Roy C. Ogle

Cell surface and transcriptional characterization of human adipose-derived adherent stromal (hADAS) cells

Adult human subcutaneous adipose tissue contains cells with intriguing multilineage developmental plasticity, much like marrow‐derived mesenchymal stem cells. Putative stem or progenitor cells from fat have been given many different names in the literature, reflecting an early and evolving consensus regarding their phenotypic characterization. The study reported here used microarrays to evaluate over 170 genes relating to angiogenesis and extracellular matrix in undifferentiated, early‐passage human adipose‐derived adherent stromal (hADAS) cells isolated from three separate donors. The hADAS populations unanimously transcribed 66% of the screened genes, and 83% were transcribed by at least two of the three populations. The most highly transcribed genes relate to functional groupings such as cell adhesion, matrix proteins, growth factors and receptors, and proteases. The transcriptome of hADAS cells demonstrated by this work reveals many similarities to published profiles of bone marrow mesenchymal stem cells (MSCs). In addition, flow analysis of over 24 hADAS cell surface proteins (n = 7 donors) both confirms and expands on the existing literature and reveals strong intergroup correlation, despite an inconsistent nomenclature and the lack of standardized protocols for cell isolation and culture. Finally, based on flow analysis and reverse transcription polymerase chain reaction studies, our results suggest that hADAS cells do not express several proteins that are implicated as markers of “stemness” in other stem cell populations, including telomerase, CD133, and the membrane transporter ABCG2.

TGF-β1, TGF-β2, and TGF-β3 Exhibit Distinct Patterns of Expression During Cranial Suture Formation and Obliteration In Vivo and In Vitro†

Cranial sutures function as bone growth centers while themselves remaining unossified. Rat frontonasal sutures become obliterated by neonatal day 21 (N21), while coronal sutures do not fuse over the life of the animal. Coronal sutures induced to undergo osseous obliteration in vitro after removal of the dura mater were found to require soluble, heparin‐binding factors present in dura mater to resist osseous obliteration. Transforming growth factor β1 (TGF‐β1), β2, and β3, heparin‐binding factors known to regulate bone cell proliferation and differentiation, were considered likely candidates. The presence and distribution of these factors in calvarial tissues both in vivo and in vitro were established by immunohistochemical analysis, while reverse transcription followed by polymerase chain reaction (RT/PCR) was employed to determine the presence of transcripts for these factors in mRNA isolated from microdissected dura mater. Results indicated that the presence of TGF‐β1 and TGF‐β2 were associated with developing coronal and frontonasal sutures, and that the continued presence of these factors was associated with osseous obliteration of the frontonasal suture. However, increased TGF‐β3 immunoreactivity was associated with the coronal suture remaining unossified. RT/PCR demonstrated the presence of transcripts for TGF‐β1, β2, and β3 in dural tissues isolated from rat calvaria. These data support the notion of a role for TGF‐βs in regulating cranial suture morphogenesis and establish the in vitro model as a valid system for examining mechanisms by which growth factors regulate both suture morphogenesis and bone growth at the suture site.

Role of Basement Membranes in Cell Differentiation

Extracellular matrices have diverse biological effects, including promoting the growth and differentiation of various cells of epithelial origin. The components of one of these matrices, the basement membrane, are discussed, as well as studies using these components alone or in combination with cells in culture. The particular response observed varies with the cell type examined and appears to be dependent on multiple interactions with components of the matrix. Potential uses for a basement membrane-derived matrix in vitro and in vivo are being developed.

Injectable Tissue-Engineered Bone Repair of a Rat Calvarial Defect

Advances in bone repair have focused on the minimally‐invasive delivery of tissue‐engineered bone (TEB). A promising injectable biopolymer of chitosan and inorganic phosphates was seeded with mesenchymal stem cells (MSCs) and a bone growth factor (BMP‐2), and evaluated in a rat calvarial critical size defect (CSD). Green fluorescent protein (GFP)‐labeled MSCs are used to evaluate patterns of cell viability and proliferation.

The cellular plasticity of human adipocytes

Little is known regarding the biology of fat considering its extensive use clinically in soft tissue implantation. Free-fat transfer is problematic the result of graft site volume loss, appearing histologically as the replacement of mature adipocytes with a fibroblast-like infiltrate. We hypothesize that these histologic changes reflect a dedifferentiation of ischemic mature adipocytes instead of, or in addition to, a more traditional response. To explore this hypothesis, we studied the in vitro morphologic changes of cultured mature human adipocytes isolated from liposuctioned adipose tissue. Most adipocytes over time lost significant amounts of intracellular lipid. Ultimately, these cells lost all lipid, appeared fibroblastic, and proliferated to confluence. Adipogenic induction of such dedifferentiated adipocytes resulted in reaccumulation of intracellular lipid. This study demonstrates that mature adipocytes can be cultured from human liposuctioned fat, they can dedifferentiate into fibroblastic cells, and the fibroblast-like cells can be expanded and turned into lipid-synthesizing adipocytes. Exploration of this cellular plasticity might ultimately yield important insights into free-fat transfer and novel tissue-engineering strategies.

Laminin Nanofiber Meshes That Mimic Morphological Properties and Bioactivity of Basement Membranes

The basement membrane protein, laminin I, has been used broadly as a planar two-dimensional film or in a three-dimensional form as a reconstituted basement membrane gel such as Matrigel to support cellular attachment, growth, and differentiation in vitro. In basement membranes in vivo, laminin exhibits a fibrillar morphology, highlighting the electrospinning process as an ideal method to recreate such fibrous substrates in vitro. Electrospinning was employed to fabricate meshes of murine laminin I nanofibers (LNFs) with fiber size, geometry, and porosity of authentic basement membranes. Purified laminin I was solubilized and electrospun in parametric studies of fiber diameters as a function of polymer solution concentration, collecting distance, and flow rate. Resulting fiber diameters ranged from 90 to 300 nm with mesh morphologies containing beads. Unlike previously described nanofibers (NFs) synthesized from proteins such as collagen, meshes of LNFs retain their structural features when wetted and do not require fixation by chemical crosslinking, which often destroys cell attachment and other biological activity. The LNF meshes maintained their geometry for at least 2 days in culture without chemical crosslinking. PC12 cells extended neurites without nerve growth factor stimulation on LNF substrates. Additionally, LNFs significantly enhance both the rate and quantity of attachment of human adipose stem cells (ASCs) compared to laminin films. ASCs were viable and maintained attachment to LNF meshes in serum-free media for at least 3 days in culture and extended neurite-like processes after 24 h in serum-free media conditions without media additives to induce differentiation. LNF meshes are a novel substrate for cell studies in vitro, whose properties may be an excellent scaffold material for delivering cells in tissue engineering applications in vivo.

Regulation of cranial suture morphogenesis.

The cranial sutures are the primary sites of bone formation during skull growth. Morphogenesis and phenotypic maintenance of the cranial sutures are regulated by tissue interactions, especially those with the underlying dura mater. Removal of the dura mater in fetuses causes abnormal suture development and premature suture obliteration. The dura mater interacts with overlying tissues of the cranial vault by providing: (1) intercellular signals, (2) mechanical signals and (3) cells, which undergo transformation and migrate to the suture. The intercellular signaling governing suture development employs the fibroblast growth factors (FGFs). In rats during formation of the sutures in the fetus, FGF-1 is localized mainly in the dura mater, while other FGFs are expressed in the overlying tissues. By birth, FGF-2 largely replaces FGF-1 in the dura mater. FGFs present in the calvaria bind either the IIIb or IIIc mRNA splice variants of the FGF receptors (FGFRs) 1, 2, or 3. Monoclonal antibodies to the b variant of FGFR2 were used to determine the distribution of FGFR2IIIb during suture development and its extracellular localization. FGFR2IIIb is present in association with mature osteoblasts and osteogenic precursor cells of the suture in the fetus. Ectodomains of FGFR2IIIb, the products of proteolytic cleavage of the receptors, were present throughout the extracellular matrix of sutures resisting obliteration (coronal and sagittal), but absent from the core of sutures undergoing normal fusion (posterior intrafrontal). This observation is consistent with a possible mechanism, in which truncated receptors bind FGFs, thus regulating free FGF available to nearby cells. Mechanical signaling in the calvaria results from tensional forces in the dura mater generated during rapid expansion of the neurocranium. Posterior intrafrontal sutures of rats, which fuse between days 16 and 24, were subjected to cyclical tensional forces in vitro. Significant delay in the timing of suture fusion and increases in the expression domains of FGFR1 and 2 were observed, demonstrating the sensitivity of suture patency to mechanical signals and a possible role of the FGF system in mediating such stimuli. Finally, cells of the dura mater beneath the intrafrontal and sagittal sutures were observed to undergo a morphological transformation to a dendritic morphology and migrate into the suture mesenchyme between days 10 and 16 of development. This process may participate in suture and bone morphogenesis and influence the patency of the sutures along the anterior-posterior axis.

Comparative effects of scaffold pore size, pore volume, and total void volume on cranial bone healing patterns using microsphere-based scaffolds

Bony craniofacial deficits resulting from injury, disease, or birth defects remain a considerable clinical challenge. In this study, microsphere-based scaffold fabrication methods were use to study the respective effects of scaffold pore size, open pore volume, and total void volume fraction on osseous tissue infiltration and bone regeneration in a critical size rat cranial defect. To compare the healing effects of these parameters, three different scaffolds types were fabricated: solid 100 microm spheres, solid 500 microm spheres, and hollow 500 microm spheres. These constructs were implanted into surgically created rat calvarial defects. By 90-days post op, results of micro computed tomography (CT) analysis showed that all scaffolds generated similar amounts of new bone which was significantly greater than untreated controls. Interestingly, the spatial distribution of new bone within the defect area varied by scaffold group. MicroCT and histological analysis demonstrated healing restricted to the dural side in the hollow 500 microm group, whereas the solid 500 microm group demonstrated healing along the dural side and within the center of the defect. Solid 100 microm groups demonstrated healing along the dural layer, periosteal layer, and within the center of the defect. These results suggest that pore size and closed void volume may both play important roles in scaffold degradation patterns and associated bone healing.

Alignment and composition of laminin–polycaprolactone nanofiber blends enhance peripheral nerve regeneration

Peripheral nerve transection occurs commonly in traumatic injury, causing deficits distal to the injury site. Conduits for repair currently on the market are hollow tubes; however, they often fail due to slow regeneration over long gaps. To facilitate increased regeneration speed and functional recovery, the ideal conduit should provide biochemically relevant signals and physical guidance cues, thus playing an active role in regeneration. To that end, laminin and laminin-polycaprolactone (PCL) blend nanofibers were fabricated to mimic peripheral nerve basement membrane. In vitro assays established 10% (wt) laminin content is sufficient to retain neurite-promoting effects of laminin. In addition, modified collector plate design to introduce an insulating gap enabled the fabrication of aligned nanofibers. The effects of laminin content and fiber orientation were evaluated in rat tibial nerve defect model. The lumens of conduits were filled with nanofiber meshes of varying laminin content and alignment to assess changes in motor and sensory recovery. Retrograde nerve conduction speed at 6 weeks was significantly faster in animals receiving aligned nanofiber conduits than in those receiving random nanofiber conduits. Animals receiving nanofiber-filled conduits showed some conduction in both anterograde and retrograde directions, whereas in animals receiving hollow conduits, no impulse conduction was detected. Aligned PCL nanofibers significantly improved motor function; aligned laminin blend nanofibers yielded the best sensory function recovery. In both cases, nanofiber-filled conduits resulted in better functional recovery than hollow conduits. These studies provide a firm foundation for the use of natural-synthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits.

Chromatin structure of the ribosomal RNA genes in Physarum polycephalum

Nucleoli were purified from the slime mold Physarum polycephalum and assayed for enrichment in ribosomal DNA by analytical ultracentrifugation. Greater than 90% of the DNA from nucleoli comprises a satellite band characteristic of Physarum ribosomal DNA (rDNA) in a CsCl equilibrium sedimentation gradient. Over 75% of the nucleolar DNA molecules are 37×106 Daltons, a further characteristic of Physarum rDNA. Nucleoli incubated with micrococcal nuclease yield a distribution of discrete DNA fragments indicative of nucleosome subunits; these digestion products are indistinguishable in size from those of total nuclear chromatin. The nucleosome DNA repeat length varied with increasing digestion from 175 down to 150 base pairs. A palindrome structure for the ribosomal nucleoprotein molecules is demonstrated by electron microscopy of actively transcribing ribosomal RNA genes, which required modification of usual methods for dispersing chromatin. In the center of each palindrome is a 6.0–6.5 μm non-transcribed “spacer” region which exhibits a beaded nucleosome structure. Transcription initiates at points on either side of the central spacer, as evidenced by 4.0–4.2 μm matrices of growing rRNA fibrils extending to each end of the palindrome. The polarity of transcription matrices, together with information about the sites of rRNA coding sequences, imply that 19S rRNA is transcribed prior to 26S rRNA.