesis in vitro and in vivo with both noggin suppression and BMP-2 supplementation. By creating a cell with noggin suppressed and by using a scaffold with increased bone morphogenetic protein-2 signaling, a more angiogenic niche can be created.

Femtosecond plasma mediated laser ablation has advantages over mechanical osteotomy of cranial bone

Although mechanical osteotomies are frequently made on the craniofacial skeleton, collateral thermal, and mechanical trauma to adjacent bone tissue causes cell death and may delay healing. The present study evaluated the use of plasma‐mediated laser ablation using a femtosecond laser to circumvent thermal damage and improve bone regeneration.

Models of cranial suture biology.

AbstractCraniosynostosis is a common congenital defect caused by premature fusion of cranial sutures. The severe morphologic abnormalities and cognitive deficits resulting from craniosynostosis and the potential morbidity of surgical correction espouse the need for a deeper understanding of the complex etiology for this condition. Work in animal models for the past 20 years has been pivotal in zadvancing our understanding of normal suture biology and elucidating pathologic disease mechanisms. This article provides an overview of milestone studies in suture development, embryonic origins, and signaling mechanisms from an array of animal models including transgenic mice, rats, rabbits, fetal sheep, zebrafish, and frogs. This work contributes to an ongoing effort toward continued development of novel treatment strategies.

Pierre Robin sequence and Treacher Collins hypoplastic mandible comparison using three-dimensional morphometric analysis.

AbstractPierre Robin sequence and Treacher Collins syndrome are both associated with mandibular hypoplasia. It has been hypothesized, however, that the mandible may be differentially affected. The purpose of this study was to therefore compare mandibular morphology in children with Pierre Robin sequence with children with Treacher Collins syndrome using three-dimensional analysis of computed tomographic scans. A retrospective analysis was performed identifying children with Pierre Robin sequence and Treacher Collins syndrome undergoing computed tomography. Three-dimensional reconstruction was performed, and ramus height, mandibular body length, and gonial angle were measured. These were then compared with those in control children with normal mandibles and with the clinical norms corrected for age and sex based on previously published measurements. Mandibular body length was found to be significantly shorter for children with Pierre Robin sequence, whereas ramus height was significantly shorter for children with Treacher Collins syndrome. This resulted in distinctly different ramus height–mandibular body length ratios. In addition, the gonial angle was more obtuse in both the Pierre Robin sequence and Treacher Collins syndrome groups compared with the controls. Three-dimensional mandibular morphometric analysis in patients with Pierre Robin sequence and Treacher Collins syndrome thus revealed distinctly different patterns of mandibular hypoplasiarelative to normal controls. These findings underscore distinct considerations that must be made in surgical planning for reconstruction.