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Ca(2+)-deposition in cell matrix correlates significantly with osteocalcin-expression in osteogenic differentiated ATSC: Even in a coculture system with HUVEC.

Scheller K, Frerich B - J Oral Maxillofac Pathol (2013)

Bottom Line: Ca(2+)-deposition in the cell matrix and osteocalcin-expression correlated significantly (P = 0.030) during osteogenic differentiation (n = 7).The time of starting the coculture did not influence the differentiation.Measurement of the Ca(2+)-deposition correlates significantly to the osteogenic differentiation and osteocalcin-expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Surgery and Facial Plastic Surgery, University of Halle-Wittenberg, Halle, Germany.

ABSTRACT

Background: Tissue engineering offers the means for replacing or repairing diseased organs within the patient's body. The current problem in its clinical use is sufficient and fast revascularization of the transplanted tissues. The idea of bone-reconstruction deals with three-dimensional bone equivalents that are composed of endothelial cells (ECs) and adipose tissue derived stromal cells (ATSCs) showing osteogenic differentiation.

Materials and methods: ATSC were isolated, cultivated until third passage and osteogenically differentiated by 1.25-dihydroxycholecalciferol. Coculture systems with human umbilical vein endothelial cells (HUVEC) were performed. Osteogenic differentiation was analyzed in FACS-analyses (n = 7), by the measurement of Ca(2+)-deposition in the cell matrix (marker for osteogenic differentiation) and the expression of alkaline phosphatase (AP).

Results: Ca(2+)-deposition in the cell matrix and osteocalcin-expression correlated significantly (P = 0.030) during osteogenic differentiation (n = 7). The osteogenic cell differentiated ATSC in the coculture system (n = 6) even showed a clear increase of Ca(2+)-deposition. The time of starting the coculture did not influence the differentiation. Measurement of the Ca(2+)-deposition correlates significantly to the osteogenic differentiation and osteocalcin-expression.

Conclusion: ATSC are a promising source for bone tissue engineering. They can be differentiated into osteoblasts in a coculture system with HUVEC without the loss of any differentiation capacity. For bone tissue-equivalent fabrication, this is an encouraging procedure that is feasible and provides fast revascularization of the bone-equivalents.

No MeSH data available.


(a) Negative staining for osteocalcin in undifferentiated adipose tissue derived mesenchymal stromal cells (ATSC) (IHC stain, ×200), (b) Cell confluence of human umbilical vein endothelial cells (HUVEC) monitored by phase-contrast microscopy (×100). There was a positive expression of stem cell specific marker SH2 (c) (IHC stain, ×100) and SH3 (d) (IHC stain, ×100) in these undifferentiated ATSC. Osteogenic differentiation showed a positive reaction to Von Kossa (e) (Von Kossa stain, ×100), (f) silver staining (silver nitrate, ×100), (g) osteocalcin protein (IHC stain, ×100) and a positive reaction to enzyme alkaline phosphatase (h) (colorimetric enzyme assay, ×100)
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Figure 1: (a) Negative staining for osteocalcin in undifferentiated adipose tissue derived mesenchymal stromal cells (ATSC) (IHC stain, ×200), (b) Cell confluence of human umbilical vein endothelial cells (HUVEC) monitored by phase-contrast microscopy (×100). There was a positive expression of stem cell specific marker SH2 (c) (IHC stain, ×100) and SH3 (d) (IHC stain, ×100) in these undifferentiated ATSC. Osteogenic differentiation showed a positive reaction to Von Kossa (e) (Von Kossa stain, ×100), (f) silver staining (silver nitrate, ×100), (g) osteocalcin protein (IHC stain, ×100) and a positive reaction to enzyme alkaline phosphatase (h) (colorimetric enzyme assay, ×100)

Mentions: Preparation and cultivation of ATSC is according to methods described before.[67] Small pieces of subcutaneous AT (<0.5 cm3) from the lateral thigh of seven different donors (n = 7) were acquired during elective surgery (Department of Oral and Maxillofacial Surgery,). Informed consent was obtained. The AT was minced with sterile scissors and subjected to collagenase digestion (collagenase type II, Boehringer, Mannheim, Germany). The suspension was centrifuged (300 g/10 min) and plated in tissue culture flasks (Greiner, Frickenhausen, Germany). Cells were cultured in 5% humidified CO2 atmosphere at 37°C. “Standard” culture medium (Iscove's modified Dulbecco's medium IMDM/HAM F12 1:1) supplemented with 10% NCS (neonatal calf serum; all from Life Technology, Paisley, Scotland). It was changed every second day and used as control. After cell-colonization, the complete surface of the first culture flask were brought into suspension by trypsination (0.25% trypsin, 1 mM EDTA) and distributed in four new flasks (1st passage). Subsequently, they were split (1:4/5 ratio) and amplified up to the 3rd passage. The undifferentiated cells were negative for osteocalcin [Figure 1a].


