Three-dimensional Collagen Sponges Research Articles

Effect of strain on human dermal fibroblasts in a three-dimensional collagen sponge

Our aim was to design a simple compression system and investigate the influence of mechanical stress on skin-like structures. Many mechanical compression studies have employed intricate culture systems, so the relationship between extracellular matrix material and the response of skin cells to mechanical stress remains unknown. Our approach uses only glass vials, 6-well plates and standard laboratory equipment. We examined the influence of mechanical stress on human skin fibroblasts embedded within a collagen sponge. The results show that mechanical compression increases MMP-1 and MMP-2 release by the cells into the the cell culture. Our results suggest that pressure on the skin may affect extracellular matrix degradation through some as yet unidentified pathways and that IL-6 mRNA expression may be involved in this effect. Using our approach, the effects of static mechanical stress on protein expression by cells in the culture medium and in sponges can be easily examined, and therefore this system will be useful for further analyses of skin responses to mechanical stress.

Effects of 50 Hz electromagnetic fields on human epidermal stem cells cultured on collagen sponge scaffolds

Purpose: This study is to investigate the effects of electromagnetic fields (EMF) on proliferation of epidermal stem cells (ESC), which could present a viable clinical option for skin tissue engineering. Materials and methods: The ESC obtained from human foreskin were grafted into type-I three-dimensional collagen sponge scaffolds, and then were exposed with EMF (frequency 50 Hz, intensity 5 mT) for 14 d (30 min per d). Meanwhile, the control group was set under the same conditions without EMF. The effects of EMF on growth and proliferation of ESC were analyzed with staining of hematoxylin and eosin (H&E) and 4′,6-diamidino-2-phenylindole (DAPI) under microscope or scanning electron microscope. The data of DAPI staining for 2 d, 7 d, 10 d and 14 d were collected respectively to investigate the cells proliferation. Results: ESC cultured in collagen sponge scaffolds could be steady grown and EMF could promote ESC proliferation compared with control (P < 0.05). Conclusions: EMF could significantly promote proliferation of ESC, which leads to a promising clinical option for skin tissue engineering.

Spatially Guided Angiogenesis by Three-Dimensional Collagen Scaffolds Micropatterned with Vascular Endothelial Growth Factor

Successful regeneration of large and highly functionalized tissue and organs depends on the ability to guide blood vessel formation with three-dimensional scaffolds. Angiogenic growth factors have the potential to stimulate blood vessels in scaffolds. However, simply incorporating angiogenic growth factors in a random fashion may lead to uncontrolled blood vessel generation, which ultimately results in poor blood vessel network function and uneven growth of engineered tissue. To control and guide the formation of a blood vessel network in porous scaffolds, we prepared collagen sponges with micropatterned vascular endothelial growth factor (VEGF). VEGF was micropatterned in three-dimensional collagen sponges using micropatterned collagen/VEGF ice lines, which were prepared by a dispersing machine. The VEGF-micropatterned collagen sponges were implanted subcutaneously in nude mice. Following 6 weeks of implantation, the VEGF-micropatterned collagen sponges induced the formation of micropatterned blood vessel networks. More blood vessels were observed in the regions in which VEGF was immobilized than those without VEGF. The micropattern of VEGF determined the micropattern of the regenerated blood vessel network. The spatial immobilization of VEGF in three-dimensional porous scaffolds may be useful to stimulate guided blood vessel formation in a variety of tissue-engineering applications.

Differentiation of PC12 cells in three‐dimensional collagen sponges with micropatterned nerve growth factor

Micropatterning of biological cues is important for the guided formation of neuronal outgrowth and neuronal differentiation. Nerve growth factor (NGF) was micropatterned in a three-dimensional collagen sponges by using micropatterned ice lines that were composed of collagen and NGF. The micropatterned ice lines were prepared by a dispersing machine. PC12 cells were cultured in the NGF-micropatterned collagen sponges and showed micropatterned neurite outgrowth. The neurite outgrowth followed the micropattern of NGF with more neurite outgrowth in the collagen/NGF lines than in the regions between the collagen/NGF lines. The micropattern of the NGF and the neurite network of the PC12 cells can be manipulated by controlling the micropattern of the NGF. The three-dimensional porous scaffolds prepared by this method will have a potential application for the regeneration and repair of the nervous system.

