Journal: Stem Cells Translational Medicine

Article Title: A Selective Cell Population from Dermis Strengthens Bone Regeneration

doi: 10.5966/sctm.2015-0426

Figure Lengend Snippet: Characterization of BmprIB+ dermal cells. (A): Immunohistochemical staining of BmprIB in human foreskin. (B): Percentage evaluation of BmprIB expression in freshly isolated human dermal cells by flow cytometry. Histograms of BmprIB expression (left; 3.5% ± 0.4%) and isotype control (right). (C): Cell morphology under phase‐contrast microscopy. (D): The proliferative potential was assessed with the Alamar Blue assay in usDCs and BmprIB+ cells. Cell numbers were quantified by the absorbance wavelength at 570 nm using a spectrophotometer at 24, 48, 72, and 96 hours. Data represented the mean of three individuals and each of them was the mean of triplicate experiments. The osteogenic potential of BmprIB+ cells was demonstrated by ALP staining (E) and ARS staining (F) . (G): The mRNA expression levels of ALP (at day 7) and OCN , OPN , and BSP (at day 21) were analyzed by real‐time polymerase chain reaction after osteogenic induction. Data represent the mean ± SD ( n = 3). ∗, p < .05 indicates statistical significance. Scale bars = 50 µm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S stain; BmprIB, bone morphogenetic protein receptor type IB; usDC, unsorted dermal cell.

Article Snippet: For magnetic‐activated cell sorting, the cell suspensions were centrifuged and resuspended in phosphate‐buffered saline (PBS; Sigma‐Aldrich) containing 0.5% bovine serum albumin (BSA; Sigma‐Aldrich), labeled with phycoerythrin (PE)‐conjugated anti‐human BmprIB antibody (FAB5051P; R&D Systems, Minneapolis, MN, https://www.rndsystems.com ), and further incubated with anti‐PE microbeads (catalog no. 130‐048‐801; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com ).

Techniques: Immunohistochemical staining, Staining, Expressing, Isolation, Flow Cytometry, Control, Microscopy, Alamar Blue Assay, Spectrophotometry, Real-time Polymerase Chain Reaction

Journal: Stem Cells Translational Medicine

Article Title: A Selective Cell Population from Dermis Strengthens Bone Regeneration

doi: 10.5966/sctm.2015-0426

Figure Lengend Snippet: Dermal cell/coral scaffold compatibility. (A): Gross view of a coral scaffold with the size of 4‐mm diameter by 1‐mm thickness (top panel); two‐dimensional (left) and three‐dimensional images (right) of a coral scaffold by µCT scanning (bottom panel). (B): The cell proliferation analysis of the BmprIB+ cells and usDCs on the coral scaffold was assessed with the Alamar Blue assay at 12, 24, 36, 48, 60, 72, 84, and 96 hours. Data represent the mean of three individuals and each of them was the mean of triplicate experiments. (C): Scanning electron microscopy evaluation of the adherence and matrix deposition of BmprIB+ cells on the coral scaffold 1, 3, and 7 days after seeding. Scale bar = 20 µm. The coral alone without cell seeding served as a control. Scale bar = 100 µm. (D): Osteogenic potential of BmprIB+ cells and usDCs on the coral scaffold was evaluated according to the ALP activity and osteocalcin content at days 1, 3, 7, 14, 21, and 28 after osteogenic induction, respectively. Data represent the mean of three individuals and each of them was the mean of triplicate experiments. Abbreviations: 2D, two‐dimensional; ALP, alkaline phosphatase; BmprIB, bone morphogenetic protein receptor type IB; μCT, microcomputed tomography; usDC, unsorted dermal cell.

Article Snippet: For magnetic‐activated cell sorting, the cell suspensions were centrifuged and resuspended in phosphate‐buffered saline (PBS; Sigma‐Aldrich) containing 0.5% bovine serum albumin (BSA; Sigma‐Aldrich), labeled with phycoerythrin (PE)‐conjugated anti‐human BmprIB antibody (FAB5051P; R&D Systems, Minneapolis, MN, https://www.rndsystems.com ), and further incubated with anti‐PE microbeads (catalog no. 130‐048‐801; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com ).

