David E. Sahar

Applications of a mouse model of calvarial healing: Differences in regenerative abilities of juveniles and adults

Young children are capable of healing large calvarial defects, whereas adults lack this endogenous osseous tissue-engineering capacity. Despite the important clinical implications, little is known about the molecular and cell biology underlying this differential ability. Traditionally, guinea pig, rabbit, and rat models have been used to study the orchestration of calvarial healing. To harness the research potential of knockout and transgenic mice, the authors developed a mouse model for calvarial healing. Nonsuture-associated parietal defects 3, 4, and 5 mm in diameter were made in both juvenile (6-day-old, n = 15) and adult (60-day-old, n = 15) mice. Calvariae were harvested after 8 weeks and analyzed radiographically and histologically. Percentage of healing was quantified using Scion Image software analysis of calvarial radiographs. A significant difference in the ability to heal calvarial defects was seen between 6-day-old and 60-day-old mice when 3-, 4-, or 5-mm defects were created. The authors’ analysis revealed that juvenile mice healed a significantly greater percentage of their calvarial defects than adult mice (juvenile mean percentage of healing: 3-mm defects, 59 percent; 4-mm defects, 65 percent; 5-mm defects, 44 percent; adult mean percentage of healing: <5 percent in all groups; p < 0.05). All three defect sizes were found to be critical in the adult, whereas significant healing was seen regardless of the size of the defect in juvenile mice. The establishment of this model will facilitate further, detailed evaluation of the molecular biology underlying the different regenerative abilities of juvenile versus adult mice and enhance research into membranous bone induction by making available powerful tools such as knockout and transgenic animals.

Effect of Endothelial Differentiated Adipose-Derived Stem Cells on Vascularity and Osteogenesis in Poly (D, L-Lactide) Scaffolds In Vivo

Abstract Prevascularization of engineered bony constructs can potentially improve in vivo viability. However, the effect of endothelial cells on osteogenesis is unknown when placed in poly(D,L-lactide) (PLA) scaffolds alone. Adipose-derived stem cells (ASCs) have the ability to differentiate into both osteoblasts and endothelial cells by culture in specific media. We hypothesized that ASC-derived endothelial cells would improve vascularity with minimal contribution to bone formation when placed in scaffold alone. ASCs were successfully differentiated into endothelial cells (ASC-Endo) and osteoblasts (ASC-Osteo) using media supplemented with vascular endothelial growth factor and bone morphogenic protein 2, respectively. Tissue-engineered constructs were created with PLA matrices containing no cells (control), undifferentiated ASCs (ASCs), osteogenic-differentiated ASCs (ASC-Osteo), or endothelial differentiated ASCs (ASC-Endo), and these constructs were evaluated in critical-size Lewis rat calvarial defect model (n = 34). Eight weeks after implantation, the bone volume and microvessel population of bony constructs were evaluated by micro–computed tomography analysis and histologic staining. Bone volumes for ASCs and ASC-Osteo constructs, 0.7 and 0.91 mm3, respectively, were statistically greater than that for ASC-Endo, 0.28 mm3 (P < 0.05). There was no statistical difference between the PLA control (0.5 mm3) and ASC-Endo (0.28 mm3) constructs in bone formation. The percent area of microvessels within constructs was highest in the ASC-Endo group, although it did not reach statistical significance (0.065). Prevascularization of PLA scaffold with ASC-Endo cells will not increase bone formation by itself but may be used as a cell source for improving vascularization and potentially improving existing osteoblast function.

The Oncologic Safety of Breast Fat Grafting and Contradictions Between Basic Science and Clinical Studies: A Systematic Review of the Recent Literature.

AbstractFat grafting is increasingly popular and is becoming a common practice in plastic surgery for postmastectomy breast reconstruction and aesthetic breast augmentation; however, concerns over the oncologic safety remains a controversial and hot topic among scientists and surgeons. Basic science and laboratory research repeatedly show a potentially dangerous effect of adipose-derived stem cells on breast cancer cells; however, clinical research, although limited, continually fails to show an increase in breast cancer recurrence after breast fat grafting, with the exception of 1 small study on a subset patient population with intraepithelial neoplasm of the breast. The aim of this review is to summarize the recent conflicting basic science and clinical data to better understand the safety of breast fat grafting from an oncological perspective.

In vitro murine posterior frontal suture fate is age-dependent: Implications for cranial suture biology

In CD-1 mice, the posterior frontal suture (analogous to the human metopic suture) fuses while all other cranial sutures remain patent. In an in vitro organ culture model, the authors previously demonstrated that posterior frontal sutures explanted immediately before the onset of suture fusion (at 25 days old) mimic in vivo physiologic fusion. In the first portion of this study, the authors defined how early in development the posterior frontal suture fuses in their tension-free, serum-free organ culture system by serially analyzing posterior frontal suture fusion from calvariae explanted at different stages of postnatal development. Their results revealed a divergence of suture fate leading to abnormal patency or physiologic fusion between the first and second weeks of life, respectively, despite viability and continued growth of the calvarial explants in vitro. From these data, the authors postulated that the gene expression patterns present in the suture complex at the time of explant may determine whether the posterior frontal suture fuses or remains patent in organ culture. Therefore, to elucidate potentially important differences in gene expression within this “window of opportunity,” they performed a cDNA microarray analysis on 5-day-old and 15-day-old posterior frontal and sagittal whole suture complexes corresponding to the age ranges for unsuccessful (1 to 7 days old) and successful (14 to 21 days old) in vitro posterior frontal suture fusion. Overall, their microarray results reveal interesting differential expression patterns of candidate genes in different categories, including angiogenic cytokines and mechanosensitive genes potentially important in cranial suture biology.