Engineering a joint: a chimeric construct with bovine chondrocytes in a devitalized chick knee.

This study assessed the feasibility of a devitalized knee as a scaffold for an engineered chimeric joint. Embryonic chick knees (19 days old), devitalized by lyophilization or multiple freeze-thaw cycles, were tested as scaffolds for repopulation with bovine articular chondrocytes (bACs). bACs were seeded into porous three-dimensional collagen sponges and were cultured for 1 day before fabrication of chimeric constructs. A pair of cell-seeded sponges was inserted into the joint space to contact preshaved articular surfaces. In some constructs, a sterile membrane of expanded polytetrafluoroethylene (ePTFE) was inserted between the collagen sponges. Histologic analysis showed that at 1 week, sponges with bACs were adherent to the shaved articular surfaces of the joint with accumulation of metachromatic extracellular matrix. Penetration of bACs and neomatrix into the devitalized matrix appeared to begin in preexistent epiphyseal canals and was observed to some extent in all specimens. Membranes of ePTFE maintained a joint space at 2 and 3 weeks, whereas there was fusion across the two sponges in many specimens lacking the membrane. Gene expression analysis demonstrated that lyophilization, but not multiple freeze-thaw cycles, completely devitalized the chick knees. These studies identified several design parameters crucial for successful engineering of a chimeric joint.

Ascorbate modulation of bovine chondrocyte growth, matrix protein gene expression and synthesis in three-dimensional collagen sponges

This report completes a previous study on the growth and metabolism of fetal bovine epiphyseal chondrocytes cultured, within native or cross-linked collagen sponges carried out without the addition of fresh ascorbate. At low initial cell density (2.3×10 6 cells/cm 3) cell proliferation and a low matrix deposition were observed, whereas at high initial cell density (2.3×10 7 cells/cm 3) there was an absence of cell proliferation, but the deposition of a cartilage-like matrix was measured. In both cases, only traces of type I collagen (marker of chondrocyte dedifferentiation) were detected. In this report, we observed, after 1 month in culture with ascorbate, in both type of scaffolds and initial cell densities, an increase in cell proliferation (2-fold) and in expression of genes encoding for collagen types I, II, X and MMP-2 and -13, but no change in the level of matrix deposition (collagen and GAG). With regard to the proteins present, the main differences with or without ascorbate concerned the increase of neosynthesised type I collagen (up to 35% of the total collagen deposited in the sponge) and of the MMP-2 active form. In conclusion, these results show that ascorbate is an important factor to consider when preparing cartilage constructs for its action on chondrocyte phenotype modulation and proliferation.

Age-related decline in the osteogenic potential of human bone marrow cells cultured in three-dimensional collagen sponges.

Studies with human and animal culture systems indicate that a sub-population of bone marrow stromal cells has the potential to differentiate into osteoblasts. There are conflicting reports on the effects of age on human marrow-derived osteogenic cells. In this study, we used a three dimensional (3D) culture system and quantitative RT-PCR methods to test the hypothesis that the osteogenic potential of human bone marrow stromal cells decreases with age. Marrow was obtained from 39 men aged 37 to 86 years, during the course of total hip arthroplasty. Low-density mononuclear cells were seeded onto 3D collagen sponges and cultured for 3 weeks. Histological sections of sponges were stained for alkaline phosphatase activity and were scored as positive or negative. In the group < or = 50 years, 7 of 11 samples (63%) were positive, whereas only 5 of 19 (26%) of the samples in the group > or = 60 years were positive (p = 0.0504). As revealed by RT-PCR, there was no expression of alkaline phosphatase or collagen type I mRNA before culture, however there were strong signals after 3 weeks, an indication of osteoblast differentiation in vitro. We performed a quantitative, competitive RT-PCR assay with 8 samples (age range 38-80) and showed that the group < or = 50 years had 3-fold more mRNA for alkaline phosphatase than the group > or = 60 years (p = 0.021). There was a significant decrease with age (r = - 0.78, p = 0.028). These molecular and histoenzymatic data indicate that the osteogenic potential of human bone marrow cells decreases with age.