Ca(2+)-deposition in cell matrix correlates significantly with osteocalcin-expression in osteogenic differentiated ATSC: Even in a coculture system with HUVEC.

Scheller K, Frerich B - J Oral Maxillofac Pathol (2013)

(a) Negative staining for osteocalcin in undifferentiated adipose tissue derived mesenchymal stromal cells (ATSC) (IHC stain, ×200), (b) Cell confluence of human umbilical vein endothelial cells (HUVEC) monitored by phase-contrast microscopy (×100). There was a positive expression of stem cell specific marker SH2 (c) (IHC stain, ×100) and SH3 (d) (IHC stain, ×100) in these undifferentiated ATSC. Osteogenic differentiation showed a positive reaction to Von Kossa (e) (Von Kossa stain, ×100), (f) silver staining (silver nitrate, ×100), (g) osteocalcin protein (IHC stain, ×100) and a positive reaction to enzyme alkaline phosphatase (h) (colorimetric enzyme assay, ×100)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3927332&req=5

Figure 1: (a) Negative staining for osteocalcin in undifferentiated adipose tissue derived mesenchymal stromal cells (ATSC) (IHC stain, ×200), (b) Cell confluence of human umbilical vein endothelial cells (HUVEC) monitored by phase-contrast microscopy (×100). There was a positive expression of stem cell specific marker SH2 (c) (IHC stain, ×100) and SH3 (d) (IHC stain, ×100) in these undifferentiated ATSC. Osteogenic differentiation showed a positive reaction to Von Kossa (e) (Von Kossa stain, ×100), (f) silver staining (silver nitrate, ×100), (g) osteocalcin protein (IHC stain, ×100) and a positive reaction to enzyme alkaline phosphatase (h) (colorimetric enzyme assay, ×100)
Mentions: Preparation and cultivation of ATSC is according to methods described before.[67] Small pieces of subcutaneous AT (<0.5 cm3) from the lateral thigh of seven different donors (n = 7) were acquired during elective surgery (Department of Oral and Maxillofacial Surgery,). Informed consent was obtained. The AT was minced with sterile scissors and subjected to collagenase digestion (collagenase type II, Boehringer, Mannheim, Germany). The suspension was centrifuged (300 g/10 min) and plated in tissue culture flasks (Greiner, Frickenhausen, Germany). Cells were cultured in 5% humidified CO2 atmosphere at 37°C. “Standard” culture medium (Iscove's modified Dulbecco's medium IMDM/HAM F12 1:1) supplemented with 10% NCS (neonatal calf serum; all from Life Technology, Paisley, Scotland). It was changed every second day and used as control. After cell-colonization, the complete surface of the first culture flask were brought into suspension by trypsination (0.25% trypsin, 1 mM EDTA) and distributed in four new flasks (1st passage). Subsequently, they were split (1:4/5 ratio) and amplified up to the 3rd passage. The undifferentiated cells were negative for osteocalcin [Figure 1a].

Bottom Line: Ca(2+)-deposition in the cell matrix and osteocalcin-expression correlated significantly (P = 0.030) during osteogenic differentiation (n = 7).The time of starting the coculture did not influence the differentiation.Measurement of the Ca(2+)-deposition correlates significantly to the osteogenic differentiation and osteocalcin-expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral and Maxillofacial Surgery and Facial Plastic Surgery, University of Halle-Wittenberg, Halle, Germany.

ABSTRACT

Background: Tissue engineering offers the means for replacing or repairing diseased organs within the patient's body. The current problem in its clinical use is sufficient and fast revascularization of the transplanted tissues. The idea of bone-reconstruction deals with three-dimensional bone equivalents that are composed of endothelial cells (ECs) and adipose tissue derived stromal cells (ATSCs) showing osteogenic differentiation.

Materials and methods: ATSC were isolated, cultivated until third passage and osteogenically differentiated by 1.25-dihydroxycholecalciferol. Coculture systems with human umbilical vein endothelial cells (HUVEC) were performed. Osteogenic differentiation was analyzed in FACS-analyses (n = 7), by the measurement of Ca(2+)-deposition in the cell matrix (marker for osteogenic differentiation) and the expression of alkaline phosphatase (AP).

Results: Ca(2+)-deposition in the cell matrix and osteocalcin-expression correlated significantly (P = 0.030) during osteogenic differentiation (n = 7). The osteogenic cell differentiated ATSC in the coculture system (n = 6) even showed a clear increase of Ca(2+)-deposition. The time of starting the coculture did not influence the differentiation. Measurement of the Ca(2+)-deposition correlates significantly to the osteogenic differentiation and osteocalcin-expression.

Conclusion: ATSC are a promising source for bone tissue engineering. They can be differentiated into osteoblasts in a coculture system with HUVEC without the loss of any differentiation capacity. For bone tissue-equivalent fabrication, this is an encouraging procedure that is feasible and provides fast revascularization of the bone-equivalents.

No MeSH data available.