Comparison of AlloDerm and AlloMax tissue incorporation in rats.

BackgroundHuman acellular dermal matrices (HADMs) are used in a variety of settings. AlloMax is a new HADM currently being used for breast reconstruction and hernia repair. We compared the in vivo tissue integration of AlloMax to AlloDerm, a well-studied HADM, in rats. MethodsWe implanted AlloDerm and AlloMax patches into subcutaneous pockets on the backs of 32 male Sprague-Dawley rats. The animals were killed after either 4 or 8 weeks, and the patches were recovered and stained for histopathologic analyses. Microscopic end points included patch thickness, vascularization, tissue in-growth, fibroblast proliferation, and inflammation. ResultsAll animals completed the study without complications or infection. There were no significant differences in graft thicknesses at 4 and 8 weeks. Microscopically, at 4 weeks, AlloDerm sections had significantly more microvessels than AlloMax (P = 0.02). This disparity increased by 8 weeks (P < 0.01). Similarly, we found greater tissue in-growth and fibroblast proliferation in AlloDerm than AlloMax sections at 4 (P < 0.01) and at 8 (P < 0.01) weeks. Inflammatory infiltrates consisted of lymphocytes, histiocytes, eosinophils, and plasma cells. Deep graft infiltration by predominately lymphocytic inflammatory cells was significantly higher in AlloDerm than AlloMax grafts at 4 (P = 0.01) and 8 (P = 0.02) weeks. Graft necrosis was uncommon, but marginal fibrosis was similar in both. ConclusionsAlloDerm grafts had greater neovascularization, tissue infiltration, fibroblast proliferation, and inflammatory reaction than AlloMax grafts when placed subcutaneously in rats. AlloDerm may be better incorporated than AlloMax when placed in vivo.

Donor-Derived, Liver-Specific Protein Expression after Bone Marrow Transplantation

Background. Bone marrow transplantation (BMT) may represent a novel mechanism to deliver a functional gene to a deficient liver. Bone marrow-derived hepatocytes are rare and without a defined contribution to liver function. Consequently, the clinical significance of BMT to treat liver disease is unclear. We sought to quantify bone marrow-derived hepatocyte protein expression after BMT and determine whether the process is inducible with liver injury. Methods. Mice transgenic for human alpha-1 antitrypsin (hAAT) under a hepatocyte-specific promoter were used as bone marrow donors. Adenoviral transduction of modified urokinase plasminogen activator (Ad-muPA) was used to induce liver injury. Eight weeks after lethal irradiation and BMT, recipients were stratified into two groups: BMT alone (n=5) and BMT + Ad-muPA (n=10). Both groups of animals were bled before (t=0) and at 2, 4, 8, and 16 weeks after Ad-muPA administration, and the serum samples were assessed for hAAT by enzyme-linked immunosorbent assay. Results. Transgenic donor mice expressed 5 to 10 mg/mL of hAAT. Recipients of BMT alone expressed less than 80 ng/mL of hAAT over all time periods. Animals receiving BMT + Ad-muPA showed sustained and stable hAAT expression of approximately 200 ng/mL. Differences were statistically significant at each time point. Conclusion. Serum protein levels from liver-specific transgene expression are detectable and persist after BMT. Expression is low, but inducible with liver injury. We are currently developing strategies to augment donor-derived, liver-specific protein expression after BMT.

Single-stage reconstruction of achilles tendon rupture with flexor hallucis longus tendon transfer and simultaneous free radial fasciocutaneous forearm flap.

AbstractReconstruction of the Achilles tendon is challenging but critical for successful ambulation. The goals of Achilles tendon reconstruction are to restore power of plantar flexion, restore normal range of movement of the ankle joint, and ensure durable and pliable soft tissue coverage. We present a dehiscence of the Achilles tendon and partial soft tissue loss secondary to infection. Simultaneous Achilles tendon reconstruction with flexor hallucis longus (FHL) tendon transfer and soft tissue reconstruction with free radial forearm flap was performed as a single-stage procedure. The FHL transfer provided good restoration of plantar flexion while the free radial flap provided stable coverage over the Achilles tendon allowing normal footwear. This single-stage reconstruction provides excellent functional and aesthetic results minimizing the number of procedures and patient recovery period.