Early Shifts in Gene Expression during Chondroinduction of Human Dermal Fibroblasts

Treatment options for damaged articular cartilage are limited because of that tissue's poor capacity for repair. Possible approaches to this problem are to stimulate cartilage matrix production in situ or to engineer replacement tissue. Both of these approaches would benefit from a detailed understanding of the molecular mechanisms of chondroblast differentiation. In previous studies, we described a novel in vitro model of postnatal chondroblast differentiation. That model of induced chondrogenesis was used to test the hypothesis that cellular interactions with demineralized bone powder (DBP) would induce specific, early shifts in gene expression, prior to the expression of cartilage matrix genes. Differentially expressed genes were identified by representational difference analysis of human dermal fibroblasts cultured for 3 days with DBP in three-dimensional collagen sponges. Genes that were upregulated by DBP comprised several functional classes, including cytoskeletal elements, protein synthesis and trafficking, and transcriptional regulation. Kinetic analysis of gene expression over 21 days showed that vigilin was transiently upregulated on day 3. In contrast, expression of cartilage signature genes continued to increase. These results are an important step toward complete characterization of the mechanisms by which DBP induces chondroblastic differentiation in postnatal cells.

Effects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges.

Various culture systems have been used for examining the anabolic and catabolic functions of isolated chondrocytes as well as for tissue engineering purposes. Perfusion or frequent medium change is beneficial for three-dimensional (3D) cultures of many cell types. In this study, bovine articular chondrocytes (bACs) were grown in 3D collagen sponges with or without medium perfusion (0.33 mL/min) for up to 15 days. The influence of medium perfusion was evaluated using markers of cartilage matrix accumulation, synthesis, and gene expression. Metachromatic matrix, collagen type II, and hyaluronan accumulated around the cells within the collagen sponges. Sulfated glycosaminoglycans (S-GAGs) that accumulated in the sponge exposed to nonperfused control were 130% of that in the perfused sponge at day 7. S-GAG accumulation after 15 days in the nonperfused control was 230% more than at day 7 (p < 0.01). (35)S-sulfate incorporation during the final 18 h of culture in the sponge exposed to nonperfusion was 180% greater than that in the perfused sponge (p < 0.01). Quantitative analyses show that at day 7, aggrecan and collagen type II gene expression were 350% and 240% greater, respectively, in the nonperfused culture than in the perfused one. These results indicate that perfused conditions that are beneficial for other cell types inhibit chondrogenesis by articular chondrocytes in 3D culture.

Age-Related Decline in Osteoprotegerin Expression by Human Bone Marrow Cells Cultured in Three-Dimensional Collagen Sponges

With advancing age, an increase in bone resorption relative to bone formation results in bone loss. Bone marrow stromal cells and their products support osteoclastogenesis from hematopoietic progenitors. Another of their products, osteoprotegerin (OPG), blocks the osteoclast–stimulatory effects of OPG ligand. We tested the hypothesis that with advancing age there is a decrease in OPG expression by human bone marrow cells. Bone marrow cells were obtained from 18 subjects (age range 38–84 years). Expression of mRNA transcripts of OPG was assessed by quantitative competitive RT-PCR. Median number of OPG transcripts in the younger group was 0.3 zetptomoles (range 0.01 to 1.30) and was higher than in the older group's median of 0.06 (range 0 to 0.5; p < 0.05). The decline in the expression of OPG with age may increase the capacity of stromal/osteoblast cells to support osteoclastogenesis.

Medium perfusion enhances osteogenesis by murine osteosarcoma cells in three-dimensional collagen sponges.

In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis.