Collagen Binding Proteins Derived from the Embryonic Fibroblast Cell Surface Recognize Arginine-Glycine-Aspartic Acid

Several cell surface proteins (Mr = 120,000, 90,000, 63,000 and 47,000) apparently integral to embryonic fibroblast plasma membranes were extracted with detergent and isolated by collagen affinity chromatography. Certain of these proteins (Mr = 120,000, 90,000, and 47,000) were specifically eluted from collagen affinity columns by synthetic peptides containing the amino acid sequence arginyl-glycyl-aspartic acid (RGD). These data show that a number of collagen binding proteins exist on the embryonic fibroblast cell surface. Some of the proteins may be collagen receptors binding to RGD sequences in the collagen molecule while at least one of the proteins (Mr = 63,000) recognizes features other than RGD

Laminin Potentiates Differentiation of PCC4uva Embryonal Carcinoma into Neurons

The embryonal carcinoma PCC4uva differentiates into neurons in response to treatment with retinoic acid and dbcAMP. We used this in vitro model system to study the effects of laminin on early neural differentiation. Laminin substrata markedly potentiate neural differentiation of retinoic acid and dbcAMP-treated cultures. Only laminin induced more rapid neural cell body clustering, neurite growth and neurite fasciculation as compared to type IV collagen, type I collagen, and fibronectin substrata. Exogenous laminin substrata promoted greater cell attachment, cellular spreading and growth to confluence than type IV collagen, type I collagen, fibronectin and glass substrata. Laminin-induced effects were inhibited by addition of laminin antibodies or the synthetic laminin-derived peptide Ile-Gly-Ser-Arg-NH2 (YIGSR-NH2). Treatment with YIGSR-NH2 also inhibited neural differentiation in the absence of exogenous laminin substrata, whereas synthetic peptides containing the RGD sequence and a control peptide YIGSK-NH2 showed no inhibitory effects. These results are consistent with the hypothesis that specific interactions between an early differentiating cell population(s) and extracellular laminin are required during neural differentiation

Preferential Lineage-Specific Differentiation of Osteoblast-Derived Induced Pluripotent Stem Cells into Osteoprogenitors

While induced pluripotent stem cells (iPSCs) hold great clinical promise, one hurdle that remains is the existence of a parental germ-layer memory in reprogrammed cells leading to preferential differentiation fates. While it is problematic for generating cells vastly different from the reprogrammed cells\u27 origins, it could be advantageous for the reliable generation of germ-layer specific cell types for future therapeutic use. Here we use human osteoblast-derived iPSCs (hOB-iPSCs) to generate induced osteoprogenitors (iOPs). Osteoblasts were successfully reprogrammed and demonstrated by endogenous upregulation of Oct4, Sox2, Nanog, TRA-1-81, TRA-16-1, SSEA3, and confirmatory hPSC Scorecard Algorithmic Assessment. The hOB-iPSCs formed embryoid bodies with cells of ectoderm and mesoderm but have low capacity to form endodermal cells. Differentiation into osteoprogenitors occurred within only 2-6 days, with a population doubling rate of less than 24 hrs; however, hOB-iPSC derived osteoprogenitors were only able to form osteogenic and chondrogenic cells but not adipogenic cells. Consistent with this, hOB-iOPs were found to have higher methylation of PPAR gamma but similar levels of methylation on the RUNX2 promoter. These data demonstrate that iPSCs can be generated from human osteoblasts, but variant methylation patterns affect their differentiation capacities. Therefore, epigenetic memory can be exploited for efficient generation of clinically relevant quantities of osteoprogenitor cells

Embryonic Chicken Fibroblast Collagen Binding Proteins: Distribution, Role in Substratum Adhesion, and Relationship to Integrins

Collagen binding proteins (CBP) are hydrophobic, cell surface polypeptides, isolated by collagen affinity chromatography. Antibodies to CBPs inhibit the attachment of embryonic chicken heart fibroblasts to native type I collagen fibrils in a dose-dependent manner. The CBP antibodies also induce rounding and detachment of cells adherent to a planar substratum. This process of antibody-mediated substratum detachment resulted in a clustering of CBP and cell-associated extracellular matrix at the cell surface, and the rearrangement of filamentous actin. Other functional studies showed that cells grown within a three-dimensional gel of type I collagen cannot be immunostained at the cell surface with CBP antibodies. However, treatment of cultures with purified collagenase, unmasks immunoreactive sites and permits strong cell surface immunolabeling. This result suggests that collagen sterically blocks antibody access to CBP. Finally, we show that antibodies to CBP recognize purified avian integrin beta subunits; and that antibodies to avian integrins recognize a 100,000 Mr CBP. These data demonstrate that chicken embryonic fibroblasts possess surface polypeptides that mediate adhesion to type I collagen, and suggest that two of these proteins are related to the integrin family