Techniques: Alamar Blue Assay, Electron Microscopy, Control, Activity Assay, Tomography

Journal: Stem Cells Translational Medicine

Article Title: A Selective Cell Population from Dermis Strengthens Bone Regeneration

doi: 10.5966/sctm.2015-0426

Figure Lengend Snippet: Evaluation of early‐stage osteogenic reconstruction. (A): A full‐thickness defect 4 mm in diameter was created by a hollow drill on the right side of the calvarial bone and repaired by coral alone, usDCs/coral, or BmprIB+ cells per coral in each group (top panel). Representative images of two‐dimensional (left) and three dimensional (right) µCT scanning were taken 24 hours after the surgery (bottom panel). (B): Histological analyses including H&E staining and Masson trichrome staining were used to evaluate new bone formation 6 weeks postimplantation. The black arrows indicate both ends of the defect. Scale bar = 1 mm. (C): Immunostaining was used to evaluate osteogenic marker expression, including osteocalcin, osteopontin, bone sialoprotein, and vascular marker CD31 in newly formed bone. Scale bar = 50 µm. (D): Histomorphometric analysis was used to quantify the percentage of positive staining area or vascular count. Data represent the average ± SD ( n = 6). ∗, p < .05 indicates statistical significance. Abbreviations: BmprIB, bone morphogenetic protein receptor type IB; H&E, hematoxylin and eosin; usDC, unsorted dermal cell.

Article Snippet: For magnetic‐activated cell sorting, the cell suspensions were centrifuged and resuspended in phosphate‐buffered saline (PBS; Sigma‐Aldrich) containing 0.5% bovine serum albumin (BSA; Sigma‐Aldrich), labeled with phycoerythrin (PE)‐conjugated anti‐human BmprIB antibody (FAB5051P; R&D Systems, Minneapolis, MN, https://www.rndsystems.com ), and further incubated with anti‐PE microbeads (catalog no. 130‐048‐801; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com ).

Techniques: Staining, Immunostaining, Marker, Expressing

Journal: Stem Cells Translational Medicine

Article Title: A Selective Cell Population from Dermis Strengthens Bone Regeneration

doi: 10.5966/sctm.2015-0426

Figure Lengend Snippet: Microcomputed tomography (µCT) evaluation of late‐stage osteogenic reconstruction. (A): The defect was repaired by engineered bone on the right side of the mouse cranium 24 weeks postimplantation. Black arrows mark the repaired defect. (B): Representative three‐dimensional µCT images of parietal bones from each group 24 weeks postimplantation. Scale bar = 5 mm (left) and 1 mm (right). (C): The opaque percentage of each group was evaluated. (D): Other important bone structure parameters, such as BV (mm 3 ), BV/TV ratio (%), the Tb.N (1/mm), the Tb.Th (mm), the Tb.Sp, and Conn.D, were compared among these three implant groups with coral, usDCs/coral, and BmprIB/coral. Data represent the mean ± SD ( n = 6). ∗, p < .05 indicates statistical significance. Abbreviations: BmprIB, bone morphogenetic protein receptor type IB; BV, bone volume; BV/TV, ratio of bone volume to total volume; Conn.D, connectivity density; Tb.N, number of trabeculae; Tb.Sp, trabecular spacing; Tb.Th, thickness of the trabecular structure; usDC, unsorted dermal cell.

Article Snippet: For magnetic‐activated cell sorting, the cell suspensions were centrifuged and resuspended in phosphate‐buffered saline (PBS; Sigma‐Aldrich) containing 0.5% bovine serum albumin (BSA; Sigma‐Aldrich), labeled with phycoerythrin (PE)‐conjugated anti‐human BmprIB antibody (FAB5051P; R&D Systems, Minneapolis, MN, https://www.rndsystems.com ), and further incubated with anti‐PE microbeads (catalog no. 130‐048‐801; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com ).

Techniques: Tomography

Journal: Stem Cells Translational Medicine

Article Title: A Selective Cell Population from Dermis Strengthens Bone Regeneration

doi: 10.5966/sctm.2015-0426

Figure Lengend Snippet: Histological evaluation of late‐stage osteogenic reconstruction. (A): Goldner trichrome staining was used for plastic‐embedded, undecalcified, 24‐week regenerated bone. Mature bone matrix stained green and immature new bone matrix stained red. (B): Hematoxylin and eosin staining was also used to evaluate 24‐week regenerated bone. The black arrows indicate both ends of the defect. Scale bar = 1 mm. Abbreviations: BmprIB, bone morphogenetic protein receptor type IB; usDC, unsorted dermal cell.

Article Snippet: For magnetic‐activated cell sorting, the cell suspensions were centrifuged and resuspended in phosphate‐buffered saline (PBS; Sigma‐Aldrich) containing 0.5% bovine serum albumin (BSA; Sigma‐Aldrich), labeled with phycoerythrin (PE)‐conjugated anti‐human BmprIB antibody (FAB5051P; R&D Systems, Minneapolis, MN, https://www.rndsystems.com ), and further incubated with anti‐PE microbeads (catalog no. 130‐048‐801; Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com ).

Techniques: Staining