Three-dimensional composite of demineralized bone powder and collagen for in vitro analysis of chondroinduction of human dermal fibroblasts

Biomaterials ◽  
1996 ◽  
Vol 17 (18) ◽  
pp. 1819-1825 ◽  
Author(s):  
Shuichi Mizuno ◽  
Julie Glowacki
Keyword(s):  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
Bone Powder ◽  
Vitro Analysis ◽  
In Vitro Analysis ◽  
Demineralized Bone

scholarly journals Validation of in vitro assays in three-dimensional human dermal constructs

2018 ◽  
Vol 41 (11) ◽  
pp. 779-788 ◽  
Author(s):  
Ayesha Idrees ◽  
Valeria Chiono ◽  
Gianluca Ciardelli ◽  
Siegfried Shah ◽  
Richard Viebahn ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
In Vitro Assays ◽  
Dimensional System ◽  
Type I ◽  
Human Dermal Fibroblasts ◽  
Cell Viability Assay ◽  
Normal Human ◽  
Normal Human Dermal Fibroblasts

Three-dimensional cell culture systems are urgently needed for cytocompatibility testing of biomaterials. This work aimed at the development of three-dimensional in vitro dermal skin models and their optimization for cytocompatibility evaluation. Initially “murine in vitro dermal construct” based on L929 cells was generated, leading to the development of “human in vitro dermal construct” consisting of normal human dermal fibroblasts in rat tail tendon collagen type I. To assess the viability of the cells, different assays CellTiter-Blue®, RealTime-Glo™ MT, and CellTiter-Glo® (Promega) were evaluated to optimize the best-suited assay to the respective cell type and three-dimensional system. Z-stack imaging (Live/Dead and Phalloidin/DAPI-Promokine) was performed to visualize normal human dermal fibroblasts inside matrix revealing filopodia-like morphology and a uniform distribution of normal human dermal fibroblasts in matrix. CellTiter-Glo was found to be the optimal cell viability assay among those analyzed. CellTiter-Blue reagent affected the cell morphology of normal human dermal fibroblasts (unlike L929), suggesting an interference with cell biological activity, resulting in less reliable viability data. On the other hand, RealTime-Glo provided a linear signal only with a very low cell density, which made this assay unsuitable for this system. CellTiter-Glo adapted to three-dimensional dermal construct by optimizing the “shaking time” to enhance the reagent penetration and maximum adenosine triphosphate release, indicating 2.4 times higher viability value by shaking for 60 min than for 5 min. In addition, viability results showed that cells were viable inside the matrix. This model would be further advanced with more layers of skin to make a full thickness model.

scholarly journals Expression of pro-inflammatory markers by human dermal fibroblasts in a three-dimensional culture model is mediated by an autocrine interleukin-1 loop

2004 ◽  
Vol 379 (2) ◽  
pp. 351-358 ◽  
Author(s):  
Daniela KESSLER-BECKER ◽  
Thomas KRIEG ◽  
Beate ECKES
Keyword(s):  
Keratinocyte Growth Factor ◽  
Interleukin 1 ◽  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
Inflammatory Processes ◽  
Cox 2 ◽  
Extracellular Matrix Interactions

In vivo, fibroblasts reside in connective tissues, with which they communicate in a reciprocal way. Such cell–extracellular matrix interactions can be studied in vitro by seeding fibroblasts in collagen lattices. Depending upon the mechanical properties of the system, fibroblasts are activated to assume defined phenotypes. In the present study, we examined a transcriptional profile of primary human dermal fibroblasts cultured in a relaxed collagen environment and found relative induction (>2-fold) of 393 out of approx. 7100 transcripts when compared with the same system under mechanical tension. Despite down-regulated proliferation and matrix synthesis, cells did not become generally quiescent, since they induced transcription of numerous other genes including matrix metalloproteinases (MMPs) and growth factors/cytokines. Of particular interest was the induction of gene transcripts encoding pro-inflammatory mediators, e.g. cyclo-oxygenase-2 (COX-2), and interleukins (ILs)-1 and -6. These are apparently regulated in a hierarchical fashion, since the addition of IL-1 receptor antagonist prevented induction of COX-2, IL-1 and IL-6, but not that of MMP-1 or keratinocyte growth factor (KGF). Our results suggest strongly that skin fibroblasts are versatile cells, which adapt to their extracellular environment by displaying specific phenotypes. One such phenotype, induced by a mechanically relaxed collagen environment, is the ‘pro-inflammatory’ fibroblast. We propose that fibroblasts that are embedded in a matrix environment can actively participate in the regulation of inflammatory processes.

scholarly journals In-vitro analysis of maxillary first molars morphology using three dimensional Micro-CT imaging: considerations for restorative dentistry

2019 ◽  
pp. 75-81 ◽  
Author(s):  
Ismail Hakki Baltacioglu ◽  
◽  
Gulbike Demirel ◽  
Mehmet Eray Kolsuz ◽  
Kaan Orhan ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Ct Imaging ◽  
Micro Ct ◽  
Restorative Dentistry ◽  
Vitro Analysis ◽  
In Vitro Analysis

A Collagen/DBP Sponge System Designed for in Vitro Analysis of Chondroinduction

1991 ◽  
Vol 252 ◽  
Author(s):  
Shuichi Mizuno ◽  
Chris Lycette ◽  
Charlene Quinto ◽  
Julie Glowacki
Keyword(s):  
Type I Collagen ◽  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Cellular Mechanisms ◽  
Fresh Tissue ◽  
Freeze Dried ◽  
Bone Powder

ABSTRACTIn response to subcutaneous implants of demineralized bone powder (DBP), cells are attracted to the DBP, are converted to chondroblasts, and produce a cartilage matrix that is resorbed and replaced by bone. In order to define the cellular mechanisms of this induction, we developed a collagen sponge model for simulating the in vivo environment and for promoting the ingrowth and viability of cells cultured in them in vitro. Reconstituted pepsin–digested type I collagen from bovine hide was neutralized. Rat DBP (75–250 εm) was added into the collagen mixture (20 mg/ml). In order to simulate the connective tissue environment, modified chondroitin sulfate, heparan sulfate, or hyaluronic acid was added into the mixture. Aliquots (0.2 ml) were placed in 3/8 inch diameter molds and freeze-dried. Human dermal fibroblasts were cultured from minced fresh tissue and inoculated at 1.5 × 105 cells/sponge. Fifteen hours later, some sponges were transferred to medium which contained growth factors (PDGF or TGF-β). At intervals, samples were examined histologically. The inoculated cells attached to the collagen fibers and migrated into the sponge. Eventually the sponges contracted and acquired an oval shape. Cells on or near DBP were ovoid or stellate in shape. Cell morphology was modulated by glycosaminoglycan composition of the sponge. Increasing doses of PDGF or TGF-β promoted cellularity within the sponges. In conclusion, this system simulates the in vivo environment but allows accessibility for analysis. This three-dimensional matrix culture system will enable investigation of mechanisms of chondroinduction by morphogenic material.

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