Insulinlike Growth Factor 1- and 2-Augmented Collagen Gel Repair of Facial Osseous Defects

BACKGROUND: Defects of the facial bone structure are common problems for the facial plastic surgeon. Native type 1 collagen gels (T1CGs) have been shown to mediate repair of facial critical-size defects in rat models. OBJECTIVE: To evaluate the efficacy of T1CG augmented with insulinlike growth factor (IGF) 1, IGF-2, and a combination of IGF-1 and IGF-2 on the repair of facial critical-size defects in a rodent model. METHODS: Twenty-four retired male breeder Sprague-Dawley rats were divided into 4 groups of 6 animals. Facial critical-size defects were created by removing the nasalis bones with a bone-cutting drill. Defects were treated with 300 pg of type 1 collagen gel (T1CG), T1CG augmented with 3 microg of IGF-1, T1CG augmented with 3 microg of IGF-2, or T1CG augmented with a combination of 3 microg of IGF-1 and 3 microg of IGF-2. After 30 days the animals were examined at necropsy with precise planimetry, histological analysis of new bone growth, and radiodensitometric analysis of bone thickness. RESULTS: Radiodensitometric measurements showed that IGF-2 augmentation resulted in greatest osseous healing, with measurements being statistically significant over those of all other groups (P\u3c or = .03). Combination IGF-1 and IGF-2 had osseous healing that was intermediate between IGF-1 augmentation and IGF-2 augmentation alone, with measurements being statistically significant over those of unaugmented gels (P CONCLUSION: Collagen gels augmented with IGF significantly enhance the osteoconductive repair of nasal critical-size defects in a rodent model, with IGF-2 showing highest efficacy

Accessible Bioprinting: Adaptation of a Low-Cost 3D-Printer for Precise Cell Placement and Stem Cell Differentiation

The precision and repeatability offered by computer-aided design and computer-numerically controlled techniques in biofabrication processes is quickly becoming an industry standard. However, many hurdles still exist before these techniques can be used in research laboratories for cellular and molecular biology applications. Extrusion-based bioprinting systems have been characterized by high development costs, injector clogging, difficulty achieving small cell number deposits, decreased cell viability, and altered cell function post-printing. To circumvent the high-price barrier to entry of conventional bioprinters, we designed and 3D printed components for the adaptation of an inexpensive \u27off-the-shelf\u27 commercially available 3D printer. We also demonstrate via goal based computer simulations that the needle geometries of conventional commercially standardized, \u27luer-lock\u27 syringe-needle systems cause many of the issues plaguing conventional bioprinters. To address these performance limitations we optimized flow within several microneedle geometries, which revealed a short tapered injector design with minimal cylindrical needle length was ideal to minimize cell strain and accretion. We then experimentally quantified these geometries using pulled glass microcapillary pipettes and our modified, low-cost 3D printer. This systems performance validated our models exhibiting: reduced clogging, single cell print resolution, and maintenance of cell viability without the use of a sacrificial vehicle. Using this system we show the successful printing of human induced pluripotent stem cells (hiPSCs) into Geltrex and note their retention of a pluripotent state 7 d post printing. We also show embryoid body differentiation of hiPSC by injection into differentiation conducive environments, wherein we observed continuous growth, emergence of various evaginations, and post-printing gene expression indicative of the presence of all three germ layers. These data demonstrate an accessible open-source 3D bioprinter capable of serving the needs of any laboratory interested in 3D cellular interactions and tissue engineering

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 lamininpolycaprolactone (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 naturalsynthetic blend electrospun nanofibers to enhance existing hollow nerve guidance conduits

Bioactive Lipid Coating of Bone Allografts Direct Engraftment and Fate Determination of Bone Marrow-Derived Cells in Rat GFP Chimeras