Unique Modulation of Cadherin Expression Pattern during Posterior Frontal Cranial Suture Development and Closure

Cranial suture development involves coordinated expression of multiple genes and tissue contribution from neural crest cells and paraxial mesoderm for timely sutural morphogenesis. Transcription factors, growth factors, and neural crest determinant genes play critical roles in calvarial growth ensuring normal development of the underlying brain. In vitro studies have implicated cell-cell adhesion molecules as a driving force behind suture closure. We performed cDNA microarray to study differential expression of adhesion molecules during the timing of suture closure in a mouse model where only the posterior frontal (PF) suture closes. Our results indicate increased expression of E-cadherin during the period of PF suture closure. Quantitative RT-PCR analysis of E- and N-cadherin in PF closing suture revealed a biphasic expression of N-cadherin, the first phase coinciding with cellular condensation preceding chondrogenesis followed by a second phase coinciding with E-cadherin co-expression and suture closure. Furthermore, expression analysis of the N-cadherin and E-cadherin transcriptional repressors Wnt7a and Snail indicate a specific temporal regulation of these genes, suggesting their potential role as regulators of both E- and N-cadherin during the PF suture development and closure. Finally, given the in vitro evidence of fibroblast growth factor (FGF)-2 as a potential regulator of E- and N-cadherin we investigated the expression of E-cadherin during PF suture closure in Fgf-2 deficient mice. In contrast to in vitrodata previously reported, E-cadherin expression is normal in these animals, and PF suture closure occurs properly, probably due to potential redundancy of FGF ligands ensuring normal temporal expression of E-cadherin and PF suture closure.

The key components of Schwann cell-like differentiation medium and their effects on gene expression pattern of adipose-derived stem cells.

BackgroundSchwann cell-like cells differentiated from adipose-derived stem cells may have an important role in peripheral nerve regeneration. Herein, we document the individual effects of growth factors in Schwann cell-like differentiation medium. MethodsThere were 6 groups in the study. In the control group, we supplemented the rat adipose–derived stem cells with normal cell culture medium. In group 1, we fed the cells with Schwann cell-like differentiation medium (normal cell culture medium supplemented with platelet-derived growth factor, basic fibroblast growth factor, forskolin, and glial growth factor). In the other groups, we removed the components of the medium one at a time from the differentiation medium so that group 2 lacked glial growth factor, group 3 lacked forskolin, group 4 lacked basic fibroblast growth factor, and group 5 lacked platelet-derived growth factor. We examined the expression of the Schwann cell–specific genes with quantitative reverse transcription polymerase chain reaction and immunofluorescence staining in each group. ResultsGroups 3 and 4, lacking forskolin and basic fibroblast growth factor, respectively, had the highest expression levels of integrin-&bgr;4, and p75. Group 1 showed a 3.2-fold increase in the expression of S100, but the expressions of integrin-&bgr;4 and p75 were significantly lower compared to groups 3 and 4. Group 2 [glial growth factor (−)] did not express significant levels of Schwann cell–specific genes. The gene expression profile in group 4 most closely resembled Schwann cells. Immunofluorescence staining results were parallel with the quantitative real-time polymerase chain reaction results. ConclusionsGlial growth factor is a key component of Schwann cell-like differentiation medium.

Comparison of Endothelial Differentiation Capacities of Human and Rat Adipose-Derived Stem Cells.

Background: The authors compared the endothelial differentiation capacities of human and rat adipose-derived stem cells to determine whether human adipose-derived stem cells can be a source of endothelial cells clinically. Methods: Human and rat adipose-derived stem cells were harvested and characterized with flow cytometry and trilineage differentiation. Cells from passages III through V were fed with endothelial cell differentiation medium for up to 3 weeks. Cells were harvested after 1, 2, and 3 weeks, and endothelial differentiation was evaluated with quantitative reverse-transcriptase polymerase chain reaction, flow cytometry, and angiogenic sprouting assays. Results: Both human and rat adipose-derived stem cells were CD90+, CD44+, and CD31− before differentiation. The cells were successfully differentiated into adipogenic, osteogenic, and chondrogenic lineages. Expression of endothelial cell–specific genes peaked at the second week of differentiation in both human and rat cells. The fold changes in expression of CD31, vascular endothelial growth factor receptor-1, nitric oxide synthase, and von Willebrand factor genes at week 2 were 0.4 ± 0.1, 34.7 ± 0.3, 2.03 ± 0.25, and 12.5 ± 0.3 respectively, in human adipose-derived stem cells; and 1.5 ± 1.01, 21.6 ± 1.7, 17.9 ± 0.6, and 11.2 ± 1.3, respectively, in rat cells. The percentages of CD31+ cells were 0.2, 0.64, and 1.6 in human cell populations and 0.5, 5.91, and 11.5 in rat cell populations at weeks 1, 2, and 3, respectively. Rat adipose-derived stem cell–derived endothelial cells displayed enhanced sprouting capability compared with the human cells. Conclusions: Human adipose-derived stem cells responded less strongly to EGM-2MV endothelial differentiation medium than did the rat cells. Still, the human cells have the potential to become a clinical source of endothelial cells with modifications in the differentiation conditions.