Bone grafting procedures are performed to treat wounds incurred during wartime trauma, accidents, and tumor resections. Endogenous mechanisms of repair are often insufficient to ensure integration between host and donor bone and subsequent restoration of function. We investigated the role that bone marrow-derived cells play in bone regeneration and sought to increase their contributions by functionalizing bone allografts with bioactive lipid coatings. Polymer-coated allografts were used to locally deliver the immunomodulatory small molecule FTY720 in tibial defects created in rat bone marrow chimeras containing genetically-labeled bone marrow for monitoring cell origin and fate. Donor bone marrow contributed significantly to both myeloid and osteogenic cells in remodeling tissue surrounding allografts. FTY720 coatings altered the phenotype of immune cells two weeks post-injury, which was associated with increased vascularization and bone formation surrounding allografts. Consequently, degradable polymer coating strategies that deliver small molecule growth factors such as FTY720 represent a novel therapeutic strategy for harnessing endogenous bone marrow-derived progenitors and enhancing healing in load-bearing bone defects

The Characterization of Ribosomal RNA Gene Chromatin from Physarum Polycephalum

We have isolated ribosomal RNA gene (rDNA) chromatin from Physarum polycephalum using a nucleolar isolation procedure that minimizes protein loss from chromatin and, subsequently, either agarose gel electrophoresis or metrizamide gradient centrifugation to purify this chromatin fraction (Amero, S. A., Ogle, R. C., Keating, J. L., Montoya, V. L., Murdoch, W. L., and Grainger, R. M. (1988) J. Biol. Chem. 263, 10725-10733). Metrizamide-purified rDNA chromatin obtained from nucleoli isolated according to the new procedure has a core histone/DNA ratio of 0.77:1. The major core histone classes comigrate electrophoretically with their nuclear counterparts on Triton-acid-urea/sodium dodecyl sulfate two-dimensional gels, although they may not possess the extent of secondary modification evident with the nuclear histones. This purified rDNA chromatin also possesses RNA polymerase I activity, and many other nonhistone proteins, including two very abundant proteins (26 and 38 kDa) that may be either ribonucleoproteins or nucleolar matrix proteins. Micrococcal nuclease digestion of the metrizamide-purified rDNA chromatin produces particles containing 145-base pair DNA fragments identical in length to those in total chromatin and which contain both transcribed and nontranscribed rDNA sequences. Some smaller fragments (30, 70, and 110 base pairs) are also seen, but their sequence content is not known. These particles sediment uniformly at 11 S in sucrose gradients containing 15 mM NaCl, and at 4-11 S in gradients containing 0.35 M NaCl. Particles enriched in gene or nontranscribed spacer sequences are not resolved in these sucrose gradients or in metrizamide gradients. Our findings suggest that the rDNA chromatin fraction we have identified contains transcriptionally active genes and that an organized, particle-containing structure exists in active rDNA chromatin

The Purification of Ribosomal RNA Gene Chromatin from Physarum Polycephalum

We have undertaken the purification of ribosomal RNA gene (rDNA) chromatin from the slime mold Physarum polycephalum, in order to study its chromatin structure. In this organism rDNA exists in nucleoli as highly repeated minichromosomes, and one can obtain crude chromatin fractions highly enriched in rDNA from isolated nucleoli. We first developed a nucleolar isolation method utilizing polyamines as stabilization agents that results in a chromatin fraction containing far more protein than is obtained by the more commonly used divalent cation isolation methods. The latter method appears to result in extensive histone loss during chromatin isolations. Two methods were then used for purifying rDNA chromatin from nucleoli isolated by the polyamine procedure. We found that rDNA chromatin migrates as a single band in agarose gels, well separated from other components in the chromatin preparation. Although the utility of this technique is somewhat limited by low yields and by progressive stripping of protein from rDNA chromatin, it can provide useful information about rDNA chromatin protein composition. The application of this technique to the fractionation of gene and spacer chromatin fragments produced by restriction enzyme digestion is discussed. We also found that rDNA chromatin, if RNase-treated, bands discretely in metrizamide equilibrium density gradients with a density lighter than that of non-nucleolar chromatin. These characteristics suggest that we have identified a transcriptionally active rDNA chromatin fraction which possesses a lower protein to DNA ratio than does non-nucleolar chromatin. This technique yields sufficient purified rDNA chromatin for further biochemical studies and does not cause extensive protein stripping. The procedures developed here should be applicable to the analysis of a variety of chromatin fractions in other systems